Effect of lipoic acid combined with paclitaxel on breast cancer cells.

PubMed

Li, B J; Hao, X Y; Ren, G H; Gong, Y

2015-12-22

Breast cancer is the most common gynecologic tumor globally that threatens women's health. Lipoic acid is a type of antioxidant that can alleviate oxidative stress damage. Studies showed that lipoic acid could inhibit the proliferation of tumor cells in cervical cancer and colon cancer. This paper intends to explore the combined effect of lipoic acid and paclitaxel on breast cancer cells. Breast cancer MCF-7 cells were divided into four groups: control group, lipoic acid group, paclitaxel group, and a combination group. MTT was applied to detect the drugs' influence on breast cancer cell proliferation. A colony formation test was used to determine the effects on breast cancer cell clone formation rate. Western blot was performed to detect the effects on nuclear factor (NF)-κB. Lipoic acid alone can inhibit tumor cell proliferation and clone formation with time dependence. Compared with the control, paclitaxel alone can significantly suppress tumor cell proliferation and clone formation (P < 0.05). Lipoic acid and paclitaxel in combination obviously strengthened their individual inhibitory effects on tumor cells (P < 0.05). Compared with the paclitaxel alone group, the combination group exhibited more remarkable inhibitory effect (P < 0.05). Lipoic acid alone or combined with paclitaxel can inhibit NF-κB expression and inhibit breast cancer cell proliferation.

Acetaminophen and Metamizole Induce Apoptosis in HT 29 and SW 480 Colon Carcinoma Cell Lines In Vitro.

PubMed

Bundscherer, Anika C; Malsy, Manuela; Gruber, Michael A; Graf, Bernhard M; Sinner, Barbara

2018-02-01

The perioperative phase is supposed to be a period with high vulnerability for cancer dissemination. Acetaminophen and metamizole are common analgesics administered during this phase. We investigated the effect of acetaminophen, metamizole and 4-methylaminoantipyrine (MAA) on proliferation and apoptosis of colon carcinoma cell lines (SW 480 and HT 29). Proliferation was detected by cell proliferation ELISA BrdU, and apoptosis by Annexin V staining. Cytochrome c and caspase 3, 8 and 9 expression levels were detected by western blot. Acetaminophen, metamizole or MAA caused slight changes in proliferation. Acetaminophen, metamizole or the combination increased apoptosis in both cell lines. All agents decreased caspase 3 and 8 expression in SW480. Acetaminophen decreased caspase 9 expression in both cell lines. In clinically relevant doses, acetaminophen and/or metamizole induce apoptosis in both colon cancer cell lines. Both mitochondrial and death receptor pathways might be involved in acetaminophen-induced apoptosis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica

Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition,more » EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.« less

[Overexpression of inhibitor of β-catenin and T cell factor (ICAT) promotes proliferation and migration of cervical cancer Caski cells].

PubMed

Jiang, Yayun; Wang, Ting; Wang, Jinshu; Xia, Jing; Gou, Liyao; Liu, Mengyao; Zhang, Yan

2016-11-01

Objective To investigate the effect of overexpressed inhibitor of β-catenin and T cell factor (ICAT) on the proliferation and migration of human cervical cancer Caski cells. Methods Caski cells were transfected with ICAT recombinant adenovirus (AdICAT). The levels of ICAT mRNA and protein were detected by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively. Effect of ICAT overexpression on proliferation, cell cycle and migration in Caski cells was respectively evaluated by MTT assay, flow cytometry and Transwell TM migration assays. Results The expression of ICAT remarkably increased in Caski cells after AdICAT infection. Overexpression of ICAT promoted Caski cells' proliferation, arrested the cell cycle in the S phase and enhanced cell migration. Conclusion Overexpression of ICAT can promote the proliferation and migration of Caski cervical cancer cells.

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animalsmore » examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication.« less

Infectious mononucleosis accompanied by clonal proliferation of EBV-infected cells and infection of CD8-positive cells.

PubMed

Arai, Ayako; Yamaguchi, Takeshi; Komatsu, Honami; Imadome, Ken-Ichi; Kurata, Morito; Nagata, Kaoru; Miura, Osamu

2014-01-01

A 22-year-old male was admitted for a sustained fever of 2 months, lymphadenopathy, and liver dysfunction. Anti-VCA-IgM antibody was positive, with elevated Epstein-Barr virus (EBV)-DNA load in the peripheral blood. Liver biopsy revealed infiltration of CD8-positive and EBV-positive cells. Most peripheral blood mononuclear cells (PBMCs) were also positive for CD8, and showed detectable levels of EBV-DNA. Monoclonal proliferation of EBV-infected cells was detected in the PBMCs by Southern blotting for EBV-terminal repeat (EBV-TR). Although EBV-positive T-cell lymphoproliferative disease (EBV-T-LPD) was suspected, the symptoms spontaneously resolved within 12 months. Anti-VCA-IgM antibody and the clonal band of EBV-TR were negative 1 year after the onset, while anti-EBNA antibody was positive. The final diagnosis was thus confirmed as infectious mononucleosis (IM). Our results indicate that EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells may be temporally detected in IM. EBV-T-LPDs should be carefully excluded in such cases.

Osthole inhibits proliferation and induces apoptosis in human osteosarcoma cells.

PubMed

Ding, Yong; Lu, Xiongwei; Hu, Xiaopeng; Ma, Jie; Ding, Huan

2014-02-01

The purpose of this study was to investigate the effect of osthole on osteosarcoma cell proliferation and apoptosis. Cell counting Kit-8 assay was performed to establish the effects of osthole on osteosarcoma MG-63 cell proliferation. Annexin V-FITC/PI was performed to analyze the apoptotic rate of the cells. The inhibitory effects of osthole on the expression of BCL-2, BAX, and caspase-3 were detected by Western blotting. Osthole inhibited the growth of human osteosarcoma MG-63 cells by inhibiting cell proliferation and induced cell apoptosis. Western blotting demonstrated that osthole downregulated the expressions of BCL-2 and caspase-3 and upregulated the expression of BAX in human osteosarcoma cells. Osthole can inhibit osteosarcoma cell proliferation and induced apoptosis effectively in a dose-dependent manner through downregulating the expression of BCL-2 and caspase-3 proteins levels and upregulating the expression of BAX proteins levels.

Detection of p53 mutations in proliferating vascular cells in glioblastoma multiforme.

PubMed

Kawasoe, Takuma; Takeshima, Hideo; Yamashita, Shinji; Mizuguchi, Sohei; Fukushima, Tsuyoshi; Yokogami, Kiyotaka; Yamasaki, Kouji

2015-02-01

Glioblastoma multiforme (GBM), one of the most aggressive tumors in humans, is highly angiogenic. However, treatment with the angiogenesis inhibitor bevacizumab has not significantly prolonged overall patient survival times. GBM resistance to angiogenesis inhibitors is attributed to multiple interacting mechanisms. Although mesenchymal transition via glioma stem-like cells has attracted attention, it is considered a poor biomarker. There is no simple method for differentiating tumor-derived and reactive vascular cells from normal cells. The authors attempted to detect the mesenchymal transition of tumor cells by means of p53 and isocitrate dehydrogenase 1 (IDH1) immunohistochemistry. Using antibody against p53 and IDH1 R132H, the authors immunohistochemically analyzed GBM tissue from patients who had undergone surgery at the University of Miyazaki Hospital during August 2005-December 2011. They focused on microvascular proliferation with a p53-positive ratio exceeding 50%. They compared TP53 mutations in original tumor tissues and in p53-positive and p53-negative microvascular proliferation cells collected by laser microdissection. Among 61 enrolled GBM patients, the first screening step (immunostaining) identified 46 GBMs as p53 positive, 3 of which manifested areas of prominent p53-positive microvascular proliferation (>50%). Histologically, areas of p53-positive microvascular proliferation tended to be clustered, and they coexisted with areas of p53-negative microvascular proliferation. Both types of microvascular proliferation cells were clearly separated from original tumor cells by glial fibrillary acidic protein, epidermal growth factor receptor, and low-/high-molecular-weight cytokeratin. DNA sequencing analysis disclosed that p53-positive microvascular proliferation cells exhibited TP53 mutations identical to those observed in the original tumor; p53-negative microvascular proliferation cells contained a normal allele. Although immunostaining indicated that 3 (2 primary and 1 secondary) of the 61 GBMs were positive for IDH1, no tumors contained microvascular proliferation cells positive for IDH1 R132H. Some microvascular proliferation clusters in GBM result from mesenchymal transition. The identification of useful markers might reveal this phenomenon as an infrequent event in GBMs.

Dose-related cell proliferation in medaka (Oryzias latipes) after N-nitrosodiethylamine exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortego, L.S.; Hawkins, W.E.; Walker, W.W.

1994-12-31

Cell proliferation is important in toxic and carcinogenic mechanisms. Carcinogens such as N-nitrosodiethylamine (DEN) that cause necrotizing injury stimulate cell proliferation as part of an injury-repair mechanism. A stimulus to cell division in an organ with a low rate of cell division, such as the liver, may initiate or enhance the carcinogenicity of a chemical. The authors examined the effect of DEN exposure on cell proliferation in the liver of medaka (Oryzias latipes). Two age groups (6 and 56 days post-hatch) were exposed to DEN continuously at 5 doses (0.0, 2.5, 5.0, 10.0, and 20.0 ppm) for 28 days. Cellmore » proliferation was measured using the proliferating cell nuclear antigen (PCNA) assay two months post-initiation of DEN exposure. The assay involves monoclonal antibody detection of PCNA, an auxiliary protein of DNA polymerase delta which is, expressed during cell division. Results suggested that cell proliferation paralleled the DEN dose and that age at initiation of exposure did not affect this relationship. The increase in cell proliferation appeared to be a sustained response from that initiated during DEN exposure. The study suggests that cell proliferation in medaka is an important component in carcinogenesis and is related to carcinogen exposure dose.« less

Cytokinetics of adult rat SVZ after EAE.

PubMed

Sajad, Mir; Chawla, Raman; Zargan, Jamil; Umar, Sadiq; Sadaqat, Mir; Khan, Haider A

2011-01-31

Cytokinetics regulating cell cycle division can be modulated by several endogenous factors. EAE (experimental autoimmune encephalomyelitis) increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S phase labeling with 5-bromo-2-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after EAE. 20% of the SVZ cell population was proliferating in adjuvant control rats. However, EAE significantly increased them up to 27% and these cells had a cell cycle length (TC) of 15.6h, significantly (P<0.05) shorter than the 19 h TC in non EAE SVZ cells. Few TUNEL (+) cells were detected in the SVZ cells of adjuvant controls. EAE increased (P<0.05) TUNEL (+) nuclei in SVZ suggesting early stage progenitor cell death. Cell cycle phase analysis revealed that EAE substantially shortened the length of the G1 phase (9.6h) compared with the G1 phase of 12.25 h in adjuvant control SVZ cells (P<0.05). This reduction in G1 contributes to EAE-induced reduction of TC because no significant changes were detected on the length of S, G2 and M phases between the two groups. Our results show a surge in proliferating progenitor cells in the SVZ with concomitant increase in apoptotic cell death after EAE. Furthermore, increase in the SVZ proliferation contributes to EAE-induced neurogenesis and this increase is regulated by shortening the G1 phase. Our investigation suggests the activation of quiescent cells in SVZ to generate actively proliferating progenitors. Moreover, the increase in the cell death in proliferating population may contribute towards negative regulation of proliferative cell number and hence diminished regenerative capacity of CNS following EAE. Copyright © 2010 Elsevier B.V. All rights reserved.

Feasibility of obtaining breast epithelial cells from healthy women for studies of cellular proliferation.

PubMed

Miller, N A; Thomas, M; Martin, L J; Hedley, D W; Michal, S; Boyd, N F

1997-05-01

Increased dietary fat intake and rate of breast epithelial cell proliferation have each been associated with the development of breast cancer. The goal of this study was to measure the effect of a low fat, high carbohydrate diet on the rate of breast epithelial cell proliferation in women at high risk for breast cancer. Women were recruited from the intervention and control groups of a randomized low fat dietary intervention trial, breast epithelial cells were obtained by fine needle aspiration, and cell proliferation was assessed in these samples using immunofluorescent detection of Ki-67 and PCNA. The effects of needle size and study group on cell yield and cytologic features of the cells were also examined. Fifty three women (20 in the intervention group and 33 in the control group) underwent the biopsy procedure. Slides from 38 subjects were stained for Ki-67 and from 14 subjects for PCNA. No cell proliferation (fluorescence) was detected for either Ki-67 or PCNA in any of the slides. Epithelial cell yield and number of stromal fragments were greater with a larger needle size. Numbers of stromal fragments and bipolar naked nuclei were greater in the low fat as compared to the control group but no differences in epithelial cell yield were observed between the two groups. This study confirms that fine needle aspiration biopsy is a feasible method of obtaining epithelial cells from women without discrete breast masses, but suggests that cell proliferation cannot be assessed using Ki-67 and PCNA in such samples.

Successful Immunosuppressive Therapy for Severe Infectious Mononucleosis in a Patient with Clonal Proliferation of EBV-infected CD8-positive Cells.

PubMed

Hosoi, Hiroki; Sonoki, Takashi; Murata, Shogo; Mushino, Toshiki; Kuriyama, Kodai; Nishikawa, Akinori; Hanaoka, Nobuyoshi; Ohshima, Koichi; Imadome, Ken-Ichi; Nakakuma, Hideki

2015-01-01

A 30-year-old woman was diagnosed with severe infectious mononucleosis (IM). The Epstein-Barr virus (EBV) had infected both CD19- and CD8-positive cells, and clonal proliferation of EBV-infected cells and T-cells was detected. Although we suspected malignant lymphoma, her condition improved following immunosuppressive therapy. A similar case was recently reported; therefore, this case is the second case of IM with EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells. Our results demonstrate that the clonal proliferation of EBV-infected cells is not always an indication for chemotherapy in the primary infection phase and that monitoring the EBV viral load is useful for therapeutic decision-making.

Silencing of CXCR4 inhibits tumor cell proliferation and neural invasion in human hilar cholangiocarcinoma.

PubMed

Tan, Xin-Yu; Chang, Shi; Liu, Wei; Tang, Hui-Huan

2014-03-01

To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

A simple method to assess in vivo proliferation in lung vasculature with EdU: the case of MMC-induced PVOD in rat.

PubMed

Fabrice, Antigny; Benoît, Ranchoux; Valérie, Nadeau; Lau, Edmund; Sébastien, Bonnet; Frédéric, Perros

2015-01-01

5-Ethynyl-2'-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a "terminal" state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

PubMed

Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

2017-10-01

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

[Effect of Magnolol on Proliferation and Apoptosis of HL-60 Cells and Its Molecular Mechanism].

PubMed

Fang, Ke; Yuan, Xiao-Fen; Liao, Qiong; Zhang, Zhi-Yong; Song, Guan-Hua; Guo, Qiang; Ren, Xia; Jiang, Guo-Sheng

2016-04-01

To investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism. MTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol (5, 10, 20, 40, 80 and 160 µg/ml). The morphological changes of HL-60 cells were examined by light microscopy, and DAPI staining was performed to observe the nuclear morphology of HL-60 cells. The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining. RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2. Western blot was performed to detect the protein expression of caspase family. The magnolol inhibited HL-60 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time- dependent manner (P < 0.05). HL-60 cells became small, even apoptotic bodies appeared after treatment with magnolol. In addition, nuclear condensation or fragmentation could be observed, which is the typical morphological features of apoptosis. When HL-60 cells were treated with 40 µg/ml of magnolol for 24 h, the ratio of early apoptotic cells reached to (11.7 ± 2.4) %, which was significant different from control (1.4 ± 1.1) % (P < 0.05). RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX, whereas down-regulated the expression of BCL-2. Western blot results showed that the cleavages of caspase-3, -8 and -9 were significantly enhanced by magnolol. The magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through up-regulation of BAX, down-regulation of BCL-2 and the activation of caspases.

Curcumin reverses irinotecan resistance in colon cancer cell by regulation of epithelial-mesenchymal transition.

PubMed

Zhang, Chunhong; Xu, Yangjie; Wang, Haowen; Li, Gang; Yan, Han; Fei, Zhenghua; Xu, Yunsheng; Li, Wenfeng

2018-04-01

The objective of this study was to investigate the effect and the mechanism by which curcumin reverses irinotecan-induced chemotherapy resistance in colon cancer. Construction of irinotecan-resistant colon cancer model LoVo/CPT-11R cells was performed by increasing drug concentration. The Cell Counting Kit-8 assay was used to detect inhibition of proliferation; cell morphology was observed by an optical microscope. Quantitative RT-PCR and western blotting were performed to detect molecular marker expressions during epithelial-mesenchymal transition (EMT); drug-resistant cells were treated with curcumin at different concentrations and Cell Counting Kit-8 was reperformed to detect cell proliferation after treatments. Drug-resistant cells were then divided into four groups: control group, irinotecan group, curcumin group, and irinotecan+curcumin group; quantitative RT-PCR and western blotting were performed to detect molecular marker expressions during epithelial-mesenchymal transition. Flow cytometry was used to detect cell apoptosis after grouping, and apoptosis-related protein was detected by western blotting. LoVo/CPT-11R cells could survive in culture medium containing irinotecan at 60 μg/ml and the drug-resistance index was 5.69; the drug-resistant cells had a larger volume than normal cells and were poorly connected to each other. E-cadherin expression was downregulated, whereas vimentin and N-cadherin expressions were upregulated. After curcumin treatment, drug-resistant cell proliferation was significantly inhibited; in the curcumin+irinotecan treatment group, E-cadherin expression was upregulated, whereas vimentin and N-cadherin expressions were downregulated. Curcumin could significantly increase cell apoptosis. EMT is involved in the development of irinotecan resistance and curcumin can reverse this drug resistance through reversion of the EMT process.

Lidocaine inhibits the proliferation of lung cancer by regulating the expression of GOLT1A.

PubMed

Zhang, Lei; Hu, Rong; Cheng, Yanyong; Wu, Xiaoyang; Xi, Siwei; Sun, Yu; Jiang, Hong

2017-10-01

Lidocaine is the most commonly used local anaesthetic in clinical and can inhibit proliferation, suppress invasion and migration and induce apoptosis in human lung adenocarcinoma (LAD) cells. However, its specific downstream molecular mechanism is unclear. LAD cell lines, A549 and H1299 cells, were treated with lidocaine. The proliferation was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assay. The expression level of related proteins was detected by real-time quantitative PCR (qPCR) and Western blot assay. The results indicated that lidocaine dose-dependently suppressed the proliferation of A549 and H1299 cells. In the LAD patients' samples, GOLT1A was upregulated and involved in the poor prognosis and higher grade malignancy. Additionally, GOLT1A mediates the function of lidocaine on repressing proliferation by regulating the cell cycle in A549 cells. Our findings suggest that lidocaine downregulates the GOLT1A expression to repress the proliferation of lung cancer cells. © 2017 John Wiley & Sons Ltd.

[Effect of CP Metronomic Chemotherapy on RPMI 8226 Cell Proli-feration and Notch1/NF-κB Signaling Pathway In Vitro].

PubMed

Guo, Lie-Ping; Zhou, Fan; Shi, Hao-Tian; Chen, Hai-Min; Lin, Chen-Hui; Chen, Xiao-Ling; Hou, Jian

2016-10-01

To investigate the effect of metronomic chemotherapy of low dose phosphoramide combined with prednisolone (CP metronomic chemotherapy) on proliferation and apoptosis of RPMI 8226 cells, and to explore its regulating effect on Notch1/NF-κB signaling pathways. Experiment was divided into the DMSO control group, and the phosphoramide mustard (PM) group, the prednisolone group, the phosphoramide mustard plus prednisolone group (the CP group). RPMI 8226 cells were treated with different drugs, CCK-8 method was used to detect cell proliferation, flow cytometry was used to detect the cell cycle and apoptosis, reverse transcription PCR was used to detect Notch1 and NF-κB mRNA expression level. Compared with DMSO control group, RPMI8226 cell proliferation inhibition rate in all the PM, prednisolone and CP groups increased significantly with prolonging of time (r of 0.994,0.996,0.999, respectively, P<0.001). And at the same time, the inhibitory rate of cell proliferation was significantly different; the cell inhibitory rate in PM group was lowest, that in CP group was highgest, that in prednissone group was intermediate (P<0.01). After 48 hours, compared with the DMSO control group, the G 1 /G 0 cell proportion in treatment group increased significantly, S phase cell proportion decreased significantly, especially in PM and CP groups. The G 2 /M phase cell proportion increased in PM group, while reduced in the prednisolone and the CP groups. After 48 hours, compared with the DMSO control group, RPMI 8226 cell apoptosis rate increased as follow: in PM, pre-dnisolone and CP group(P<0.01). After 48 hours, compared with the DMSO control group, Notch1 and NF-κB mRNA expression in the prednisolone, the PM and the CP group decreased significantly(P<0.001). CP metronomic chemotherapy can significantly reduce RPMI 8226 cell proliferation, promote RPMI 8226 cell apoptosis, arrest RPMI 8226 cells mainly in the G 1 /G 0 phase, and significantly reduce Notch1 and NF-κB expression levels. It is suggested that Notch1/NF-κB signaling pathways is involved in CP metronomic chemotherapy for MM.

Low-intensity pulsed ultrasound promotes Schwann cell viability and proliferation via the GSK-3β/β-catenin signaling pathway

PubMed Central

Ren, Cong; Chen, Xiaohui; Du, Ning; Geng, Shuo; Hu, Yingying; Liu, Xin; Wu, Xianxian; Lin, Yuan; Bai, Xue; Yin, Wenzhe; Cheng, Shi; Yang, Lei; Zhang, Yong

2018-01-01

Background: It has been reported that ultrasound enhances peripheral nerve regeneration, but the mechanism remains elusive. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance proliferation and alter protein production in various types of cells. In this study, we detected the effects of LIPUS on Schwann cells. Material and methods: Schwann cells were separated from new natal Sprague-Dawley rat sciatic nerves and were cultured and purified. The Schwann cells were treated by LIPUS for 10 minutes every day, with an intensity of 27.37 mW/cm2. After treatment for 5 days, MTT, EdU staining, and flow cytometry were performed to examine cell viability and proliferation. Neurotrophic factors, including FGF, NGF, BDNF, and GDNF, were measured by western blot and real-time PCR. GSK-3β, p-GSK-3β, β-catenin and Cyclin D1 protein levels were detected using a western blot analysis. The expression of Cyclin D1 was also detected by immunofluorescence. Results: MTT and EdU staining showed that LIPUS increased the Schwann cells viability and proliferation. Compared to the control group, LIPUS increased the expression of growth factors and neurotrophic factors, including FGF, NGF, BDNF, GDNF, and Cyclin D1. Meanwhile, GSK-3β activity was inhibited in the LIPUS group as demonstrated by the increased level of p-GSK-3β and the ratio of the p-GSK-3β/GSK-3β level. The mRNA and protein expressions of β-catenin were increased in the LIPUS group. However, SB216763, a GSK-3β inhibitor, reversed the effects of LIPUS on Schwann cells. Conclusion: LIPUS promotes Schwann cell viability and proliferation by increasing Cyclin D1 expression via enhancing the GSK-3β/β-catenin signaling pathway.

Effect of extracorporeal shock wave on proliferation and differentiation of equine adipose tissue-derived mesenchymal stem cells in vitro

PubMed Central

Raabe, O; Shell, K; Goessl, A; Crispens, C; Delhasse, Y; Eva, A; Scheiner-Bobis, G; Wenisch, S; Arnhold, S

2013-01-01

Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with different pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded to ESWT by significant activation (phosphorylation) of Erk1/2, detected in western blots. No significant effects on the differentiation potential of the ASCs were evident. Taken together, the present results show significant effects of shock waves on stem cells in vitro. PMID:23671817

A long noncoding RNA UCA1 promotes proliferation and predicts poor prognosis in glioma.

PubMed

Zhao, W; Sun, C; Cui, Z

2017-06-01

Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma.

Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines

PubMed Central

Zhao, Wei-Xiu; Wu, Zhen-Ming; Liu, Wei

2017-01-01

ABSTRACT Notch signaling pathways play important roles in cell fate and many diseases, including preeclampsia, the dysregulation of which may be the main cause of maternal mortality. This study aimed to investigate the roles of Notch2 and Notch3 in proliferation and invasion in trophoblast cell lines (BeWo and JAR). Small hairpin RNAs targeting Notch2/Notch3 and Notch2/Notch3-overexpression vectors were designed, constructed and transfected into BeWo and JAR cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were then used to detect Notch2 and Notch3 mRNA and protein levels, and confirm the efficiency of silence and overexpression. Flow cytometry assays were conducted to evaluate the cell cycle of the two cell lines, and transwell assays were used to detect migration and invasion. Western blot analysis was also performed to show the alteration of the cell lines' physiological activities at protein level. When Notch2 was downregulated in BeWo cells, proliferation was dramatically promoted, while migration and invasion were significantly inhibited. When Notch2 was upregulated in JAR cells, proliferation was inhibited, but migration and invasion were promoted. After overexpression of Notch3 in BeWo cells, proliferation was downregulated, but migration and invasion were both upregulated. By contrast, the silencing of Notch3 expression in JAR cells significantly enhanced proliferation, but suppressed migration and invasion. These data indicated that Notch2 and Notch3 mediate the invasion and migration of BeWo and JAR cells, and may play a potential role in early onset severe preeclampsia. PMID:28606936

Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines.

PubMed

Zhao, Wei-Xiu; Wu, Zhen-Ming; Liu, Wei; Lin, Jian-Hua

2017-08-15

Notch signaling pathways play important roles in cell fate and many diseases, including preeclampsia, the dysregulation of which may be the main cause of maternal mortality. This study aimed to investigate the roles of Notch2 and Notch3 in proliferation and invasion in trophoblast cell lines (BeWo and JAR). Small hairpin RNAs targeting Notch2/Notch3 and Notch2/Notch3-overexpression vectors were designed, constructed and transfected into BeWo and JAR cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were then used to detect Notch2 and Notch3 mRNA and protein levels, and confirm the efficiency of silence and overexpression. Flow cytometry assays were conducted to evaluate the cell cycle of the two cell lines, and transwell assays were used to detect migration and invasion. Western blot analysis was also performed to show the alteration of the cell lines' physiological activities at protein level.When Notch2 was downregulated in BeWo cells, proliferation was dramatically promoted, while migration and invasion were significantly inhibited. When Notch2 was upregulated in JAR cells, proliferation was inhibited, but migration and invasion were promoted. After overexpression of Notch3 in BeWo cells, proliferation was downregulated, but migration and invasion were both upregulated. By contrast, the silencing of Notch3 expression in JAR cells significantly enhanced proliferation, but suppressed migration and invasion. These data indicated that Notch2 and Notch3 mediate the invasion and migration of BeWo and JAR cells, and may play a potential role in early onset severe preeclampsia. © 2017. Published by The Company of Biologists Ltd.

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

PubMed

Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

2014-01-01

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.

[Anti-proliferation Effect of Taraxacum mongolicum Extract in HepG2 Cells and Its Mechanism].

PubMed

Guo, Jun-bin; Ye, Hai-hong; Chen, Jian-feng

2015-10-01

To study the anti-proliferation effect of Taraxacum mongolicum extract in HepG2 cells and its mechanism. The total proteins of HepG2 cells treated with Taraxacum mongolicum extract were. extracted and mitochondria-mediated apoptosis-related proteins (Survivin, Mcl-1, BCL-xL, BCL-2, Smac, BAX, Bad, Cytochrome c and Caspase-3/7/9) were detected by Western blot. Taraxacum mongolicum extract obviously inhibited the proliferation of HepG2 cells and the expression of anti-apoptotic proteins (Survivin, BCL-xL and BCL-2), increased the expression of pro-apoptotic proteins (Smac and Caspase-3/7/9), and promoted the release of Cytochrome c from mitochondria to cytoplasm in HepG2 cells. The effects were in a dose-independent mode. Taraxacum mongolicum extract can inhibit the proliferation of HepG2 cells and the anti-proliferation mechanism is related to mitochondria-mediated apoptosis.

Repair of tracheal epithelium by basal cells after chlorine-induced injury

PubMed Central

2012-01-01

Background Chlorine is a widely used toxic compound that is considered a chemical threat agent. Chlorine inhalation injures airway epithelial cells, leading to pulmonary abnormalities. Efficient repair of injured epithelium is necessary to restore normal lung structure and function. The objective of the current study was to characterize repair of the tracheal epithelium after acute chlorine injury. Methods C57BL/6 mice were exposed to chlorine and injected with 5-ethynyl-2′-deoxyuridine (EdU) to label proliferating cells prior to sacrifice and collection of tracheas on days 2, 4, 7, and 10 after exposure. Airway repair and restoration of a differentiated epithelium were examined by co-localization of EdU labeling with markers for the three major tracheal epithelial cell types [keratin 5 (K5) and keratin 14 (K14) for basal cells, Clara cell secretory protein (CCSP) for Clara cells, and acetylated tubulin (AcTub) for ciliated cells]. Morphometric analysis was used to measure proliferation and restoration of a pseudostratified epithelium. Results Epithelial repair was fastest and most extensive in proximal trachea compared with middle and distal trachea. In unexposed mice, cell proliferation was minimal, all basal cells expressed K5, and K14-expressing basal cells were absent from most sections. Chlorine exposure resulted in the sloughing of Clara and ciliated cells from the tracheal epithelium. Two to four days after chlorine exposure, cell proliferation occurred in K5- and K14-expressing basal cells, and the number of K14 cells was dramatically increased. In the period of peak cell proliferation, few if any ciliated or Clara cells were detected in repairing trachea. Expression of ciliated and Clara cell markers was detected at later times (days 7–10), but cell proliferation was not detected in areas in which these differentiated markers were re-expressed. Fibrotic lesions were observed at days 7–10 primarily in distal trachea. Conclusion The data are consistent with a model where surviving basal cells function as progenitor cells to repopulate the tracheal epithelium after chlorine injury. In areas with few remaining basal cells, repair is inefficient, leading to airway fibrosis. These studies establish a model for understanding regenerative processes in the respiratory epithelium useful for testing therapies for airway injury. PMID:23170909

A novel estrogen receptor GPER mediates proliferation induced by 17β-estradiol and selective GPER agonist G-1 in estrogen receptor α (ERα)-negative ovarian cancer cells.

PubMed

Liu, Huidi; Yan, Yan; Wen, Haixia; Jiang, Xueli; Cao, Xuefeng; Zhang, Guangmei; Liu, Guoyi

2014-05-01

G protein-coupled estrogen receptor (GPER) is recently identified as a membrane-associated estrogen receptor that mediates non-genomic effects of estrogen. Our previous immunohistochemistry study found an association between GPER and the proliferation of epithelial ovarian cancer. However, the contributions and mechanisms of GPER in the proliferation of ovarian cancers are not clear. We have examined the role of GPER in estrogen receptor α (ERα)-negative/GPER positive OVCAR5 ovarian cancer cell line. MTT assay was used to detect cell proliferation. BrdU incorporation assay was used to measure the cells in S-phase. Protein expression of marker genes of proliferation, cell cycle and apoptosis were examined by Western blot. The results showed that 17β-estradiol and selective GPER agonist G-1 stimulated the proliferation of OVCAR5 cells and increased the cells in S-phase. Both ligands upregulated the protein levels of c-fos and cyclin D1. Small interfering RNA targeting GPER or G protein inhibitor pertussin toxin (PTX) inhibited basal cell proliferation and attenuated 17β-estradiol- or G-1-induced cell proliferation. GPER mediated cell growth was also associated with the apoptosis of OVCAR5 cells. These findings suggest that GPER has an important function in the proliferation of ovarian cancer cells lacking ERα. GPER might be a promising therapeutic target in ovarian cancer. © 2014 International Federation for Cell Biology.

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke

2008-03-21

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation.more » Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.« less

[Effect of rapamycin on proliferation of rat heart valve interstitial cells in vitro].

PubMed

Tan, Yan; Wang, Ji-Ye; Yi, Ren-Liang; Qiu, Jian

2016-04-01

To investigate the effect of rapamycin on the proliferation of rat valvular interstitial cells in primary culture. The interstitial cells isolated from rat aortic valves were cultured and treated with rapamycin, and the cell growth and cell cycle changes were analyzed using MTT assay and flow cytometry, respectively. RT-PCR was used to detect mRNA expression levels of S6 and P70S6K in cells, and the protein expressions level of S6, P70S6K, P-S6, and P-P70S6K were detected using Western blotting. Rat aortic valvular interstitial cells was isolated successfully. The rapamycin-treated cells showed a suppressed proliferative activity (P<0.05), but the cell cycle distribution remained unaffected. Rapamycin treatment resulted in significantly decreased S6 and P70S6K protein phosphorylation level in the cells (P<0.05). The mechanism by which rapamycin inhibits the proliferation of valvular interstitial cells probably involves suppression of mTOR to lower S6 and P70S6K phosphorylation level but not direct regulation of the cell cycle.

Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma

PubMed Central

Tan, Xin-Yu; Chang, Shi; Liu, Wei

2014-01-01

Background/Aims To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. Methods An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. Results The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. Conclusions CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA. PMID:24672662

Hepatitis B virus replication is upregulated in proliferated peripheral blood lymphocytes.

PubMed

Yan, Qin; Lan, Ying-Hua; Huang, Yan-Xin; Fan, Rong-Shan; Liu, Lan; Song, Shu-Peng; Li, Yong-Guo

2016-04-01

Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin‑2 (IL‑2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT‑PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL‑2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.

S-phase arrest after vincristine treatment may promote hepatitis B virus replication

PubMed Central

Xu, Lei; Tu, Zeng; Xu, Ge; Hu, Jie-Li; Cai, Xue-Fei; Zhan, Xing-Xing; Wang, Yu-Wei; Huang, Yuan; Chen, Juan; Huang, Ai-Long

2015-01-01

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication. PMID:25663769

[Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

PubMed

Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

2016-12-01

Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

PubMed

Yin, Haoyuan; Shao, Ying; Chen, Xuan

2017-01-01

To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P < 0.05). Downregulated the expression of CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

Regulators of Intestinal Epithelial Migration in Sepsis.

PubMed

Meng, Mei; Klingensmith, Nathan J; Liang, Zhe; Lyons, John D; Fay, Katherine T; Chen, Ching-Wen; Ford, Mandy L; Coopersmith, Craig M

2018-02-08

The gut is a continuously renewing organ, with cell proliferation, migration and death occurring rapidly under basal conditions. Since the impact of critical illness on cell movement from crypt base to villus tip is poorly understood, the purpose of this study was to determine how sepsis alters enterocyte migration. Wild type, transgenic and knockout mice were injected with 5-bromo-2'deoxyuridine (BrdU) to label cells in S phase before and after the onset of cecal ligation and puncture and were sacrificed at pre-determined endpoints to determine distance proliferating cells migrated up the crypt-villus unit. Enterocyte migration rate was decreased from 24-96 hours following sepsis. BrdU was not detectable on villi 6 days after sham laparotomy, meaning all cells had migrated the length of the gut and been exfoliated into its lumen. However, BrdU positive cells were detectable on villi 10 days after sepsis. Multiple components of gut integrity altered enterocyte migration. Sepsis decreased crypt proliferation, which further slowed enterocyte transit as mice injected with BrdU after the onset of sepsis (decreased proliferation) had slower migration than mice injected with BrdU prior to the onset of sepsis (normal proliferation). Decreasing intestinal apoptosis via gut-specific overexpression of Bcl-2 prevented sepsis-induced slowing of enterocyte migration. In contrast, worsened intestinal hyperpermeability by genetic deletion of JAM-A increased enterocyte migration. Sepsis therefore significantly slows enterocyte migration, and intestinal proliferation, apoptosis and permeability all affect migration time, which can potentially be targeted both genetically and pharmacologically.

miR-885-5p upregulation promotes colorectal cancer cell proliferation and migration by targeting suppressor of cytokine signaling.

PubMed

Su, Meng; Qin, Baoli; Liu, Fang; Chen, Yuze; Zhang, Rui

2018-07-01

The aim of the present study was to investigate the role of microRNA (miR)-885-5p in colorectal cancer cell proliferation and migration, and to determine the possible underlying molecular mechanisms. The expression of miR-885-5p in colorectal cancer tissue and cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of three suppressor of cytokine signaling (SOCS) factors were detected by RT-qPCR and western blotting. The effects of miR-885-5p on tumor cell proliferation and migration were studied using MTT and Transwell assays, respectively. Additionally, the expression levels of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, E-cadherin, vimentin and Snail) were detected by RT-qPCR and western blot analysis. Furthermore, the target of miR-885-5p was predicted and confirmed using a luciferase reporter assay. miR-885-5p was demonstrated to be upregulated and SOCS was downregulated in colorectal cancer tissue, and cells. miR-885-5p suppression significantly inhibited tumor cell proliferation and migration, promoted E-cadherin expression, and inhibited the expression levels of N-cadherin, vimentin and Snail. Further studies showed that SOCS5, SOCS6 and SOCS7 were direct targets of miR-885-5p. The results suggest that miR-885-5p suppression inhibited cell proliferation and migration, and the EMT process by targeting SOCS5, SOCS6 and SOCS7 genes in colorectal cancer. miR-885-5p and SOCS may be used for the diagnosis and treatment of colorectal cancer.

VNN3, a potential novel biomarker for benzene toxicity, is involved in 1, 4-benzoquinone induced cell proliferation.

PubMed

Sun, Pengling; Guo, Xiaoli; Chen, Yujiao; Zhang, Wei; Duan, Huawei; Gao, Ai

2018-02-01

Benzene is widely employed in the field of production, and its toxicity on biological systems has received increasing attention. Cell proliferation is a major life characteristic of living organisms. KLF15 and NOTCH1 are mature and classical genes in cell proliferation studies, particularly in the area of tumor investigation. The aim of this study was to investigate the effect and mechanism of VNN3 on cell proliferation induced by 1,4-benzoquinone (1,4-BQ), an important metabolite of benzene, and obtain a sensitive biomarker for the hazard screening and health care of benzene exposure. Normally growing AHH-1 cells were cultured in vitro and were incubated with different concentrations of 1,4-BQ (0, 10, 20, and 40 μM) for 24 h. A CCK-8 assay was used to assess the cell viability, whereas EdU was used to detect the cell proliferation of AHH-1 cells. The expression of VNN3, KLF15 and NOTCH1 was detected by real-time PCR. Moreover, a lentiviral model was constructed in AHH-1 cells to interfere with VNN3 expression. The results showed that 1,4-BQ clearly increased the expression of VNN3. Moreover, 1,4-BQ dose-dependently inhibited cell proliferation and caused increased KLF15 expression; in contrast, the NOTCH1 expression decreased in AHH-1 cells. Furthermore, following interference with the VNN3 expression, the cell proliferation inhibition and the expression of KLF15 and NOTCH1 were rescued. To further investigate the action of VNN3 in benzene hematotoxicity, we assessed it in benzene-exposed workers. The results showed that there was a remarkable correlation between the VNN3 expression and hemogram, which included RBC, NEUT and HGB. In addition, analysis of the KLF15 and NOTCH1 expression showed that the VNN3 expression was related to cell proliferation, which was consistent with the in vitro results. In conclusion, VNN3 influences cell proliferation induced by 1,4-BQ by regulating the expression of KLF15 and NOTCH1. VNN3 may represent a potential biomarker of benzene toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Moclobemide up-regulates proliferation of hippocampal progenitor cells in chronically stressed mice.

PubMed

Li, Yun-feng; Zhang, You-zhi; Liu, Yan-qin; Wang, Heng-lin; Yuan, Li; Luo, Zhi-pu

2004-11-01

To explore the action mechanism of antidepressants. The PC12 cell proliferation was detected by flow cytometry. The proliferation of hippocampal progenitor cells and level of brain-derived neurotrophic factor (BDNF) were measured by immunohistochemistry. Treatment with N-methylaspartate (NMDA) 600 micromol/L for 3 d significantly decreased the percentage of S-phase in PC12 cells, while in the presence of classical antidepressant, moclobemide (MOC) 2 and 10 micromol/L, the percentage in S-phase increased. Furthermore, the proliferation of progenitor cells in hippocampal dentate gyrus (subgranular zone), as well as the level of BDNF in hippocampus significantly decreased in chronically stressed mice, while chronic administration with MOC 40 mg/kg (ip) up-regulated the progenitor cell proliferation and BDNF level in the same time course. Up-regulation of the proliferation of hippocampal progenitor cells is one of the action mechanisms for MOC, which may be closely related to the elevation of BDNF level at the same time. These results also extend evidence for our hypothesis that up-regulation of the hippocampal neurogenesis is one of the common mechanisms for antidepressants.

Endogenous Hydrogen Sulfide Enhances Cell Proliferation of Human Gastric Cancer AGS Cells.

PubMed

Sekiguchi, Fumiko; Sekimoto, Teruki; Ogura, Ayaka; Kawabata, Atsufumi

2016-01-01

Hydrogen sulfide (H2S), the third gasotransmitter, is endogenously generated by certain H2S synthesizing enzymes, including cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) from L-cysteine in the mammalian body. Several studies have shown that endogenous and exogenous H2S affects the proliferation of cancer cells, although the effects of H2S appear to vary with cell type, being either promotive or suppressive. In the present study, we determined whether endogenously formed H2S regulates proliferation in human gastric cancer AGS cells. CSE, but not CBS, was expressed in AGS cells. CSE inhibitors, DL-propargylglycine (PPG) and β-cyano-L-alanine (BCA), significantly suppressed the proliferation of AGS cells in a concentration-dependent manner. CSE inhibitors did not increase lactate dehydrogenase (LDH) release in the same concentration range. The inhibitory effects of PPG and BCA on cell proliferation were reversed by repetitive application of NaHS, a donor of H2S. Interestingly, nuclear condensation and fragmentation were detected in AGS cells treated with PPG or BCA. These results suggest that endogenous H2S produced by CSE may contribute to the proliferation of gastric cancer AGS cells, most probably through anti-apoptotic actions.

The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

DTIC Science & Technology

2014-10-01

Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation, Senescence. 16. SECURITY CLASSIFICATION OF: 17...Tyrosine Kinase, Her/ErbB2 signaling, Rab Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation...lines included RCP condition to internalization and detection of E-cadherin, a well-known adherent junction and epithelial mesenchymal transition

[Effects of non-saccharomyces albicans metabolic products on the proliferation of human umbilical vein endothelial cell ECV304].

PubMed

Chen, Bin; Che, Tuanjie; Bai, Decheng; He, Xiangyi

2013-04-01

To evaluate the effects of non-Saccharomyces albicans metabolic products on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro. The parallel dilution supernatant of Saccharomyces tropicalis, Saccharomyces krusei and Saccharomyces glabrata were prepared, and 1, 4, 16-fold(s) diluted concentration and control group were set up. The line of human umbilical vein endothelial cell ECV304 was cultured in vitro and treated by non-Saccharomyces albicans supernatant. The proliferous effect of ECV304 induced by non-Saccharomyces albicans supernatant after 24, 48, 72 h was detected by the methods of MTT, and the changes of cell density and cycle after 48 h were investigated by inverted microscope and flow cytometry. At the 24th hour, all of the higher concentration (1-fold) of non-Saccharomyces albicans supernatant and the 4-folds diluted Saccharomyces krusei could promote ECV304 proliferation(P < 0.05). After adding various non-Saccharomyces albicans supernatant at 48h and 72th hour, Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant significantly increased proliferation rate of ECV304, while Saccharomyces tropicalis supernatant group showed no significant change no matter which concentration was tested. At 48th hour after adding the non-Saccharomyces albicans supernatant, the ECV304 cells density treated by Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant were significantly higher under the inverted microscope. The G0/G1 population of ECV304 cells decreased while cell proliferation index (PI) increased after incubated with Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant for 48 hours (P < 0.05). Saccharomyces tropicalis group showed no significant change (P > 0.05). The metabolic products of Sacharoymces krusei and Saccharomyces glabrata could induce proliferation of ECV304 cell, which suggests non-Saccharomyces albicans should be undergone more attention clinically in detection and treatment.

[Study on thaspine in inducing apoptosis of A549 cell].

PubMed

Zhang, Yan-min; He, Lang-chong

2007-04-01

To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. Thaspine has the effects of anti-tumor and inducing apoptosis.

Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

PubMed

Yang, X Q; Yang, J; Wang, R; Zhang, S; Tan, Q W; Lv, Q; Meng, W T; Mo, X M; Li, H J

2015-12-02

The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

Adipose-derived stem cell-derived microvesicle-released miR-210 promoted proliferation, migration and invasion of endothelial cells by regulating RUNX3.

PubMed

Zheng, Zeqi; Liu, Lijuan; Zhan, Yuliang; Yu, Songping; Kang, Ting

2018-06-18

To explore the potential mechanism of miRNA released from adipose-derived stem cell (ADSC)-derived micro vesicle (MV) on the modulation of proliferation, migration and invasion of endothelial cells. miR-210 level was detected by qT-PCR. Alix, VEGF and RUNX3 expressions were detected by Western blot. The proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) were observed by MTT assay and Transwell assay. Luciferase reporter gene assay was conducted to validate the targeting activity of MVs-released miR-210 on RUNX3. Hypoxia significantly increased the expression of MVs-released miR-210. MVs released from ADSCs in hypoxic group significantly promoted the proliferation, migration and invasion of HUVECs. Overexpression of miR-210 significantly upregulated VEGF expression, and promoted the proliferation, migration and invasion of HUVECs. Besides, RUNX3 was identified as the direct of miR-210 in HUVECs. Overexpression of miR-210 decreased RUNX3 expression and promoted the proliferation, migration and invasion of HUVECs, while overexpression of RUNX3 inhibited these promotion effects. In vivo experiment showed that MVs derived from ADSCs under hypoxia increased miR-210 level and capillary density, and inhibition of miR-210 decreased capillary density. We also found MVs downregulated RUNX3 expression, and inhibition of miR-210 upregulated RUNX3 expression. miR-210 released from ADSCs-derived MVs promoted proliferation, migration and invasion of HUVECs by targeting RUNX3, which revealed one of the mechanisms of ADSCs-derived MVs on the promotion of proliferation, migration and invasion of HUVECs.

[Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

PubMed

Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

2017-08-01

Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

miR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1.

PubMed

Zhang, Peng; Sun, Hongwei; Yang, Bo; Luo, Wenzheng; Liu, Zengjin; Wang, Junkuan; Zuo, Yuchao

2017-08-01

Aberrant DNA methylation is associated with tumor onset and progression. Study has verified that the DNA methylation of miR-152 was mediated in many tumors, but whether it involved in glioblastomas was still unclear. This study enrolled 20 patients with glioma to analyze the expression pattern of miR-152. Real-time PCR and western blot were used to detect the mRNA or protein expression level, respectively. The relationship between miR-152 and runx2 was detected by Luciferase reporter assay. The methylation level of miR-152 was determined by methylation-specific PCR. Cell proliferation and apoptosis were detected by MTT and Annexin-FITC/PI assay. The expression of miR-152 was down-regulated while the expression of DNMT1 was up-regulated in both glioma tissue and cell lines. MiR-152 was hypermethylated and its expression was negatively correlated with DNMT in glioma cell lines. DNMT1 knockdown promoted the expression of miR-152, however, DNMT1 overexpression suppressed the expression of miR-152. MiR-152 overexpression promoted glioma cell apoptosis while miR-152 knockdown promoted cell proliferation. MiR-152 targets Runx2 to regulate its expression, Runx2 overexpression abolished the effects of miR-152 overexpression. MiR-152 regulated cell proliferation and apoptosis of glioma mediated by Runx2, while the mechanism of down regulated miR-152 in glioma tissues and cells was its hypermethylation. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis.

PubMed

Husari, Ayman; Hülter-Hassler, Diana; Steinberg, Thorsten; Schulz, Simon Daniel; Tomakidi, Pascal

2018-01-01

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound β-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/β-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/β-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage. Copyright © 2017 Elsevier B.V. All rights reserved.

MAGE-A inhibits apoptosis in proliferating myeloma cells through repression of Bax and maintenance of survivin.

PubMed

Nardiello, Tricia; Jungbluth, Achim A; Mei, Anna; Diliberto, Maurizio; Huang, Xiangao; Dabrowski, Ania; Andrade, Valéria C C; Wasserstrum, Rebecca; Ely, Scott; Niesvizky, Ruben; Pearse, Roger; Coleman, Morton; Jayabalan, David S; Bhardwaj, Nina; Old, Lloyd J; Chen-Kiang, Selina; Cho, Hearn Jay

2011-07-01

The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.

MAGE-A inhibits apoptosis in proliferating myeloma cells through repression of Bax and maintenance of survivin

PubMed Central

Nardiello, Tricia; Jungbluth, Achim A.; Mei, Anna; DiLiberto, Maurizio; Huang, Xiangao; Dabrowski, Ania; Andrade, Valéria C. C.; Wasserstrum, Rebecca; Ely, Scott; Niesvizky, Ruben; Pearse, Roger; Coleman, Morton; Jayabalan, David S.; Bhardwaj, Nina; Old, Lloyd J.; Chen-Kiang, Selina; Cho, Hearn Jay

2011-01-01

Purpose The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67+ malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. Experimental Design The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups; newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by shRNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. Results MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared to newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A demonstrated that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of pro-apoptotic Bax expression and by reduction of survivin expression through both p53-dependent and independent mechanisms. Conclusions These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms. PMID:21565982

[Effect of Ca(OH)2 on the cytotoxicity of lipopolysaccharide extracted from Porphyromonas endodontalis in vitro].

PubMed

Guo, Jia-jie; Qiu, Li-hong; Yu, Ya-qiong; Xu, Li-ya; Fan, Yun-qian; Zhong, Ming

2014-02-01

To detect the degradation of Ca(OH)2 on lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.e) in vitro and estimate the influence of P.e LPS pretreated with Ca(OH)2 on the proliferation of MC3T3-E1 cells. The effect of Ca(OH)2 on MC3T3-E1 cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Then P.e LPS was treated with Ca(OH)2 for 30 mins or 60 mins at 37 degrees centigrade in vitro and the activity of P.e LPS was evaluated by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) test. Finally, MC3T3-E1 cells were exposed to P.e LPS pretreated with 15% Ca(OH)2 for 1, 3 and 5 d, and the cell proliferation was measured using the MTT assay comparing with the P.e LPS control group. SPSS 13.0 software package was used for statistical analysis. Compared with the negative control, exposing cells to 5%, 10% and 15% Ca(OH)2 had greatly promoted MC3T3-E1 cell proliferation. P.e LPS treated with 10% and 15% Ca(OH)2 both presented the best results by CE TAL and significant difference compared with P.e LPS control group. When 10 μg/mL P.e LPS was pretreated with 15% Ca(OH)2, no inhibition of MC3T3-E1 cell proliferation was noted. Ca(OH)2 detoxifies P.e LPS in vitro, mitigates the impact of P.e LPS on MC3T3-E1 cell proliferation. Supported by Science and Technology Projects of Liaoning Province (2011225020).

Downregulation of Id1 by small interfering RNA in gastric cancer inhibits cell growth via the Akt pathway

PubMed Central

YANG, GUANG; ZHANG, YAN; XIONG, JIANJUN; WU, JING; YANG, CHANGFU; HUANG, HONGBING; ZHU, ZHENYU

2012-01-01

Inhibitor of differentiation or DNA binding (Id1) is a member of the helix-loop-helix transcription factor family that is overexpressed in various types of cancer, including gastric carcinoma. Previous studies showed that Id1 is a prognostic marker in patients with gastric cancer. However, the role of Id1 in the proliferation of human gastric cancer cells has yet to be clarified. In the present study, we downregulated the Id1 gene in SGC-7901 gastric cancer cells by RNA interference, and we also constructed a recombinant plasmid-expressing Id1 to investigate its effects on the proliferation of SGC-7901 cells. Results showed that the downregulation of Id1 inhibited proliferation of SGC-7901 cells, while the upregulation of Id1 had no effect on SGC-7901 cell proliferation. The potential mechanism was also investigated. The changes of certain proteins associated with cell proliferation, apoptosis and the cell cycle were detected by western blotting. Furthermore, we demonstrated a positive correlation between Id1 and phospho-Akt expression in SGC-7901 cells. PMID:22245935

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

PubMed Central

Mainou-Fowler, T; Copplestone, J A; Prentice, A G

1995-01-01

AIMS--To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS--The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS--Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS--B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis. Images PMID:7629299

MicroRNA-145 Inhibits Cell Migration and Invasion and Regulates Epithelial-Mesenchymal Transition (EMT) by Targeting Connective Tissue Growth Factor (CTGF) in Esophageal Squamous Cell Carcinoma.

PubMed

Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Wang, Xiao-Jing; Zhang, Bing; Chen, Hua

2016-10-23

BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (P<0.05). We found significantly more positively expressed CTGF protein in ESCC tissues was than in normal adjacent esophageal tissues (P<0.01). Dual luciferase reporter gene assay showed that miR-145 can specifically bind with the 3'UTR of CTGF and significantly inhibit the luciferase activity by 55% (P<0.01). Up-regulation of miR-145 or down-regulation of CTGF can suppress the proliferation, migration, invasion, and EMT process of ESCC cells. CONCLUSIONS MiR-145 was significantly down-regulated in ESCC tissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

BAG3 promotes the phenotypic transformation of primary rat vascular smooth muscle cells via TRAIL.

PubMed

Fu, Yao; Chang, Ye; Chen, Shuang; Li, Yuan; Chen, Yintao; Sun, Guozhe; Yu, Shasha; Ye, Ning; Li, Chao; Sun, Yingxian

2018-05-01

Under normal physiological condition, the mature vascular smooth muscle cells (VSMCs) show differentiated phenotype. In response to various environmental stimuluses, VSMCs convert from the differentiated phenotype to dedifferentiated phenotype characterized by the increased ability of proliferation/migration and the reduction of contractile ability. The phenotypic transformation of VSMCs played an important role in atherosclerosis. Both Bcl-2-associated athanogene 3 (BAG3) and tumor necrosis factor-related apopt-osis inducing ligand (TRAIL) involved in apoptosis. The relationship between BAG3 and TRAIL and their effects the proliferation and migration in VSMCs are rarely reported. This study investigated the effects of BAG3 on the phenotypic modulation and the potential underlying mechanisms in primary rat VSMCs. Primary rat VSMCs were extracted and cultured in vitro. Cell proliferation was detected by cell counting, real-time cell analyzer (RTCA) and EdU incorporation. Cell migration was detected by wound healing, Transwell and RTCA. BAG3 and TRAIL were detected using real-time PCR and western blotting and the secreted proteins in the cultured media by dot blot. The expression of BAG3 increased with continued passages in cultured primary VSMCs. BAG3 promoted the proliferation and migration of primary rat VSMC in a time-dependent manner. BAG3 significantly increased the expression of TRAIL while had no effects on its receptors. TRAIL knockdown or blocking by neutralizing antibody inhibited the proliferation of VSMCs induced by BAG3. TRAIL knockdown exerted no obvious influence on the migration of VSMCs. Based on this study, we report for the first time that BAG3 was expressed in cultured primary rat VSMCs and the expression of BAG3 increased with continued passages. Furthermore, BAG3 promoted the proliferation of VSMCs via increasing the expression of TRAIL. In addition, we also demonstrated that BAG3 promoted the migration of VSMCs independent of TRAIL upregulation.

[Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists].

PubMed

Zheng, Yan-min; Xie, Li-qun; Li, Xuan; Zhao, Jun-yan; Chen, Xiao-yi; Chen, Li; Zhou, Jing; Li, Fei

2009-12-01

To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH(2) upon the proliferation of hepatoma cells and its intracellular signaling mechanism. PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH(2), trypsin, reverse PAR-2 agonist peptide VKGILS-NH(2) or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/beta-actin) were 0.70 +/- 0.04 and 0.99 +/- 0.05 respectively in cells treated with trypsin and SLIGKV-NH(2). They were both significantly higher than that in the control group (0.35 +/- 0.05, F = 135.534, P < 0.01). Percent G(0)/G(1) phase of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly lower than those in the control group [(56.11 +/- 0.85)%, (57.85 +/- 0.46)% vs (79.12 +/- 0.67)%, both P < 0.01] Percent S phase, G(2)/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly elevated (P < 0.01). The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH(2) were significantly blocked by pretreatment with PD98059 (P < 0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH(2) and control group (P > 0.05). PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH(2) promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

The role of MiR-324-3p in polycystic ovary syndrome (PCOS) via targeting WNT2B.

PubMed

Jiang, Y-C; Ma, J-X

2018-06-01

To investigate the expression of microRNA-324-3p (miR-324-3p) in polycystic ovary syndrome (PCOS) and its effects on the proliferation and apoptosis of ovarian granulosa cells. A total of 60 Sprague-Dawley (SD) rats were randomly divided into normal group (n=30) and experimental group (n=30). Rats in the experimental group were intramuscularly injected with dehydroepiandrosterone (DHEA) (6 mg/100 g of body weight) and 0.2 mL oil for injection, while those in normal group were intramuscularly injected with 0.2 mL oil for injection. The ovarian tissues of PCOS model rats were removed to extract the total ribose nucleic acid (RNA). The expression of miR-324-3p was detected via reverse transcription-polymerase chain reaction (RT-PCR). Primary ovarian granulosa cells were isolated and cultured, and NC-miRNA and miR-324-3p mimic were transfected into cells. After 48 h, cell proliferation and apoptosis were detected via cell counting kit 8 (CCK-8) and flow cytometry assay, respectively. The targeted molecule of miR-324-3p was explored using bioinformatics, and dual-luciferase assay was performed to verify the effect of miR-324-3p on WNT2B expression. Granulosa cells were co-transfected with WNT2B-small-interfering RNA (siRNA) and miR-324-3p mimic, and then cell proliferation and apoptosis were detected via CCK-8 and flow cytometry assay, respectively. The expression of miR-324-3p in ovarian tissues of PCOS group was significantly lower than that of normal group (p < 0.01). After transfection with miR-324-3p mimic into granulosa cells, cell proliferation was significantly inhibited and cell apoptosis was promoted (p < 0.01). MiR-324-3p exerted its effect on granulosa cells by directly targeting WNT2B. Silencing WNT2B expression could reverse the effects of miR-324-3p on proliferation and apoptosis of granulosa cells (p < 0.05). The expression of miR-324-3p in the ovary of PCOS rats is decreased significantly. Overexpression of miR-324-3p can reduce the proliferation and induce the apoptosis of granulosa cells via targeting of WNT2B.

[Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines].

PubMed

Ma, Li; Han, Xiao-hong; Wang, Shuai; Wang, Jian-fei; Shi, Yuan-kai

2012-09-25

To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

PubMed

Yin, Jiuheng; Sheng, Baifa; Han, Bin; Pu, Aimin; Yang, Kunqiu; Li, Ping; Wang, Qimeng; Xiao, Weidong; Yang, Hua

2016-05-01

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation. © 2016 International Federation for Cell Biology.

Aberrant expression of the tight junction molecules claudin-1 and zonula occludens-1 mediates cell growth and invasion in oral squamous cell carcinoma.

PubMed

Babkair, Hamzah; Yamazaki, Manabu; Uddin, Md Shihab; Maruyama, Satoshi; Abé, Tatsuya; Essa, Ahmed; Sumita, Yoshimasa; Ahsan, Md Shahidul; Swelam, Wael; Cheng, Jun; Saku, Takashi

2016-11-01

We reported that altered cell contact mediated by E-cadherin is an initial event in the pathogenesis of oral epithelial malignancies. To assess other effects of cell adhesion, we examined the expression levels of tight junction (TJ) molecules in oral carcinoma in situ (CIS) and squamous cell carcinoma (SCC). To identify changes in the expression of TJ molecules, we conducted an analysis of the immunohistochemical profiles of claudin-1 (CLDN-1) and zonula occludens-1 (ZO-1) in surgical specimens acquired from patients with oral SCC containing foci of epithelial dysplasia or from patients with CIS. We used immunofluorescence, Western blotting, reverse-transcription polymerase chain reaction, and RNA interference to evaluate the functions of CLDN-1 and ZO-1 in cultured oral SCC cells. TJ molecules were not detected in normal oral epithelial tissues but were expressed in SCC/CIS cells. ZO-1 was localized within the nucleus of proliferating cells. When CLDN-1 expression was inhibited by transfecting cells with specific small interference RNAs, SCC cells dissociated, and their ability to proliferate and invade Matrigel was inhibited. In contrast, although RNA interference-mediated inhibition of ZO-1 expression did not affect cell morphology, it inhibited cell proliferation and invasiveness. Our findings indicated that the detection of TJ molecules in the oral epithelia may serve as a marker for the malignant phenotype of cells in which CLDN-1 regulates proliferation and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

Dual effects exerted in vitro by micromolar concentrations of deoxynivalenol on undifferentiated caco-2 cells.

PubMed

Manda, Gina; Mocanu, Mihaela Andreea; Marin, Daniela Eliza; Taranu, Ionelia

2015-02-16

Contamination of crops used for food and feed production with Fusarium mycotoxins, such as deoxynivalenol (DON), raise important health and economic issues all along the food chain. Acute exposure to high DON concentrations can alter the intestinal barrier, while chronic exposure to lower doses may exert more subtle effects on signal transduction pathways, leading to disturbances in cellular homeostasis. Using real-time cellular impedance measurements, we studied the effects exerted in vitro by low concentrations of DON (0.37-1.50 μM), relevant for mycotoxin-contaminated food, on the proliferation of undifferentiated Caco-2 cells presenting a tumorigenic phenotype. A 1.5 μM concentration of DON maintained cell adherence of non-proliferating Caco-2 cells, whilst arresting the growth of actively proliferating cells compared with control Caco-2 cells in vitro. At 0.37 μM, DON enhanced Caco-2 cell metabolism, thereby triggering a moderate increase in cell proliferation. The results of the current study suggested that low concentrations of DON commonly detected in food may either limit or sustain the proliferation of colon cancer cells, depending on their proliferation status and on DON concentration. Soluble factors released by Lactobacillus strains can partially counteract the inhibitory action of DON on actively proliferating colon cancer cells. The study also emphasized that real-time cellular impedance measurements were a valuable tool for investigating the dynamics of cellular responses to xenobiotics.

Automated mitosis detection of stem cell populations in phase-contrast microscopy images.

PubMed

Huh, Seungil; Ker, Dai Fei Elmer; Bise, Ryoma; Chen, Mei; Kanade, Takeo

2011-03-01

Due to the enormous potential and impact that stem cells may have on regenerative medicine, there has been a rapidly growing interest for tools to analyze and characterize the behaviors of these cells in vitro in an automated and high throughput fashion. Among these behaviors, mitosis, or cell division, is important since stem cells proliferate and renew themselves through mitosis. However, current automated systems for measuring cell proliferation often require destructive or sacrificial methods of cell manipulation such as cell lysis or in vitro staining. In this paper, we propose an effective approach for automated mitosis detection using phase-contrast time-lapse microscopy, which is a nondestructive imaging modality, thereby allowing continuous monitoring of cells in culture. In our approach, we present a probabilistic model for event detection, which can simultaneously 1) identify spatio-temporal patch sequences that contain a mitotic event and 2) localize a birth event, defined as the time and location at which cell division is completed and two daughter cells are born. Our approach significantly outperforms previous approaches in terms of both detection accuracy and computational efficiency, when applied to multipotent C3H10T1/2 mesenchymal and C2C12 myoblastic stem cell populations.

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.

We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwovenmore » scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.« less

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

PubMed

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Long non-coding RNA taurine-upregulated gene 1 predicts unfavorable prognosis, promotes cells proliferation, and inhibits cells apoptosis in epithelial ovarian cancer.

PubMed

Li, Tong-Huai; Zhang, Jing-Jing; Liu, Shao-Xiao; Chen, Yan

2018-05-01

The aim of this study was to evaluate the correlation of long non-coding RNAs (lncRNAs) taurine-upregulated gene 1 (TUG1) with clinicopathological characteristics as well as overall survival (OS) in epithelial ovarian cancer (EOC) patients, and investigate its function in EOC cells proliferation and apoptosis in vitro.LncRNA TUG1 expressions were detected in tumor tissues and paired adjacent tissues obtained from 96 EOC patients. Blank mimic, lncRNA TUG1 mimic, blank inhibitor, and lncRNA TUG1 inhibitor plasmids were transfected into SKOV3 cells. CKK-8, annexin V-FITC-propidium iodide, qPCR and western blot assays were performed to detect cells proliferation, cells apoptosis, RNA expression, and protein expression, respectively.LncRNA TUG1 expression was higher in tumor tissue compared to paired adjacent tissue (P < .001), and it was positively correlated with pathological grade (P = .022), tumor size (P = .011) and FIGO stage (P < .001). Kaplan-Meier curve showed that lncRNA TUG1 high expression was associated with worse OS (P = .003). Multivariate Cox analysis indicated that lncRNA TUG1 high expression (vs. low expression) (P = .035) was independently predictive factor for shorter OS. In vitro, cells proliferation was promoted after treatment with lncRNA TUG1 mimic and was suppressed after treatment with lncRNA TUG1 inhibitor. In addition, cells apoptosis rate was decreased in lncRNA TUG1 mimic group compared to NC1 mimic, and increased in lncRNA TUG1 inhibitor group compared to NC2 inhibitor.In conclusion, lncRNA TUG1 is positively correlated with advanced disease and poor prognosis, and it promotes cells proliferation and inhibits cells apoptosis in EOC cells.

Fenofibrate suppressed proliferation and migration of human neuroblastoma cells via oxidative stress dependent of TXNIP upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Cunjin; Shi, Aiming; Cao, Guowen

2015-05-15

There are no appropriate drugs for metastatic neuroblastoma (NB), which is the most common extra-cranial solid tumor for childhood. Thioredoxin binding protein (TXNIP), the endogenous inhibitor of ROS elimination, has been identified as a tumor suppressor in various solid tumors. It reported that fenofibrate exerts anti-tumor effects in several human cancer cell lines. However, its detail mechanisms remain unclear. The present study assessed the effects of fenofibrate on NB cells and investigated TXNIP role in its anti-tumor mechanisms. We used MTT assay to detect cells proliferation, starch wound test to investigate cells migration, H{sub 2}DCF-DA to detect intracellular ROS, siRNAmore » to interfere TXNIP and peroxisome proliferator-androgen receptor-alpha (PPAR-α) expression, western blot to determine protein levels, flow cytometry to analyze apoptosis. Fenofibrate suppressed proliferation and migration of NB cells, remarkably increased intracellular ROS, upregulated TXNIP expression, promoted cell apoptosis. Furthermore, inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. Our results indicated the anti-tumor role of fenofibrate on NB cells by exacerbating oxidative stress and inducing apoptosis was dependent on the upregulation of TXNIP. - Highlights: • We found that fenofibrate suppressed proliferation and migration of NB cells. • We found that fenofibrate remarkably increased intracellular ROS, upregulated TXNIP expression, and promoted cell apoptosis. • Inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. • Our results indicated the anti-tumor role of fenofibrate on NB cells was dependent on the upregulation of TXNIP.« less

[Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

PubMed

Yang, Jia-Yao; Tao, Dong-Qing; Liu, Song; Zhang, Shu; Ma, Wei; Shi, Zhao-Hong

2017-04-01

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions. Copyright© by the Chinese Pharmaceutical Association.

Irisin suppresses the migration, proliferation, and invasion of lung cancer cells via inhibition of epithelial-to-mesenchymal transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Lei; Jinan Central Hospital Affiliated to Shandong University, Jinan, 250012; Li, Huanjie

Irisin is involved in promoting metabolism, immune regulation, and affects chronic inflammation in many systemic diseases, including gastric cancer. However, the role of irisin in lung cancer is not well characterized. To determine whether irisin has a protective effect against lung cancer, we cultured A549 and NCI-H446 lung cancer cells and treated them with irisin. We detected the proliferation by MTT assay, and assessed the migration and invasion of the cells by scratch wound healing assay and Tran-swell assay. The expression levels of epithelial-to-mesenchymal transition (EMT) markers and the related signaling pathways were detected by western blot analysis. Meanwhile, anmore » inhibitor of PI3K was used to investigate the effect of irsin. Finally, the expression of Snail was detected. We demonstrated that irisin inhibits the proliferation, migration, and invasion of lung cancer cells, and has a novel role in mediating the PI3K/AKT pathway in the cells. Irisin can reverse the activity of EMT and inhibit the expression of Snail via mediating the PI3K/AKT pathway, which is a key regulator of Snail. These results revealed that irisin inhibited EMT and reduced the invasion of lung cancer cells via the PI3K/AKT/Snail pathway. - Highlights: • Irisin inhibits the proliferation of lung cancer cells. • Irisin inhibits the migration and invasion of lung cancer cells. • Irisin affects the expression of EMT markers via inhibiting the PI3K/AKT pathway in lung cancer cells. • Irisin induces Snail downregulation via PI3K/AKT pathway activation.« less

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion.

PubMed

Nakajima, Kazuyuki; Graeber, Manuel B; Sonoda, Maya; Tohyama, Yoko; Kohsaka, Shinichi; Kurihara, Tadashi

2006-08-01

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.

[The expression of Cyclin D1 modulated by somatotropin on human pancreas cancer cell lines Bxpc-3].

PubMed

Li, Fei; Liu, Da-chuan; Sun, Jia-bang

2004-04-07

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

Correlation of lung surface area to apoptosis and proliferation in human emphysema.

PubMed

Imai, K; Mercer, B A; Schulman, L L; Sonett, J R; D'Armiento, J M

2005-02-01

Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function. To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area. In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.

Substance P Promotes the Progression of Endometrial Adenocarcinoma.

PubMed

Ma, Jing; Yuan, Shifa; Cheng, Jianxin; Kang, Shan; Zhao, Wenhong; Zhang, Jie

2016-06-01

It has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma. The expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis. The expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects. Substance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

NASA Astrophysics Data System (ADS)

Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

[Protective effect of hydrogen against hyperoxia-induced type II alveolar epithelial cell injury].

PubMed

Yao, Lan; Xu, Feng; Luo, Chong; Yu, Pan; Dong, Xinxin; Sun, Xuejun; Liu, Chengjun

2013-02-01

To investigate the protective effect of hydrogen against hyperoxia-induced oxidative stress injury in premature rat type II alveolar epithelial cells (AECs). The type II AECs isolated from premature rats were randomly divided into air (21% oxygen) control group, hyperoxia (95% oxygen) control group, air + hydrogen group, and hyperoxia+ hydrogen group. The cells with hydrogen treatment were cultured in the presence of rich hydrogen. After the corresponding exposure for 24 h, the cell morphology was observed microscopically. MTT assay was used to evaluated the cell proliferation ability, and JC-1 fluorescence probe was used to detect the mitochondrial membrane potential (δφ) changes of the type II AECs. The concentration of maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity in the cell supernatant were detected using colorimetric method. No significant differences were found in cell growth or measurements between air control and air + hydrogen groups. Compared with air control group, the cells exposed to hyperoxia showed significantly suppressed proliferation, reduced mitochondrial membrane potential, increased MDA content, and decreased SOD activity. Intervention with hydrogen resulted in significantly increased cell proliferation and SOD activity and lowered MDA content, and restored the mitochondrial membrane potential in the cells with hyperoxia exposure (P<0.05). Hydrogen can significantly reduce hyperoxia-induced oxidative stress injury in premature rat type II AECs, improve the cellular antioxidant capacity, stabilize the mitochondrial membrane potential, and reduce the inhibitory effect of hyperoxia on cell proliferation.

Elucidation of proliferative capability of mononuclear tetraploid cells, emerging spontaneously from diploid cells, using image cytometry and fluorescence in situ hybridization.

PubMed

Ito, Hideaki; Oga, Atsunori; Furuya, Tomoko; Ikemoto, Kenzo; Amakawa, Genta; Chochi, Yasuyo; Kawauchi, Shigeto; Sasaki, Kohsuke

2013-06-01

Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method. In this study, two completely disomic cell lines were used, TIG-7, a fibroblast cell line and CAL-51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony-forming ability were examined. Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG-7 and CAL-51. Colonies of TIG-7 TCs were not observed, but were observed of CAL-51 TCs. Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG-7 TCs do not proliferate but that mononuclear CAL-51 TCs are able to. © 2013 Blackwell Publishing Ltd.

Divergent Kinetics of Proliferating T Cell Subsets in Simian Immunodeficiency Virus (SIV) Infection: SIV Eliminates the “First Responder” CD4+ T Cells in Primary Infection

PubMed Central

Wang, Xiaolei; Xu, Huanbin; Pahar, Bapi; Lackner, Andrew A.

2013-01-01

Although increased lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been reported in blood, there is little information on cell turnover in tissues, particularly in primary SIV infection. Here we examined the levels of proliferating T cell subsets in mucosal and peripheral lymphoid tissues of adult macaques throughout SIV infection. To specifically label cells in S-phase division, all animals were inoculated with bromodeoxyuridine 24 h prior to sampling. In healthy macaques, the highest levels of proliferating CD4+ and CD8+ T cells were in blood and, to a lesser extent, in spleen. Substantial percentages of proliferating cells were also found in intestinal tissues, including the jejunum, ileum, and colon, but very few proliferating cells were detected in lymph nodes (axillary and mesenteric). Moreover, essentially all proliferating T cells in uninfected animals coexpressed CD95 and many coexpressed CCR5 in the tissues examined. Confocal microscopy also demonstrated that proliferating cells were substantial viral target cells for SIV infection and viral replication. After acute SIV infection, percentages of proliferating CD4+ and CD8+ T cells were significantly higher in tissues of chronically infected macaques and macaques with AIDS than in those of the controls. Surprisingly, however, we found that proliferating CD4+ T cells were selectively decreased in very early infection (8 to 10 days postinoculation [dpi]). In contrast, levels of proliferating CD8+ T cells rapidly increased after SIV infection, peaked by 13 to 21 dpi, and thereafter remained significantly higher than those in the controls. Taken together, these findings suggest that SIV selectively infects and destroys dividing, nonspecific CD4+ T cells in acute infection, resulting in homeostatic changes and perhaps continuing loss of replication capacity to respond to nonspecific and, later, SIV-specific antigens. PMID:23596288

Divergent kinetics of proliferating T cell subsets in simian immunodeficiency virus (SIV) infection: SIV eliminates the "first responder" CD4+ T cells in primary infection.

PubMed

Wang, Xiaolei; Xu, Huanbin; Pahar, Bapi; Lackner, Andrew A; Veazey, Ronald S

2013-06-01

Although increased lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been reported in blood, there is little information on cell turnover in tissues, particularly in primary SIV infection. Here we examined the levels of proliferating T cell subsets in mucosal and peripheral lymphoid tissues of adult macaques throughout SIV infection. To specifically label cells in S-phase division, all animals were inoculated with bromodeoxyuridine 24 h prior to sampling. In healthy macaques, the highest levels of proliferating CD4(+) and CD8(+) T cells were in blood and, to a lesser extent, in spleen. Substantial percentages of proliferating cells were also found in intestinal tissues, including the jejunum, ileum, and colon, but very few proliferating cells were detected in lymph nodes (axillary and mesenteric). Moreover, essentially all proliferating T cells in uninfected animals coexpressed CD95 and many coexpressed CCR5 in the tissues examined. Confocal microscopy also demonstrated that proliferating cells were substantial viral target cells for SIV infection and viral replication. After acute SIV infection, percentages of proliferating CD4(+) and CD8(+) T cells were significantly higher in tissues of chronically infected macaques and macaques with AIDS than in those of the controls. Surprisingly, however, we found that proliferating CD4(+) T cells were selectively decreased in very early infection (8 to 10 days postinoculation [dpi]). In contrast, levels of proliferating CD8(+) T cells rapidly increased after SIV infection, peaked by 13 to 21 dpi, and thereafter remained significantly higher than those in the controls. Taken together, these findings suggest that SIV selectively infects and destroys dividing, nonspecific CD4(+) T cells in acute infection, resulting in homeostatic changes and perhaps continuing loss of replication capacity to respond to nonspecific and, later, SIV-specific antigens.

MicroRNA-126 enhances the sensitivity of osteosarcoma cells to cisplatin and methotrexate

PubMed Central

JIANG, LIANGDONG; HE, AIYONG; HE, XIAOJIE; TAO, CHENG

2015-01-01

The establishment of novel chemotherapy drugs for osteosarcoma is urgently required, and the mechanisms and effects of cisplatin (DDP) and methotrexate (MTX) in the current treatment of osteosarcoma have not been fully elucidated. The present study aimed to observe the effect of DDP, MTX and rapamycin on osteosarcoma cell proliferation and apoptosis, and to investigate the association between miR-126 and the effects of DDP and MTX in osteosarcoma cells. miR-126-overexpressing and -silencing lentiviral vectors were constructed, and MG63 and U-2 OS osteosarcoma cells were infected. An MTT assay was conducted to detect transfected cell proliferation, and the effects of the chemotherapy drugs on transfected cell apoptosis were detected by flow cytometry. The cell cycle of the transfected cells was analyzed via flow cytometry. As the miR-126-overexpressing and -silencing osteosarcoma cell lines were successfully constructed, it was observed that DDP and MTX inhibited osteosarcoma cell proliferation. With the decreased expression of miR-126, the sensitivity of osteosarcoma cells to DDP and MTX was reduced at the same concentration. The flow cytometry suggested that DDP and MTX could promote the apoptosis of osteosarcoma cells with overexpressed miR-126, whereas they could not significantly impact the apoptosis of the miR-126-silenced osteosarcoma cells. Meanwhile, DDP inhibited the cell cycle of the miR-126-overexpressing osteosarcoma cells. In conclusion, DDP and MTX inhibited the proliferation and promoted the apoptosis of the osteosarcoma cells, and these processes were dependent upon the expression of miR-126. PMID:26788206

Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

PubMed

Mao, Meiya; Zheng, Xiaojiao; Jin, Bohong; Zhang, Fubin; Zhu, Linyan; Cui, Lining

2017-12-01

CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

Mangiferin regulates proliferation and apoptosis in glioma cells by induction of microRNA-15b and inhibition of MMP-9 expression.

PubMed

Xiao, Jinsong; Liu, Li; Zhong, Zian; Xiao, Cheng; Zhang, Junjian

2015-06-01

Mangiferin, a flavonoid extracted from the leaves of the Anacardiaceae plant, the mango tree, has physiological activity and pharmacological effects in many aspects. The present study aimed to clarify the effect of mangiferin on proliferation and apoptosis of glioma cells and the mechanism of these curative effects of mangiferin. In this experiment, we detected the proliferation using 3-(4,5-dimethylthylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Then, cell apoptosis of U87 glioma cells was measured with the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, DAPI staining assay and the caspase-3 and caspase-9 activity assay kit. Next, quantitative real-time PCR and gelatin zymography were used to analyze the expression of microRNA-15b (miR-15b) and matrix metalloproteinase-9 (MMP-9), respectively. MMP-9 agonist, miR-15b mimics and anti-miR-15b mimics were added to the U87 glioma cells for elucidating the mechanisms involved in the curative effects of mangiferin. In the present study, mangiferin notably restrained the proliferation and increased the apoptosis of the U87 glioma cells. Meanwhile, mangiferin specifically promoted the expression of miR-15b and suppressed the level of MMP-9 in the U87 glioma cells. miR-15b regulated the expression of MMP-9 in the U87 glioma cells. MMP-9 agonist and anti-miR‑15b reduced the curative effects of mangiferin in the U87 glioma cells. In summary, mangiferin regulates proliferation and apoptosis in glioma cells by induction of miR-15b and inhibition of MMP-9 expression.

Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis

PubMed Central

WANG, QING; WANG, YAN-LIN; WANG, KAI; YANG, JIAN-LIN; CAO, CHUN-YU

2015-01-01

The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML. PMID:25435975

[Overexpression of miR-519d-3p inhibits the proliferation of DU-145 prostate cancer cells by reducing TRAF4].

PubMed

Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

2018-01-01

Objective To observe the effect of microRNA-519d-3p (miR-519d-3p) on the proliferation of prostate cancer cells and explore the possible molecular mechanism. Methods The expression level of miR-519d-3p in PC-3, DU-145, 22RV1, PC-3M, LNCaP human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time quantitative PCR. miR-519d-3p mimics or negative control microRNAs (miR-NC) was transfected into the prostate cancer cells with the lowest level of miR-519d-3p expression. Transfection efficiency was examined. The effect of miR-519d-3p on the cell cycle of prostate cancer was detected by flow cytometry. MTT assay and plate clone formation assay were used to detect its effect on the proliferation of prostate cancer cells. Bioinformatics software was used to predict and dual luciferase reporter assay was used to validate the target gene of miR-519d-3p. Real-time quantitative PCR was used to detect the expression of miR-519d-3p target gene. Western blot analysis was used to detect the expression of target gene protein and downstream protein. Results The expression of miR-519d-3p in normal prostate epithelial cells was significantly higher than that in prostate cancer cells, and the lowest was found in DU-145 cells. After transfected with miR-519d-3p mimics, the expression level of miR-519d-3p in DU-145 cells increased significantly. Bioinformatics prediction and dual luciferase reporter gene confirmed that tumor necrosis factor receptor associated factor 4 (TRAF4) was the target gene of miR-519d-3p. Overexpression of miR-519d-3p significantly reduced the expression of TRAF4 gene and its downstream TGF-β signaling pathway proteins in the prostate cancer cells. Conclusion The expression of miR-519d-3p is down-regulated in prostate cancer cells. Overexpression of miR-519d-3p can inhibit the proliferation of prostate cancer cells. The possible mechanism is that miR-519d-3p inhibits the expression of TRAF4.

Integration of Breast Cancer Secretomes with Clinical Data Elucidates Potential Serum Markers for Disease Detection, Diagnosis, and Prognosis.

PubMed

Ziegler, Yvonne S; Moresco, James J; Yates, John R; Nardulli, Ann M

2016-01-01

Cancer cells secrete factors that influence adjacent cell behavior and can lead to enhanced proliferation and metastasis. To better understand the role of these factors in oncogenesis and disease progression, estrogen and progesterone receptor positive MCF-7 cells, triple negative breast cancer MDA-MB-231, DT22, and DT28 cells, and MCF-10A non-transformed mammary epithelial cells were grown in 3D cultures. A special emphasis was placed on triple negative breast cancer since these tumors are highly aggressive and no targeted treatments are currently available. The breast cancer cells secreted factors of variable potency that stimulated proliferation of the relatively quiescent MCF-10A cells. The conditioned medium from each cell line was subjected to mass spectrometry analysis and a variety of secreted proteins were identified including glycolytic enzymes, proteases, protease inhibitors, extracellular matrix proteins, and insulin-like growth factor binding proteins. An investigation of the secretome from each cell line yielded clues about strategies used for breast cancer proliferation and metastasis. Some of the proteins we identified may be useful in the development of a serum-based test for breast cancer detection, diagnosis, prognosis, and monitoring.

[Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells from patients with aplastic anemia].

PubMed

Wang, Xin; Ma, Feng-Xia; Lu, Shi-Hong; Chi, Ying; Chen, Fang; Li, Xue; Li, Juan-Juan; Du, Wen-Jing; Feng, Ying; Cui, Jun-Jie; Song, Bao-Quan; Han, Zhong-Chao

2014-06-01

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Sesamin induces ER stress-mediated apoptosis and activates autophagy in cervical cancer cells.

PubMed

Dou, Haowen; Yang, Shasha; Hu, Yulai; Xu, Dongyuan; Liu, Lan; Li, Xiangdan

2018-05-01

Sesamin, a major lignan of sesame oil, has demonstrated anticancer properties. However, its anticancer effects on cervical cancer have not been studied. Here, we investigated the effects of sesamin on cervical cancer (HeLa) cell line and explored the underlying mechanisms. HeLa cells were cultured with sesamin. CCK-8 and scratch wound test were applied to detect the proliferation and migration ability, while flow cytometry and TUNEL staining were applied to detect apoptosis. The expression of Bax and Bcl-2 was assessed by Western blotting. Further observe the ultrastructure using transmission electron microscopy (TEM) and detect the expression of caspase-12, GRP78, GADD153, IRE1α, p-IRE1α, JNK, p-JNK, LC3I/II and beclin-1. In addition, HeLa cells were treated with 3-MA (an autophagy inhibitor) and/or sesamin. Then detect the expression of LC3I/II and cell viability. CCK-8 and scratch wound test revealed that sesamin inhibits HeLa cells proliferation and migration, while flow cytometry and TUNEL staining indicated that sesamin induces apoptosis in these cells. In sesamin group, the expression of Bax, caspase-12, GRP78, GADD153, p-IRE1α, p-JNK, LC3I/II and beclin-1 was up-regulated while Bcl-2 was down-regulated compared to control group. Further research revealed that sesamin also induces Hela cells autophagy and inhibition of autophagy increases cell viability of sesamin-treated HeLa cells. Sesamin inhibits proliferation/migration of HeLa cells and induces ER stress-mediated apoptosis through IRE1α/JNK pathway, and that it activates autophagy and autophagic death in these cells, further validate the anticancer effect of sesamin. Copyright © 2018 Elsevier Inc. All rights reserved.

Neuronal models for evaluation of proliferation in vitro using high content screening.

PubMed

Mundy, William R; Radio, Nicholas M; Freudenrich, Theresa M

2010-04-11

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation. Published by Elsevier Ireland Ltd.

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

PubMed Central

Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

2013-01-01

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. PMID:24074569

[Saponin 6 of Anemone Taipaiensis inhibits proliferation and induces apoptosis of U87 MG cells].

PubMed

Ji, Chenchen; Cheng, Guang; Tang, Haifeng; Zhang, Yun; Hu, Yiyang; Zheng, Minhua; Fei, Zhou

2015-04-01

To investigate the effect of saponin 6 of Anemone Taipaiensis on the proliferation of human U87 MG glioma cells and the possible mechanism. U87 MG cells were treated with different concentrations of saponin 6 (0.0, 1.6, 3.2, 6.4, 12.8 μg/mL) for 24 hours or 48 hours. Cell viability was measured by MTT assay; the apoptosis rate was detected by flow cytometry combined with annexin V-FITC /PI staining; Western blotting was applied to determine the protein level of activated caspase-3. Compared with control groups, saponin 6 significantly inhibited U87 MG cell proliferation in a time- and dose-depended manner. Apoptosis rate of U87 MG cells and the expression of activated caspase-3 were raised with the increasing concentration of saponin 6. Saponin 6 of Anemone Taipaiensis could depress cell proliferation in a dose-depended manner, increase the expression of activated caspase-3 and promote apoptosis in U87 MG cells.

Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.

PubMed

Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

2017-02-17

We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2'-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

PubMed Central

Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

2017-01-01

We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity. PMID:28218663

Rac1b enhances cell survival through activation of the JNK2/c-JUN/Cyclin-D1 and AKT2/MCL1 pathways

PubMed Central

Wang, Hong; Wei, Si-Si; Chen, Jie; Chen, Yi-He; Xu, Wei-Ping; Jie, Qi-Qiang; Zhou, Qing; Li, Yi-Gang; Wei, Yi-Dong; Wang, Yue-Peng

2016-01-01

Rac1b is a constitutively activated, alternatively spliced form of the small GTPase Rac1. Previous studies showed that Rac1b promotes cell proliferation and inhibits apoptosis. In the present study, we used microarray analysis to detect genes differentially expressed in HEK293T cells and SW480 human colon cancer cells stably overexpressing Rac1b. We found that the pro-proliferation genes JNK2, c-JUN and cyclin-D1 as well as anti-apoptotic AKT2 and MCL1 were all upregulated in both lines. Rac1b promoted cell proliferation and inhibited apoptosis by activating the JNK2/c-JUN/cyclin-D1 and AKT2/MCL1 pathways, respectively. Very low Rac1b levels were detected in the colonic epithelium of wild-type Sprague-Dawley rats. Knockout of the rat Rac1 gene exon-3b or knockdown of endogenous Rac1b in HT29 human colon cancer cells downregulated only the AKT2/MCL1 pathway. Our study revealed that very low levels of endogenous Rac1b inhibit apoptosis, while Rac1b upregulation both promotes cell proliferation and inhibits apoptosis. It is likely the AKT2/MCL1 pathway is more sensitive to Rac1b regulation. PMID:26918455

Effect of concentrated growth factors on beagle periodontal ligament stem cells in vitro.

PubMed

Yu, Bohan; Wang, Zuolin

2014-01-01

Identifying a reliable and effective cytokine or growth factor group has been the focus of stem cell osteogenic induction studies. Concentrated growth factors (CGFs) as the novel generation of platelet concentrate products, appear to exhibit a superior clinical and biotechnological application potential, however, there are few studies that have demonstrated this effect. This study investigated the proliferation and differentiation of periodontal ligament stem cells (PDLSCs) co‑cultured with CGFs. The rate of proliferation was analyzed by cell counting and an MTT assay. Mineralization nodule counts, alkaline phosphatase activity detection, qPCR, western blot analysis and immunohistochemistry were used to analyze mineralization effects. The results showed that CGF significantly promoted the proliferation of PDLSCs, and exhibited a dose‑dependent effect on the activation and differentiation of the stem cells. The application of CGF on PDLSC proliferation and osteoinduction may offer numerous clinical and biotechnological application strategies.

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia).

PubMed

Gonzalez, C R; Muscarsel Isla, M L; Fraunhoffer, N A; Leopardo, N P; Vitullo, A D

2012-08-01

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.

Low molecular weight fraction secreted by SKOV3 cells expands peripheral CD4+CD25+ regulatory T cells and enhances their suppressive capacity.

PubMed

Li, Xiao; Wan, Xiaoyun; Mao, Yuyan; Lu, Weiguo; Xie, Xing

2010-09-01

The increase of CD4+CD25+ regulatory T cells in patients with ovarian carcinoma has been verified. Here we investigated the effects of supernatant derived from ovarian carcinoma cell SKOV3 on peripheral regulatory T cells. Supernatant from SKOV3 was collected and fractionated into three different molecular weight fractions (MWFs). The proliferation of the CD4+CD25+ regulatory T cells cultured in complete RPMI 1640 medium with the different stimulators was detected. The phenotype (GITR and CTLA-4) of natural and expanded CD4+CD25+ T cells was detected by flow cytometry. Foxp3 mRNA expression of low MWF-expanded CD4+CD25+ T cells was detected by RT-PCR. Those expanded CD4+CD25+ regulatory T cells showed enhanced capacity to suppress CD4+CD25- T proliferation and increased expression of GITR and CTLA-4. In brief, low molecular weight fraction of supernatant secreted by SKOV3 could expand peripheral CD4+CD25+ regulatory T cells and enhance their suppressive function.

Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death

PubMed Central

Yan, Bing Chun; Park, Joon Ha; Chen, Bai Hui; Cho, Jeong-Hwi; Kim, In Hye; Ahn, Ji Hyeon; Lee, Jae-Chul; Hwang, In Koo; Cho, Jun Hwi; Lee, Yun Lyul; Kang, Il-Jun; Won, Moo-Ho

2014-01-01

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperitoneal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen (NeuN; a neuronal marker) and Fluoro-Jade B (a marker for the localization of neuronal degeneration) was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67 (a marker for proliferating cells)-immunoreactive cells were reduced in number and reached the lowest level at 4 weeks. Doublecortin (DCX; a marker for newly generated neurons)-immunoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2′-deoxyuridine (BrdU)-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature BrdU/NeuN double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death. PMID:25422633

Effects of real or simulated microgravity on plant cell growth and proliferation

NASA Astrophysics Data System (ADS)

Medina, Francisco Javier; Manzano, Ana Isabel; Herranz, Raul; Dijkstra, Camelia; Larkin, Oliver; Hill, Richard; Carnero-Díaz, Eugénie; van Loon, Jack J. W. A.; Anthony, Paul; Davey, Michael R.; Eaves, Laurence

Experiments on seed germination and seedling growth performed in real microgravity on the International Space Station and in different facilities for simulating microgravity in Earth-based laboratories (Random Positioning Machine and Magnetic Levitation), have provided evidence that the absence of gravity (or the artificial compensation of the gravity vector) results in the uncoupling of cell growth and proliferation in root meristematic cells. These are two essential cellular functions that support plant growth and development, which are strictly coordinated under normal ground gravity conditions. Under conditions of altered gravity, we observe that cell proliferation is enhanced, whereas cell growth is reduced, according to different morphometric, cytological and immunocytochemical parameters. Since coordination of cell growth and proliferation are major features of meristematic cells, this observed uncoupling represents a major stress condition for these cells, inducing major alterations in the pattern of plant development. Moreover, the expression of the cyclin B1 gene, a regulator of the entry into mitosis and normally used as an indicator of cell proliferation, appears reduced in the smaller and more actively proliferating cells of samples grown under the conditions of our experiments. These results are compatible with an alteration of the regulation of the cell cycle, producing a shorter G2 period. Interestingly, while cyclin B1 expression is depleted in these conditions in root meristematic cells, it is enhanced in cotyledons of the same seedlings, as shown by qPCR and by the expression of the gus reporter gene. It is known that regulation of root growth (including regulation of root meristematic activity) is driven mainly by auxin, whereas cytokinin is the key hormone regulating cotyledon growth. Therefore, our results indicate a major role of auxin in the sensitivity to altered gravity of root meristematic cells. Auxin is crucial in maintaining the coupling of cell growth and proliferation under normal conditions and it should have a decisive influence in the uncoupling of these processes under altered gravity. Experiments to detect auxin distribution in roots under altered gravity produced by diamagnetic levitation have shown that the lateral balanced distribution of the growth regulator in the root cap is altered slightly and that the total concentration of the auxin detected in root tips is somewhat reduced. These effects are independent of the orientation of statoliths in columella cells.

Effects and mechanism of GA-13315 on the proliferation and apoptosis of KB cells in oral cancer.

PubMed

Shen, Shan; Tang, Jingxia

2017-08-01

The present study describes the effects and mechanism of GA-13315 on the proliferation and apoptosis of KB cells in oral cancer. Oral cancer is twice as common in men than women. More than 90% of oral cancers in men and 85% in women are linked to lifestyle and environmental factors. PPP2R2B methylation may be associated with survival and prognosis in patients with gliomas. In tumor cell proliferation and apoptosis, the mechanism of PPP2R2B remains unclear. In the present study, we found that PPP2R2B expression of H1299 cells is significantly decreased after being treated by GA-13315. KB cells were isolated from patients with oral cancer and treated with GA-13315 (5 µM). Cells without GA-13315 treatment served as the control group. An MTT experiment was performed to detect the post-treatment cell growth between the groups. A flow cytometry was used to detect cell apoptosis. Western blot analysis and quantitative polymerase chain reaction methods were used for detecting the expression of PPP2R2B. Compared with the control group, the cell proliferation of the treatment group slowed after being treated with GA-13315. The difference was statistically significant (P<0.05). Western blotting showed that the PPP2R2B expression of cells was reduced after being treated with GA-13315. Compared with the control group, the difference was statistically significant (P<0.05). According to results from the Transwell migration assay, the invasiveness of the KB cells of oral cancer were weakened after being treated by GA-13315. GA-13315 can accelerate the apoptosis of oral cancer cells and presents a dose correlation. The biological effect is exerted through the decrease of PPP2R2B.

[RITA combined with temozolomide inhibits the proliferation of human glioblastoma U87 cells].

PubMed

He, Xiao-Yan; Feng, Xiao-Li; Song, Xin-Pei; Zeng, Huan-Chao; Cao, Zhong-Xu; Xiao, Wei-Wei; Zhang, Bao; Wu, Qing-Hua

2016-10-20

To observe the effect of RITA, a small molecule that targets p53, combined with temozolomide (TMZ) on proliferation, colony formation and apoptosis of human glioblastoma U87 cells and explore the underlying mechanism. Cultured U87 cells were treated with RITA (1, 5, 10, 20 µmol/L), TMZ, or RITA+TMZ (half dose) for 24, 48 or 72 h. MTS assay were used to detect the cell proliferation, and the cell proliferation rate and inhibitory rate were calculated. The effect of combined treatments was evaluated by the q value. The expressions of p53, p21 and other apoptosis-associated genes were detected by qRT-PCR and Western blotting; cell apoptosis was assayed using flow cytometry with Annexin V/PI double staining; colony formation of the cells was detected with crystal violet staining. MTS assay showed that RITA at the 4 doses more potently inhibited U87 cell viability than TMZ at 72 h (P=0.000) with inhibitory rates of 25.94%-41.38% and 3.84%-8.20%, respectively. RITA combined with TMZ caused a more significant inhibition of U87 cells (29.21%-52.11%) than RITA (P<0.01) and TMZ (P=0.000) alone. At the doses above 5 µmol/L, the combined treatments with RITA+TMZ for 48 h resulted in q values exceeding 1.2 and showed an obvious synergistic effect of the drugs. Both RITA and TMZ, especially the latter, significantly increased the expressions of p53, p21, puma, and other apoptosis-associated genes to accelerate apoptosis and inhibit the growth and colony formation of U87 cells, and the effect was more obvious with a combined treatment. RITA inhibits the growth of human glioblastoma cells and enhance their sensitivity to TMZ by up-regulating p53 expression, and when combined, RITA and TMZ show a synergistic effect to cause a stronger cell inhibition.

[Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

PubMed

Li, Lixuan; Li, Jia

2015-05-01

To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

A peptide derived from alpha-fetoprotein inhibits the proliferation induced by estradiol in mammary tumor cells in culture.

PubMed

Sierralta, Walter D; Epuñan, Maria J; Reyes, José M; Valladares, Luis E; Andersen, Thomas T; Bennett, James A; Jacobson, Herbert I; Pino, Ana M

2008-01-01

This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.

[RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].

PubMed

Wei, Min; Zhang, Yan-Ling; Chen, Lan; Cai, Cui-Xia; Wang, Han-Duo

2016-02-20

To explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms. Three HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting. Transfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (P<0.01). HERC4 silencing by siRNA-3 markedly suppressed the proliferation and migration of Hela cells, increased the apoptosis rate (P<0.01) and reduced the expression levels of cyclin D1 and Bcl-2 (P<0.01). Silencing of HERC4 efficiently inhibits the proliferation, migration, and invasion of Hela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

PubMed

Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

2015-01-01

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

[Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133(+) liver cancer stem cells and increases their cisplatin chemosensitivity].

PubMed

Lan, Xi; Wang, Yong; Cao, Shu; Zou, Dongling; Li, Fang; Li, Shaolin

2012-12-01

To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line. CD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry. The sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

Selectively Reduced Intracellular Proliferation of Salmonella enterica Serovar Typhimurium within APCs Limits Antigen Presentation and Development of a Rapid CD8 T Cell Response1

PubMed Central

Albaghdadi, Homam; Robinson, Nirmal; Finlay, Brett; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Ag presentation to CD8+ T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 × 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8+ T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8+ T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8+ T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intra-cellular proliferation of virulent pathogens may be an important mechanism of immune evasion. PMID:19692639

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts.

PubMed

Will, Katja; Kalbe, Claudia; Kuzinski, Judith; Lösel, Dorothea; Viergutz, Torsten; Palin, Marie-France; Rehfeldt, Charlotte

2012-08-01

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 μg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.

Effect of low level laser therapy and high intensity laser therapy on endothelial cell proliferation in vitro: preliminary communication

NASA Astrophysics Data System (ADS)

Lukowicz, Malgorzata; Szymanska, Justyna; Goralczyk, Krzysztof; Zajac, Andrzej; Rość, Danuta

2013-01-01

Background: The main purpose of this study was to analyze the influence of power intensity and wavelength of Low Level Laser Therapy (LLLT) and HILT (High Intensity Laser Therapy) on endothelial cell proliferation. Material and methods: The tests were done on human umbilical vein endothelial cells (HUVEC). Cultures were exposed to laser irradiation of 660 nm and 670 nm at different dosages, power output was 10 - 40 mW as well as 820 nm with power 100 mW and 808 nm with power 1500 mW. Energy density was from 0.28 to 11,43 J/cm2. Cell proliferation of a control and tested culture was evaluated with a colorimetric device to detect live cells. The tests were repeated 8 times. Results: We observed good effects of LLLT on live isolated ECs and no effects in experiments on previous deep-frozen cultures. Also HILT stimulated the proliferation of HUVEC. Conclusion: Endothelial cells play a key role in vascular homeostasis in humans. We observed the stimulatory effect of LLLT and HILT on proliferation of HUVEC. Many factors influence the proliferation of EC, so is it necessary to continue the experiment with different doses, intensity and cell concentration.

A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation.

PubMed

Dawes, L J; Shelley, E J; McAvoy, J W; Lovicu, F J

2018-04-01

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and β-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hypoxia enhances periodontal ligament stem cell proliferation via the MAPK signaling pathway.

PubMed

He, Y; Jian, C X; Zhang, H Y; Zhou, Y; Wu, X; Zhang, G; Tan, Y H

2016-11-21

There is high incidence of periodontal disease in high-altitude environments; hypoxia may influence the proliferation and clone-forming ability of periodontal ligament stem cells (PDLSCs). The MAPK signaling pathway is closely correlated with cell proliferation, differentiation, and apoptosis. Thus, we isolated and cultured PDLSCs under hypoxic conditions to clarify the impact of hypoxia on PDLSC proliferation and the underlying mechanism. PDLSCs were separated and purified by the limiting dilution method and identified by flow cytometry. PDLSCs were cultured under hypoxic or normoxic conditions to observe their cloning efficiency. PDLSC proliferation at different oxygen concentrations was evaluated by MTT assay. Expression of p38/MAPK and MAPK/ERK signaling pathway members was detected by western blotting. Inhibitors for p38/MAPK or ERK were applied to PDLSCs to observe their impacts on clone formation and proliferation. Isolated PDLSCs exhibited typical stem cell morphological characteristics, strong abilities of globular clone formation and proliferation, and upregulated expression of mesenchymal stem cell markers. Stem cell marker expression was not statistically different between PDLSCs cultured under hypoxia and normoxia (P > 0.05). The clone number in the hypoxia group was significantly higher than that in the control (P < 0.05). PDLSC proliferation under hypoxia was higher than that of the control (P < 0.001). p38 and ERK1/2 phosphorylation in hypoxic PDLSCs was markedly enhanced compared to that in the control (P < 0.05). Either P38/MAPK inhibitor or ERK inhibitor treatment reduced clone formation and proliferation. Therefore, hypoxia enhanced PDLSC clone formation and proliferation by activating the p38/MAPK and ERK/MAPK signaling pathways.

Original Research: miR-194 inhibits proliferation and invasion and promotes apoptosis by targeting KDM5B in esophageal squamous cell carcinoma cells.

PubMed

Cui, Guanghui; Liu, Donglei; Li, Weihao; Li, Yuhang; Liang, Youguang; Shi, Wensong; Zhao, Song

2017-01-01

Increasing evidence suggests that miR-194 is down-regulated in esophageal squamous cell carcinoma tumor tissue. However, the role and underlying mechanism of miR-194 in esophageal squamous cell carcinoma have not been well defined. We used DIANA, TargetScan and miRanda to perform target prediction analysis and found KDM5B is a potential target of miR-194. Based on these findings, we speculated that miR-194 might play a role in esophageal squamous cell carcinoma development and progression by regulation the expression of KDM5B. We detected the expression of miR-194 and KDM5B by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot assays, respectively, and found down-regulation of miR-194 and up-regulation of KDM5B existed in esophageal squamous cell carcinoma cell lines. By detecting proliferation, invasion and apoptosis of TE6 and TE14 cells transfected with miR-194 mimics or mimic control, miR-194 was found to inhibit proliferation and invasion and promote apoptosis of esophageal squamous cell carcinoma cells. miR-194 was further verified to regulate proliferation, apoptosis and invasion of esophageal squamous cell carcinoma cells by directly targeting KDM5B. Furthermore, animal studies were performed and showed that overexpression of miR-194 inhibited the growth of esophageal squamous cell carcinoma tumors in vivo. These results confirmed our speculation that miR-194 targets KDM5B to inhibit esophageal squamous cell carcinoma development and progression. These findings offer new clues for esophageal squamous cell carcinoma development and progression and novel potential therapeutic targets for esophageal squamous cell carcinoma. © 2016 by the Society for Experimental Biology and Medicine.

TRPC1 is required for survival and proliferation of cochlear spiral ganglion stem/progenitor cells.

PubMed

Chen, Hsin-Chien; Wang, Chih-Hung; Shih, Cheng-Ping; Chueh, Sheau-Huei; Liu, Shu-Fan; Chen, Hang-Kang; Lin, Yi-Chun

2015-12-01

The present studies were designed to test the hypothesis that canonical transient receptor potential channel 1 (TRPC1) is required for the proliferation of cochlear spiral ganglion stem/progenitor cells (SPCs). TRPC1 were detected and evaluated in postnatal day 1 CBA/CaJ mice pups derived-cochlear spiral ganglion SPCs by reverse transcription-polymerase chain reaction, Western blot, immunocytochemistry, and calcium imaging. The cell viability and proliferation of the spiral ganglion SPCs following si-RNA mediated knockdown of TRPC1 or addition of TRPC channel blocker SKF9635 were compared to controls. In spiral ganglion SPCs, TRPC1 was found to be the most abundantly expressed TRPC subunit and shown to contribute to store-operated calcium entry. Silencing of TRPC1 or addition of TRPC channel blockers significantly decreased the rate of cell proliferation. The results suggest that TRPC1 might serve as an essential molecule in regulating the proliferation of spiral ganglion SPCs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The coordinated effects of Apatinib and Tripterine on the proliferation, invasiveness and apoptosis of human hepatoma Hep3B cells.

PubMed

Li, Huihui; Fan, Yichang; Yang, Fan; Zhao, Lei; Cao, Bangwei

2018-07-01

As a novel vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor, Apatinib has exhibited antitumor effects in a variety of solid tumors. Extracts of Chinese herbal medicines have emerged as a promising alternative option to increase the sensitivity of patients to chemotherapeutics while alleviating side effects. The present study aimed to investigate the effects of Apatinib and the traditional Chinese herb Tripterine on the proliferation, invasion and apoptosis of human hepatoma Hep3B cells. The expression of VEGFR-2 in Hep3B cells was detected by western blotting and immunofluorescence assays. Hep3B cells were then divided into four different groups: Control group, Apatinib group, Tripterine group and Apatinib plus Tripterine group. The proliferation, invasion and apoptosis of these four groups of Hep3B cells were assessed by MTS, wound healing and Transwell assays, and flow cytometry, respectively. Finally, the levels of the proliferation-associated proteins phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) and the apoptosis-associated proteins cleaved Caspase-3 and B-cell lymphoma-associated X protein (Bax) were detected by western blotting. The proliferation, migration and invasion of Hep3B cells were significantly inhibited by Apatinib and Tripterine, compared with the control group (P<0.01). The inhibitory effect of the combination group was markedly stronger than that of the Apatinib and Tripterine groups. The downregulation of p-Akt and p-ERK induced by Apatinib and Tripterine was further inhibited in the combination group (P<0.05), and the expression levels of Caspase-3 and Bax were also significantly increased in the combination group (P<0.05). The combination of Apatinib and Tripterine significantly inhibited the proliferation, migration and invasion ability and promoted the apoptosis of Hep3B cells by downregulating the expression of p-Akt and p-ERK, and upregulating the expression of Caspase-3 and Bax.

Chrysin and its phosphate ester inhibit cell proliferation and induce apoptosis in Hela cells.

PubMed

Zhang, Ting; Chen, Xiaolan; Qu, Lingbo; Wu, Jinglan; Cui, Ran; Zhao, Yufen

2004-12-01

To improve the biological activities of chrysin (CR), we synthesize Diethyl Chysin-7-yl phosphate (CPE: C(19)H(19)O(7)P) and tetraethyl bis-phosphoric ester of chrysin (CP: C(23)H(28)O(10)P(2)) through a simplified Atheron-Todd reaction. The interactions of the CR and CPE with lysozyme were explored by electrospray ionization mass spectrometry (ESI) and fluorescence spectrometry method. Experimental results indicate that CPE could form the noncovalent compound with lysozyme, while the interaction of the CR with lysozyme was not detected. In addition, whether and how the compounds CPE and CP affect proliferation and apoptosis in human cervical cancer Hela cells were investigated. Moreover, the effects of CPE and CP in Hela cells were compared with that of the nonmodified CR compound. The Hela cells were co-cultured with CR, CP, and CPE as experimental groups, respectively, and corresponding control groups treated without CR, CP, and CPE. The proliferation and apoptosis were detected using MTT assay, HCl denatured-methyl green-pyronin staining, PCNA immunohistochemistry and TUNEL techniques. The cell growth IC(50), relative absorbance (RA), proliferating index (PI), PCNA-IR (immunoreactivity IR) integration value (IV), and apoptosis index (AI) were calculated and their correlation was analyzed in each group. The results show that all CR, CP, and CPE could inhibit proliferation and induce apoptosis in Hela cells. Moreover, the effects of CP and CPE were more potent than that of CR. The CP and CPE were proved to be a kind of stronger apoptosis inducers than nonphosphated CR. There was a negative correlation between proliferation and apoptosis. In conclusion, the CR, CP, and CPE could effectively inhibit growth by down-regulated expression of PCNA, and induce apoptosis in Hela cells. The efficiency of the modified CP and CPE preceded nonmodified CR compounds. The CP and CPE may be a new potential anti-cancer drug for therapy of human cervical carcinoma.

p27Kip1 is expressed in proliferating cells in its form phosphorylated on threonine 187

PubMed Central

Troncone, Giancarlo; Martinez, Juan C; Iaccarino, Antonino; Zeppa, Pio; Caleo, Alessia; Russo, Maria; Migliaccio, Ilenia; Motti, Maria L; Califano, Daniela; Palmieri, Emiliano A; Palombini, Lucio

2005-01-01

Background G1/S cell cycle progression requires p27Kip1 (p27) proteolysis, which is triggered by its phosphorylation on threonine (Thr) 187. Since its levels are abundant in quiescent and scarce in cycling cells, p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research. Methods However here we showed that by using a specific phosphorylation site (pThr187) antibody, p27 is detectable also in proliferative compartments of normal, dysplastic and neoplastic tissues. Results In fact, whereas un-phosphorylated p27 and MIB-1 showed a significant inverse correlation (Spearman R = -0.55; p < 0,001), pThr187-p27 was positively and significantly correlated with MIB-1 expression (Spearman R = 0.88; p < 0,001). Thus proliferating cells only stain for pThr187-p27, whereas they are un-reactive with the regular p27 antibodies. However increasing the sensitivity of the immunocytochemistry (ICH) by the use of an ultra sensitive detection system based on tiramide signal amplification, simultaneous expression and colocalisation of both forms of p27 was shown in proliferating compartments nuclei by double immunofluorescence and laser scanning confocal microscopy studies. Conclusion Overall, our data suggest that p27 expression also occurs in proliferating cells compartments and the combined use of both regular and phospho- p27 antibodies is suggested. PMID:15725363

Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone

NASA Astrophysics Data System (ADS)

Li, K.-T.; Zhang, J.; Duan, Q.-Q.; Bi, Y.; Bai, D.-Q.; Ou, Y.-S.

2014-06-01

A giant cell tumor in bone is the common primary bone tumor with aggressive features, occurring mainly in young adults. Photodynamic therapy is a new therapeutic technique for tumors. In this study, we investigated the effects of Pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy on the proliferation of giant cell tumor cells and its mechanism of action. Cell proliferation was evaluated using an MTT assay. Cellular apoptosis was detected by Hoechst nuclear staining, and flow cytometric assay. Mitochondrial membrane potential changes and cytochrome c, caspase-9, caspase-3, and Bcl-2 expression was assessed. Finally, we found that MPPa-mediated photodynamic therapy could effectively suppress the proliferation of human giant cell tumor cells and induce apoptosis. The mitochondrial pathway was involved in the MPPa-photodynamic therapy-induced apoptosis.

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

PubMed Central

Liu, Shuying; Zhang, Zhengyu; Wang, Jingjing; Zhou, Yuhuan; Liu, Kefeng; Huang, Jintao; Chen, Dadi; Wang, Junmei; Li, Chaohong

2015-01-01

Aims This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms. Methods Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression. Results Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis. Conclusions Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels. PMID:26488175

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

NASA Astrophysics Data System (ADS)

Wu, Riqin; Zhang, Peijun; Li, Jun; Xu, Yongli

2005-03-01

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes ( P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

PubMed Central

Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

2008-01-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by β-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, β-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles. PMID:17921358

Conditional deletion of Pten causes bronchiolar hyperplasia.

PubMed

Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

2008-03-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

Two-signal electrochemical method for evaluation suppression and proliferation of MCF-7 cells based on intracellular purine.

PubMed

Li, Jinlian; Lin, Runxian; Wang, Qian; Gao, Guanggang; Cui, Jiwen; Liu, Jiguang; Wu, Dongmei

2014-07-01

Two electrochemical signals ascribed to xanthine/guanine and hypanthine/adenine in MCF-7 cells were detected at 0.726 and 1.053 V, respectively. Based on the intensity of signals, the genistein-induced proliferation and suppression of MCF-7 cells could be evaluated. The results showed that with the increase of genistein dose at the range of 10(-9) to 10(-6)M, the two electrochemical signals of MCF-7 cell suspension increased due to the proliferation, whereas the tendency at the high dosage range of more than 10(-5)M was decreased. The proliferation and cytotoxicity obtained by the electrochemical method were in agreement with those obtained by cell counting and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] method. Thus, the two-signal electrochemical method is an effective way to evaluate the effect of drugs on cell activity based on purine metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.

[Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells].

PubMed

Wang, P; Liu, S; Cheng, B; Wu, X Z; Ding, S S; Xu, L; Liu, Y; Duan, L; Sun, S Z

2017-03-08

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

NASA Astrophysics Data System (ADS)

Sun, Qingqing; Kanehira, Koki; Taniguchi, Akiyoshi

2016-01-01

This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO2 nanoparticles (TiO2-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml-1) of TiO2-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO2-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO2-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO2-PEG NPs. In conclusion, our results indicate that TiO2-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses.

[Effects of UO-126 on proliferation and fbw7 expression of HeLa cells].

PubMed

Sun, Di; Shen, Yi; Wang, Shao-hua; Xiang, Zi-wu; Xie, Ying-shan; Jiang, Xin

2010-02-01

To observe the effects of UO-126 on the expression of F-box and WD repeat domain-containing protein 7(FBW7)and on the proliferation of human cervical cancer cell lines (HeLa cells). HeLa cells were treated with different concentrations of UO-126, MTT assay was used to observe the proliferation of HeLa cells. Immunofluorescence showed the location and expression of FBW7 in HeLa cells. The mRNA and protein expression of FBW7 were detected by RT-PCR and Western blot before and after mitogen-activated protein kinases (MAPK)signal was blocked by UO-126 a MAPK inhibitor. MTT results showed that the concentration range of MAPK signaling pathway inhibitor UO-126 inhibited the proliferation of HeLa cells in a concentration-and time-dependent manner(P<0.05). Immunofluorescence showed that the expression of positive FBW7 had increased after HeLa cells were treated with UO-126. RT-PCR and Western blot exhibited that the FBW7 mRNA and protein expression had significantly increased before and after HeLa cells were treated with UO-126(P<0.05). UO-126 could inhibit HeLa cells proliferation, FBW7 lied downstream of MAPK signaling pathway.

Proliferation of the human urothelium is induced by atypical β1 -adrenoceptors.

PubMed

Winder, M; Wasén, C; Aronsson, P; Giglio, D

2015-09-01

We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively. Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β1/2/3 ), dobutamine (β1 ), salbutamol (β2 ), BRL 37344 (β3 ), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β1 , propranolol; β1/2 ). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the β1 -adrenoceptor. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β1 -adrenoceptors in both urothelial cell lines. The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation. © 2016 John Wiley & Sons Ltd.

[Effects of PCI-32765 and Dasatinib on the Acute Lymphoblastic Leukemic Cells and Their Mechanisms].

PubMed

Deng, Yuan; Tao, Shan-Dong; Zhang, Xin; Ma, Jing-Jing; He, Zheng-Mei; Chen, Yue; Deng, Zhi-Kui; Yu, Liang

2017-02-01

To investigate the effects of Btk inhibitor (PCI-32765) and BCR-ABL tyrosine kinase inhibitor (Dasatinib) on proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell lines (Sup-B15, RS4;11) and the possible mechanism. RS4;11 and Sup-B15 cells were treated with PCI-32765 and Dasatinib, the cell proliferation and apoptosis were detected by CCK-8, the Btk and other apoptotic proteins were detected by Western blot. PCI-32765 could inhibit the proliferation of RS4;11 and Sup-B15 cells in a dose-dependent manner, Sup-B15 cells were more sensitive to PCI-32765 than RS4;11 cells, their IC 50 were 3 µmol/L and 8 µmol/L respectively, the difference between them was statistically significant (P<0.05). Dasatinib also could inhibit the proliferation of RS4;11 cells and Sup-B15 cells in a dose-dependent manner. The IC 50 was 5 µmol/L and 5 nmol/L, respectively, the difference between them was statistically very significant (P<0.01), and the inhibitory effect was enhanced by the combination of Damatinib with the PCI-32765(P<0.05). The cell survival rate decreased gradually in PCI-32765 or Dasatinib alone group and the combination group at the different time-point (8, 12, 24, 36, 48 and 72 h), the 2 drugs showed a synergistic effect on cells in a time-dependent manner. After being treated with PCI-32765 and Dasatinib, the RS4;11 and Sup-B15 cells showed that cell shrinkage, increase of cytoplasmic density, nuclear pyknosis, deviation and karyorrhexis, and increase of the apoptotic cells in the combination group, while the promotive effect of low dosage dasatinib on apoptosis of RS4;11 cells was not strong. PCI-32765 and Dasatinib could decrease the expression and activity of BCR-ABL, Btk, Lyn, Src in Sup-B15 and RS4;11 cells. PCI-32765 or Dasatinib can inhibit the proliferation and induce the apoptosis of Sup-B15 and RS4;11 cells, PCI-32765 and Dasatinib displayed the synergistic effects. The possible mechanism may be related with the blocking of B cell receptor(BCR) signal pathway, thereby inhibiting the cell proliferation and promoting the cell apoptosis.

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Duchrow, Michael

2002-11-01

The Ki-67 antigen, pKi-67, is one of the most commonly used markers of proliferating cells. The protein can only be detected in dividing cells (G(1)-, S-, G(2)-, and M-phase) but not in quiescent cells (G(0)). The standard antibody to detect pKi-67 is MIB-1, which detects the so-called 'Ki-67 motif' FKELF in 9 of the protein's 16 tandem repeats. To investigate the function of these repeats we expressed three of them in an inducible gene expression system in HeLa cells. Surprisingly, addition of a nuclear localization sequence led to a complete absence of signal in the nuclei of MIB-1-stained cells. At the same time antibodies directed against different epitopes of pKi-67 did not fail to detect the protein. We conclude that the overexpression of the 'Ki-67 motif', which is present in the repeats, can lead to inability of MIB-1 to detect its antigen as demonstrated in adenocarcinoma tissue samples. Thereafter, in order to prevent the underestimation of Ki-67 proliferation indices in MIB-1-labeled preparations, additional antibodies (for example, MIB-21) should be used. Additionally, we could show in a mammalian two-hybrid assay that recombinant pKi-67 repeats are capable of self-associating with endogenous pKi-67. Speculating that the tandem repeats are intimately involved in its protein-protein interactions, this offers new insights in how access to these repeats is regulated by pKi-67 itself.

Lysophosphatidic acid (LPA) effects on endometrial carcinoma in vitro proliferation, invasion, and matrix metalloproteinase activity.

PubMed

Wang, Feng-qiang; Ariztia, Edgardo V; Boyd, Leslie R; Horton, Faith R; Smicun, Yoel; Hetherington, Jessica A; Smith, Phillip J; Fishman, David A

2010-04-01

Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation. All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 microM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay. LPA, at the concentrations of 0.1 and 1.0 microM, significantly induced the proliferation of HEC-1A cells (p<0.01). At 10 microM, LPA- induced HEC-1A proliferation to a less extent and showed no significant effect on HEC-1A invasion and migration (p>0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner. LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer. Copyright 2009. Published by Elsevier Inc.

Immunomodulator 'mushroom beta glucan' induces Wnt/β catenin signalling and improves wound recovery in tilapia and rat skin: a histopathological study.

PubMed

Hsiao, Chien-Mei; Wu, Yu-Sheng; Nan, Fan-Hua; Huang, Shih-Ling; Chen, Lynette; Chen, Shiu-Nan

2016-12-01

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β-catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

β-Catenin Dosage Is a Critical Determinant of Tracheal Basal Cell Fate Determination

PubMed Central

Brechbuhl, Heather M.; Ghosh, Moumita; Smith, Mary Kathryn; Smith, Russell W.; Li, Bilan; Hicks, Douglas A.; Cole, Brook B.; Reynolds, Paul R.; Reynolds, Susan D.

2011-01-01

The purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells. PMID:21703416

[Monocarboxylate transporter 1 enhances the sensitivity of breast cancer cells to 3-bromopyruvate in vitro].

PubMed

Li, Qi-Xiang; Zhang, Pei; Liu, Fang; Wang, Xian-Zhi; Li, Lu; Wang, Zhong-Kun; Jiang, Chen-Chen; Zheng, Hai-Lun; Liu, Hao

2017-05-20

To investigate the role of monocarboxylate transporter 1 (MCT1) in enhancing the sensitivity of breast cancer cells to 3-bromopyruvate (3-BrPA). The inhibitory effect of 3-BrPA on the proliferation of breast cancer cells was assessed with MTT assay, and brominated propidium bromide single staining flow cytometry was used for detecting the cell apoptosis. An ELISA kit was used to detect the intracellular levels of hexokinase II, lactate dehydrogenase, lactate, and adenosine triphosphate, and Western blotting was performed to detect the expression of MCT1. MDA-MB-231 cells were transiently transfected with MCT1 cDNA for over-expressing MCT1, and the effect of 3-BrPA on the cell proliferation and adenosine triphosphate level was deteced. 3-BrPA did not produce significant effects on the proliferation and apoptosis of MDA-MB-231 cells, and the cells treated with 200 µmol/L 3-BrPA for 24 h showed an inhibition rate and an apoptosis rate of only 8.72% and 7.8%, respectively. The same treatment, however, produced an inhibition rate and an apoptosis rate of 84.6% and 82.3% in MCF-7 cells, respectively. In MDA-MB-231 cells with MCT1 overexpression, 200 µmol/L 3-BrPA resulted in an inhibition rate of 72.44%, significantly higher than that in the control cells (P<0.05); treatment of the cells with 25, 50, 100, and 200 µmol/L 3-BrPA for 6 h resulted in intracellular adenosine triphosphate levels of 96.98%, 88.44%, 43.3% and 27.56% relative to the control level respectively. MCT1 can enhance the sensitivity of breast cancer cells to 3-BrPA possibly by transporting 3-BrPA into cells to inhibit cell glycolysis.

The multifunctional RNA-binding protein hnRNPK is critical for the proliferation and differentiation of myoblasts.

PubMed

Xu, Yongjie; Li, Rui; Zhang, Kaili; Wu, Wei; Wang, Suying; Zhang, Pengpeng; Xu, Haixia

2018-06-14

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcrip-tion, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired prolif-eration phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly in-creased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, my-osin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentia-tion and may be an essential regulator of myoblast function.

Effects of Lidocaine-Mediated CPEB3 Upregulation in Human Hepatocellular Carcinoma Cell Proliferation In Vitro

PubMed Central

Liu, Hongjun; Wang, Yiru; Chen, Bing

2018-01-01

Lidocaine displays antitumor activity by inducing apoptosis and suppressing tumor growth in human hepatocellular carcinoma (HepG2) cells in vitro. However, the molecular mechanism underlying lidocaine-mediated antitumor activity is unclear. In this study, HepG2 cells were treated with lidocaine, and cell proliferation and colony-forming ability were assessed. The expression level of cytoplasmic polyadenylation element binding protein 3 (CPEB3) was detected by real-time quantitative PCR and western blot. Lidocaine treatment resulted in decreased HepG2 cell viability and colony formation in a dose-dependent manner. In hepatocellular carcinoma patient samples, CPEB3 was downregulated and was associated with poor prognosis and high-grade malignancy. Additionally, CPEB3 was a critical mediator of lidocaine-induced repression of HepG2 cell proliferation. These results demonstrated that lidocaine decreased cell viability and colony-forming ability of HepG2 cells by upregulating CPEB3 expression.

MiR-7 inhibited peripheral nerve injury repair by affecting neural stem cells migration and proliferation through cdc42.

PubMed

Zhou, Nan; Hao, Shuang; Huang, Zongqiang; Wang, Weiwei; Yan, Penghui; Zhou, Wei; Zhu, Qihang; Liu, Xiaokang

2018-01-01

Objective Neural stem cells play an important role in the recovery and regeneration of peripheral nerve injury, and the microRNA-7 (miR-7) regulates differentiation of neural stem cells. This study aimed to explore the role of miR-7 in neural stem cells homing and proliferation and its influence on peripheral nerve injury repair. Methods The mice model of peripheral nerve injury was created by segmental sciatic nerve defect (sciatic nerve injury), and neural stem cells treatment was performed with a gelatin hydrogel conduit containing neural stem cells inserted into the sciatic nerve injury mice. The Sciatic Function Index was used to quantify sciatic nerve functional recovery in the mice. The messenger RNA and protein expression were detected by reverse transcription polymerase chain reaction and Western blot, respectively. Luciferase reporter assay was used to confirm the binding between miR-7 and the 3'UTR of cell division cycle protein 42 (cdc42). The neural stem cells migration and proliferation were analyzed by transwell assay and a Cell-LightTM EdU DNA Cell Proliferation kit, respectively. Results Neural stem cells treatment significantly promoted nerve repair in sciatic nerve injury mice. MiR-7 expression was decreased in sciatic nerve injury mice with neural stem cells treatment, and miR-7 mimic transfected into neural stem cells suppressed migration and proliferation, while miR-7 inhibitor promoted migration and proliferation. The expression level and effect of cdc42 on neural stem cells migration and proliferation were opposite to miR-7, and the luciferase reporter assay proved that cdc42 was a target of miR-7. Using co-transfection into neural stem cells, we found pcDNA3.1-cdc42 and si-cdc42 could reverse respectively the role of miR-7 mimic and miR-7 inhibitor on neural stem cells migration and proliferation. In addition, miR-7 mimic-transfected neural stem cells could abolish the protective role of neural stem cells on peripheral nerve injury. Conclusion MiR-7 inhibited peripheral nerve injury repair by affecting neural stem cells migration and proliferation through cdc42.

TMEM16A regulates portal vein smooth muscle cell proliferation in portal hypertension.

PubMed

Zeng, Xi; Huang, Ping; Chen, Mingkai; Liu, Shiqian; Wu, Nannan; Wang, Fang; Zhang, Jing

2018-01-01

The aim of the present study was to elucidate the effect of transmembrane protein 16A (TMEM16A) on portal vein smooth muscle cell (PVSMC) proliferation associated with portal vein remodeling in portal hypertension (PHT). Sprague-Dawley rats were subjected to bile duct ligation to establish a rat model of liver cirrhosis and PHT. Sham-operated animals served as controls. At 8 weeks after bile duct ligation, the extent of liver fibrosis and the portal vein wall thickness were assessed using hematoxylin-eosin staining. The protein expression levels of TMEM16A, extracellular signal-regulated kinase 1 and 2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) in the portal vein were detected by immunohistochemistry and western blotting. In vitro , the lentivirus vectors were constructed and transfected into PVSMCs to upregulate the expression of TMEM16A. Isolated rat primary PVSMCs were treated with a small molecule inhibitor of TMEM16A, T16A-inhA01. Cell cycle was detected by flow cytometry. The activity of TMEM16A in the portal vein isolated from bile duct ligated rats was decreased, while the expression level of p-ERK1/2 was increased. However, in vitro , upregulation of TMEM16A promoted the proliferation PVSMCs, while inhibition of TMEM16A channels inhibited the proliferation of PVSMCs. The results indicated that TMEM16A contributes to PVSMCs proliferation in vitro , but in vivo , it may be a negative regulator of cell proliferation influenced by numerous factors.

Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells

PubMed Central

Onabajo, Olusegun O.; Porter-Gill, Patricia; Paquin, Ashley; Rao, Nina; Liu, Luyang; Tang, Wei; Brand, Nathan

2015-01-01

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance. PMID:26134097

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

PubMed

Xin, Jia-Xuan; Yue, Zhen; Zhang, Shuai; Jiang, Zhong-Hua; Wang, Ping-Yu; Li, You-Jie; Pang, Min; Xie, Shu-Yang

2013-10-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

Potential involvement of leptin in carcinogenesis of hepatocellular carcinoma.

PubMed

Wang, Xiu-Jie; Yuan, Shu-Lan; Lu, Qing; Lu, Yan-Rong; Zhang, Jie; Liu, Yan; Wang, Wen-Dong

2004-09-01

To investigate the potential involvement of leptin in carcinogenesis of hepatocellular carcinoma (HCC) and to elucidate the etiology, carcinogenesis and progress of HCC. Expressions of Ob gene product, leptin and its receptor, Ob-R were investigated in 36 cases of HCC specimens and corresponding adjacent non-tumorous liver tissues with immunohistochemical staining. The effect of leptin on proliferation of Chang liver cell line and liver cancer cell line SMMC-7721 was studied with cell proliferation assay (MTT). Leptin expression was detected in 36 cases of adjacent non-tumorous liver tissues (36/36, 100%) with moderate (++) to strong (+++) intensity; and in 72.22%(26/36) of HCC with weaker (+) intensity (P<0.05). Thirty of 36 (83.33%) cases of adjacent non-tumorous liver tissues were positive for Ob-R, with moderate (++) to strong (+++) intensity. In HCC, 11/36 (30.56%) cases were positive, with weak (+) intensity (P<0.05). In cell proliferation assay, leptin inhibited the proliferation of Chang liver cells. The cell survival rate was 10-13% lower than that of the untreated cells (P>0.05). Leptin had little effect on the proliferation of liver cancer cells (P>0.05). High level expression and decreased or absent expression of leptin and its receptor in adjacent non-tumorous liver cells and HCC cells, inhibitory effect of leptin on the proliferation of normal Chang liver cells and no effect of leptin on proliferation of liver cancer cells, may provide new insights into the carcinogenesis and progression of human HCC. It could be assumed that leptin acting as an inhibitor and/or promoter, is involved in the process of carcinogenesis and progress of human HCC. Copyright 2004 The WJG Press ISSN

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques.

PubMed

Ahsan, Muhammad H; Gill, Amy F; Alvarez, Xavier; Lackner, Andrew A; Veazey, Ronald S

2013-11-01

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. © 2013 Elsevier Inc. All rights reserved.

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells.

PubMed

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group and blank group (P<0.05), demonstrating that after the down-regulation of Tiam1 gene expression, the speed of cell proliferation was inhibited. MTT assay results showed that the total growth speed in experimental group was significantly lower than that in control group and blank group (P<0.05), indicating that the proliferation activity of cholangiocarcinoma cells was inhibited after targeted inhibition of Tiam1 gene expression. Transwell detection results showed that the metastasis rate in experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that targeted inhibition of Tiam1 gene expression could significantly inhibit migration ability of RBE cells. Tiam1 expression significantly increased in cholangiocarcinoma tissues, and increased along with the degree of malignancy of cholangiocarcinoma. Targeted silencing Tiam1 expression could inhibit proliferation and migration activity of cholangiocarcinoma cells.

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells

PubMed Central

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

Objective: To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Methods: Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. Results: The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group and blank group (P<0.05), demonstrating that after the down-regulation of Tiam1 gene expression, the speed of cell proliferation was inhibited. MTT assay results showed that the total growth speed in experimental group was significantly lower than that in control group and blank group (P<0.05), indicating that the proliferation activity of cholangiocarcinoma cells was inhibited after targeted inhibition of Tiam1 gene expression. Transwell detection results showed that the metastasis rate in experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that targeted inhibition of Tiam1 gene expression could significantly inhibit migration ability of RBE cells. Conclusion: Tiam1 expression significantly increased in cholangiocarcinoma tissues, and increased along with the degree of malignancy of cholangiocarcinoma. Targeted silencing Tiam1 expression could inhibit proliferation and migration activity of cholangiocarcinoma cells. PMID:26884821

Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

PubMed

Kobayashi, Toru; Honryo, Tomoki; Agawa, Yasuo; Sawada, Yoshifumi; Tapia, Ileana; Macìas, Karla A; Cano, Amado; Scholey, Vernon P; Margulies, Daniel; Yagishita, Naoki

2015-06-01

To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

PubMed Central

Pettini, Elena; Prota, Gennaro; Ciabattini, Annalisa; Boianelli, Alessandro; Fiorino, Fabio; Pozzi, Gianni; Vicino, Antonio; Medaglini, Donata

2013-01-01

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs. PMID:24349003

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

PubMed Central

Xie, You-Li; Tao, Wen-Hui; Yang, Ti-Xiong; Qiao, Jian-Guo

2016-01-01

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma. PMID:27698776

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

PubMed

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor in a ferret

PubMed Central

INOUE, Saki; YONEMARU, Kayoko; YANAI, Tokuma; SAKAI, Hiroki

2014-01-01

A 5-year-old male ferret presented with an enlarged canalicular testis in the left inguinal region. Microscopically, the enlarged testis consisted of a diffuse intimately admixed proliferation of c-kit-positive germ cell-like and Wilms tumor-1 protein-positive Sertoli cell-like components, but no Call-Exner body was detected. In addition, the compact proliferation of steroidogenic acute regulatory protein-intense positive interstitial cells was identified in a separate peripheral area of the mass. Based on histopathological and immunohistochemical findings, the tumor was diagnosed as a mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor. PMID:25311985

miR-137 inhibits the proliferation of human non-small cell lung cancer cells by targeting SRC3

PubMed Central

Chen, Ruilin; Zhang, Yongqing; Zhang, Chengcheng; Wu, Hua; Yang, Shumei

2017-01-01

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The results of the present study demonstrate that high expression of microRNA (miR)-137 and low expression of steroid receptor coactivator-3 (SRC3) had a significant negative correlation in 40 NSCLC tissue samples. In addition, cell colony formation and proliferation was significantly reduced in miR-137-transfected A549 and NCI-H838 cells compared with scramble-transfected NSCLC cell lines. miR-137 was identified to induce G1/S cell cycle arrest and dysregulate the mRNA expression of cell cycle-associated proteins (proliferating cell nuclear antigen, cyclin E, cyclin A1, cyclin A2 and p21) in NSCLC cells. Notably, miR-137 could significantly suppress SRC3 3′ untranslated region (UTR) luciferase-reporter activity, an effect that was not detectable when the putative 3′-UTR target-site was mutated, further clarifying the molecular mechanisms underlying the role of miR-137 in NSCLC. In conclusion, the results of the present study suggest that miR-137 suppresses NSCLC cell proliferation by partially targeting SRC3. PMID:28521488

[Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

PubMed

Wu, Duan; Zhou, Jianyin; Yin, Zhenyu; Liu, Pingguo; Zhao, Yilin; Liu, Jianming; Wang, Xiaomin

2014-12-02

To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition

PubMed Central

Fu, Changlong; Zheng, Chunsong; Lin, Jie; Ye, Jinxia; Mei, Yangyang; Pan, Caibin; Wu, Guangwen; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2017-01-01

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose- and time-dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin-dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis. PMID:28358416

Decorin-Mediated Inhibition of Human Trophoblast Cells Proliferation, Migration, and Invasion and Promotion of Apoptosis In Vitro

PubMed Central

Zou, Yanfen; Yu, Xiang; Lu, Jing; Jiang, Ziyan; Zuo, Qing; Fan, Mingsong; Huang, Shiyun

2015-01-01

Preeclampsia (PE) is a unique complication of pregnancy, the pathogenesis of which has been generally accepted to be associated with the dysfunctions of extravillous trophoblast (EVT) including proliferation, apoptosis, and migration and invasion. Decorin (DCN) has been proved to be a decidua-derived TGF-binding proteoglycan, which negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast cells. In this study, we identified a higher expression level of decorin in severe PE placentas by both real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). And an inhibitory effect of decorin on proliferation, migration, and invasion and an enhanced effect on apoptosis in trophoblast cells HTR-8/SVneo and JEG-3 were validated in vitro. Also the modulations of decorin on trophoblast cells' metastasis and invasion functions were detected through regulating the matrix metalloproteinases (MMP2 and MMP9). Thus, we suggested that the contribution of decorin to the modulation of trophoblast cells might have implications for the pathogenesis of preeclampsia. PMID:26357650

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease

PubMed Central

Buxbaum, Nataliya P.; Farthing, Donald E.; Maglakelidze, Natella; Lizak, Martin; Merkle, Hellmut; Carpenter, Andrea C.; Oliver, Brittany U.; Kapoor, Veena; Castro, Ehydel; Swan, Gregory A.; dos Santos, Liliane M.; Bouladoux, Nicolas J.; Bare, Catherine V.; Flomerfelt, Francis A.; Eckhaus, Michael A.; Telford, William G.; Belkaid, Yasmine; Bosselut, Remy J.; Gress, Ronald E.

2017-01-01

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells. PMID:28614804

Stimulatory effect of icariin on the proliferation of neural stem cells from rat hippocampus.

PubMed

Fu, Xiaolong; Li, Shujun; Zhou, Shaoyu; Wu, Qin; Jin, Feng; Shi, Jingshan

2018-01-29

Icariin (ICA), a major ingredient of Epimediumbrevicornum, has various pharmacological activities including central nervous system protective functions such as the improvement of learning and memory function in mice models of Alzheimer's disease. It has been reported that ICA can promote regeneration of peripheral nerve and functional recovery. The purpose of this study was to investigate the potentiating effect of ICA on the proliferation of rat hippocampal neural stem cells, and explore the possible mechanism involved. Primary neural stem cells were prepared from the hippocampus of newly born SD rats, and cells were cultured in special stem cell culture medium. Neural stem cells were confirmed by immunofluorescence detection of nestin, NSE and GFAP expression. The effect of ICA on the growth and proliferation of the neural stem cells was evaluated by 5-ethynyl-2-deoxyuridine (EdU) labeling of proliferating cells, and photomicrographic images of the cultured neural stem cells. Further, the mechanism of ICA-induced cell proliferation of neural stem cells was investigated by analyzing the gene and protein expression of cell cycle related genes cyclin D1 and p21. The present study showed that icariin promotes the growth and proliferation of neural stem cells from rat hippocampus in a dose-dependent manner. Incubation of cells with icariin resulted in significant increase in the number of stem cell spheres as well as the increased incorporation of EdU when compared with cells exposed to control vehicle. In addition, it was found that icariin-induced effect on neural stem cells is associated with increased mRNA and protein expression of cell cycle genes cyclin D1 and p21. This study evidently demonstrates the potentiating effect of ICA on neural stem cell growth and proliferation, which might be mediated through regulation of cell cycle gene and protein expression promoting cell cycle progression.

A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

PubMed

Sierralta, Walter D; Epuñan, María J; Reyes, José M; Valladares, Luis E; Pino, Ana M

2008-01-01

A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.

Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus).

PubMed

Takahashi, H; Sakamoto, T; Narita, K

2006-06-01

In order to replace the diffusive loss of water to the surrounding environment, seawater (SW)-acclimated euryhaline fishes have gastrointestinal tracts with higher ion/water flux in concert with greater permeability, and contrast that to freshwater (FW)-acclimated fish. To understand the cellular basis for these differences, we examined cell proliferation and apoptosis in the anterior intestine of mudskipper transferred from one-third SW to FW or to SW for 1 and 7 days, and those kept out of water for 1 day. The intestinal apoptosis (indicated by DNA laddering) increased during seawater acclimation. TUNEL staining detected numerous apoptotic cells over the epithelium of SW-acclimated fish. Cell proliferation ([3H]thymidine incorporation) in the FW fish was greater than those in SW 7 days after transfer. Labeling with a Proliferating cell nuclear antigen (PCNA) antibody indicated that proliferating cells were greater in number and randomly distributed in the epithelium of FW fish, whereas in SW fish they were almost entirely in the troughs of the intestinal folds. There were no changes in cell turnover in fish kept out of water. During acclimation to different salinities, modification of the cell turnover and abundance may play an important role in regulating the permeability (and transport capacity) of the gastrointestinal tract of fish.

The local lymph node assay being too sensitive?

PubMed

Hans-Werner, Vohr; Jürgen, Ahr Hans

2005-12-01

The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skin-sensitizing potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. In this paper, we have compared both endpoints: (1) cell proliferation alone and (2) cell proliferation in combination with inflammatory (irritating) processes. It turned out that a considerable number of tests were "false positive" to the definition mentioned above. By excluding such false positive results the LLNA seems not to be more sensitive than relevant guinea pig assays. These various methods and results are described here.

[Effects of sinensetin on proliferation and apoptosis of human gastric cancer AGS cells].

PubMed

Dong, Yang; Ji, Guang; Cao, Aili; Shi, Jianrong; Shi, Hailian; Xie, Jianqun; Wu, Dazheng

2011-03-01

To study the effects and mechanisms of sinensetin on proliferation and apoptosis of human AGS gastric cancer cells. MTT assay was used to detect the growth inhibition rates of human AGS gastric cancer cells treated with sinsesectin in different concentrations and times. The cell cycle distribution was measured by flow cytometry. The apoptosis was examined by Annexin-FITC/PI staining and DNA fragment analysis. The apoptosis morphology was observed by inverted fluorescence microscope after Hoechst 33342 staining. The protein expressions of p21 and p53 were detected by western blot. MTT assay showed that sinensetin inhibited the growth of AGS gastric cancer cells in a dose- and time-dependent manner. Sinensetin blocked AGS cells in G2/ M and increased the apoptosis rates of AGS cells in a dose-dependent manner. DNA ladder was observed in cells treated with 60 micromol x L(-1) sinensetin for 48 h. The typical apoptotic morphological changes including cell nucleus shrinkage, chromatin condensation and apoptotic bodies were observed when treated with different dose of sinensetin. Western blot showed that sinensetin increased expressions of p53 and p21 in a dose-dependent manner. Sinensetin could inhibit human AGS gastric cancer cells proliferation and induce cell cycle block in G2/M phase and apoptosis. The up regulation of p53 and p21 protein might be one of the mechanisms.

Effects of TGF-β1 on the Proliferation and Apoptosis of Human Cervical Cancer Hela Cells In Vitro.

PubMed

Tao, Ming-Zhu; Gao, Xia; Zhou, Tie-Jun; Guo, Qing-Xi; Zhang, Qiang; Yang, Cheng-Wan

2015-12-01

To investigate the effects of TGF-β1 on the proliferation and apoptosis of cervical cancer Hela cells in vitro. Human cervical cancer Hela cells were cultured in vitro and divided into the experimental and control groups. In the experimental groups, Hela cells were stimulated with different concentrations of TGF-β1 (0.01, 0.1, 1, and 10 ng/mL), while Hela cells cultured in serum-free medium without TGF-β1 were used as controls. The CCK8 method was adopted to detect the effect of TGF-β1 on Hela cell proliferation, and flow cytometry was used to determine cell apoptosis 72 h after TGF-β1 treatment. Compared with the control group, the CCK-8 tests showed that different concentrations of TGF-β1 had no obvious effect on Hela cell proliferation 24 h after treatment (P > 0.05). However, upon 48 or 72 h of treatment, TGF-β1 significantly inhibited the proliferation of Hela cells in a time- and dose-dependent manner (P < 0.05). The flow cytometry results indicated that TGF-β1 influenced the apoptosis of human cervical cancer Hela cells in a dose-dependent manner after 72 h of treatment (P < 0.05). TGF-β1 significantly inhibited the growth and induced the apoptosis of human cervical Hela cells in vitro.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro.

PubMed

Huang, Jie; Xie, Liang-di; Luo, Li; Zheng, Su-Li; Wang, Hua-Jun; Xu, Chang-Sheng

2014-05-01

The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNL-HSP27-EGFP-2 (all P < 0.01). To conclude, these data indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases.

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression.

PubMed

Kraus, Dominik; Reckenbeil, Jan; Wenghoefer, Matthias; Stark, Helmut; Frentzen, Matthias; Allam, Jean-Pierre; Novak, Natalija; Frede, Stilla; Götz, Werner; Probstmeier, Rainer; Meyer, Rainer; Winter, Jochen

2016-03-01

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3β and nuclear translocation of β-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

The antidepressant effects of running and escitalopram are associated with levels of hippocampal NPY and Y1 receptor but not cell proliferation in a rat model of depression.

PubMed

Bjørnebekk, Astrid; Mathé, Aleksander A; Brené, Stefan

2010-07-01

One hypothesis of depression is that it is caused by reduced neuronal plasticity including hippocampal neurogenesis. In this study, we compared the effects of three long-term antidepressant treatments: escitalopram, voluntary running, and their combination on hippocampal cell proliferation, NPY and the NPY-Y1 receptor mRNAs, targets assumed to be important for hippocampal plasticity and mood disorders. An animal model of depression, the Flinders Sensitive Line (FSL) rat, was used and female rats were chosen because the majority of the depressed population is females. We investigated if these treatments were correlated to immobility, swimming, and climbing behaviors, which are associated with an overall, serotonergic-like and noradrenergic-like antidepressant response, in the Porsolt swim test (PST). Interestingly, while escitalopram, running and their combination increased the number of hippocampal BrdU immunoreactive cells, the antidepressant-like effect was only detected in the running group and the group with access both to running wheel and escitalopram. Hippocampal NPY mRNA and the NPY-Y1 receptor mRNA were elevated by running and the combined treatment. Moreover, correlations were detected between NPY mRNA levels and climbing and cell proliferation and NPY-Y1 receptor mRNA levels and swimming. Our results suggest that increased cell proliferation is not necessarily associated with an antidepressant effect. However, treatments that were associated with an antidepressant-like effect did regulate hippocampal levels of mRNAs encoding NPY and/or the NPY-Y1 receptor and support the notion that NPY can stimulate cell proliferation and induce an antidepressant-like response. Copyright 2009 Wiley-Liss, Inc.

[Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research].

PubMed

Li, Ling-Hui; Ding, Dao-Fang; Du, Guo-Qing; Gong, Hao; Wang, Hui-Hao; Zhan, Hong-Sheng

2013-05-01

To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism. Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week. The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner. The osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P < 0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P > 0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.

Controllable bioeffects of laser-generated intracellular microbubbles

NASA Astrophysics Data System (ADS)

Zohdy, Marwa Joy

Laser-induced optical breakdown (LIOB) is a nonlinear energy absorption process that can generate precise damage in biological tissues. With femtosecond laser pulses, disruption is highly localized with minimal thermal and mechanical effects to the surrounding region. Cavitation bubbles are produced as a result of LIOB, and these bubbles can be detected and monitored with high-frequency ultrasound. In this work, the controllable viability effects of LIOB bubbles in single cells were characterized. Using a high-frequency acoustic transducer synchronized with a 793 nm, 100 fs laser pulsed at 250 kHz, thermal effects in the vicinity of an LIOB event were directly assessed. Temperaturedependent pulse-echo displacements were calculated using phase-sensitive correlation tracking and fit to a finite-element heat transfer model to estimate thermal distribution. Results indicate a minimal temperature increase (<1 degree C) within 100 microns of a bubble created with multiple laser pulses, confirming that LIOB can be controlled to be thermally noninvasive in the bubble vicinity. Acoustically detectable microbubbles were generated in individual cells with femtosecond LIOB. By adjusting laser fluence, exposure time, and focal location, LIOB could be controlled to produce distinctly different cellular effects. Small (1-2 micron) bubbles with short lifetimes (10100 ms) could be generated in cells without affecting their viability; and, alternatively, large (510 micron) bubbles with long lifetimes (1-5 s) could be generated for selective cell killing without affecting immediately neighboring cells. Experiments were performed in Chinese hamster ovary (CHO) cells in vitro, and LIOB was detected with both optical and acoustic microscopy. A long-term proliferation assay was also performed using green-fluorescent MCA207 mouse sarcoma cells targeted for LIOB. This assay confirmed that nondestructive bubbles did not affect target cell proliferation over several generations, and that destructive bubbles could indeed eliminate target cells and prevent further proliferation with no effect on immediately neighboring cells. These studies help to outline future applications for site-activated, acoustically monitored intracellular microbubbles. Nondestructive bubbles can potentially be used for functional cell measurements without introducing exogenous agents or affecting subsequent cell proliferation, and destructive bubbles can be used for highly precise biologically-targeted cancer cell therapy with real-time acoustic validation.

[Anti-tumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line in vitro].

PubMed

Li, Maolan; Lu, Wei; Zhang, Fei; Ding, Qichen; Wu, Xiangsong; Tan, Zhujun; Wu, Wenguang; Weng, Hao; Wang, Xuefeng; Shi, Weibin; Dong, Ping; Gu, Jun; Liu, Yingbin

2014-11-04

To explore the antitumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line. DDP-PLLA-CNTs were prepared with the method of ultrasound emulsification. The morphology of DDP-PLLA-CNTs was determined by scanning electron microscope (SEM). And its drug loading and drug release curve in vitro was detected by UV-Vis-NIR spectrophotometer. CCK8 was used to test the cytotoxic effects of DDP-PLLA-CNTs at different concentrations on QBC939 cell proliferation.Flow cytometry was employed to measure the changes of apoptotic rate. With excellent controlled-release characteristic of in vitro drug release, DDP-PLLA-CNTs inhibited the proliferation and significantly increased the apoptotic rate of QBC939 cell line. DDP-PLLA-CNTs have drug sustained-release characteristics and can significantly inhibit the proliferation of QBC939 cell line.

Low-dose naltrexone targets the opioid growth factor-opioid growth factor receptor pathway to inhibit cell proliferation: mechanistic evidence from a tissue culture model.

PubMed

Donahue, Renee N; McLaughlin, Patricia J; Zagon, Ian S

2011-09-01

Naltrexone (NTX) is an opioid antagonist that inhibits or accelerates cell proliferation in vivo when utilized in a low (LDN) or high (HDN) dose, respectively. The mechanism of opioid antagonist action on growth is not well understood. We established a tissue culture model of LDN and HDN using short-term and continuous opioid receptor blockade, respectively, in human ovarian cancer cells, and found that the duration of opioid receptor blockade determines cell proliferative response. The alteration of growth by NTX also was detected in cells representative of pancreatic, colorectal and squamous cell carcinomas. The opioid growth factor (OGF; [Met(5)]-enkephalin) and its receptor (OGFr) were responsible for mediating the action of NTX on cell proliferation. NTX upregulated OGF and OGFr at the translational but not at the transcriptional level. The mechanism of inhibition by short-term NTX required p16 and/or p21 cyclin-dependent inhibitory kinases, but was not dependent on cell survival (necrosis, apoptosis). Sequential administration of short-term NTX and OGF had a greater inhibitory effect on cell proliferation than either agent alone. Given the parallels between short-term NTX in vitro and LDN in vivo, we now demonstrate at the molecular level that the OGF-OGFr axis is a common pathway that is essential for the regulation of cell proliferation by NTX.

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

PubMed Central

Sun, Qingqing; Kanehira, Koki; Taniguchi, Akiyoshi

2016-01-01

Abstract This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO2 nanoparticles (TiO2-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml–1) of TiO2-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO2-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO2-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO2-PEG NPs. In conclusion, our results indicate that TiO2-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses. PMID:27877913

Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

PubMed

Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping

2013-02-01

This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model.

PubMed

Ji, Xiang-Jun; Chen, Sui-Hua; Zhu, Lin; Pan, Hao; Zhou, Yuan; Li, Wei; You, Wan-Chun; Gao, Chao-Chao; Zhu, Jian-Hong; Jiang, Kuan; Wang, Han-Dong

2013-07-01

NF-E2-related factor 2 (Nrf2) is a pivotal transcription factor of cellular responses to oxidative stress and recent evidence suggests that Nrf2 plays an important role in cancer pathobiology. However, the underlying mechanism has yet to be elucidated, particularly in glioma. In the present study, we investigated the role of Nrf2 in the clinical prognosis, cell proliferation and tumor growth of human glioblastoma multiforme (GBM). We detected overexpression of Nrf2 protein levels in GBM compared to normal brain tissues. Notably, higher protein levels of Nrf2 were significantly associated with poorer overall survival and 1-year survival for GBM patients. Furthermore, we constructed the plasmid Si-Nrf2 and transduced it into U251MG cells to downregulate the expression of Nrf2 and established stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed cell proliferation in vitro and tumor growth in mouse xenograft models. We performed immunohistochemistry staining to detect the protein levels of Nrf2, Ki-67, caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki-67 were much lower in the Si-Nrf2 group compared to the Si-control group. In addition, the number of caspase-3-positive cells was significantly increased in the Si-Nrf2 group. By analysis of microvessel density (MVD) assessed by CD31, the MVD value in the Si-Nrf2 group decreased significantly compared to the Si-control group. These findings indicate that the knockdown of Nrf2 may suppress tumor growth by inhibiting cell proliferation, increasing cell apoptosis and inhibiting angiogenesis. These results highlight the potential of Nrf2 as a candidate molecular target to control GBM cell proliferation and tumor growth.

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

PubMed

Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

2017-04-01

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in the formation and development of hepatocellular carcinoma and that peroxisome proliferator-activated receptor gamma coactivator-1 alpha may be a potential therapeutic target for hepatocellular carcinoma.

[Inhibitory effects of Porphyromonas endodontalis lipopolysaccharides on proliferation and differentiation of mouse osteoblast].

PubMed

Li, Ren; Qiu, Li-hong; Yang, Di; Xue, Ming; Zhong, Ming

2011-03-01

To observe the effect of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on osteoblast cell proliferation and the activity of alkaline phosphatase (ALP) and interleukin (IL)-6 secretion and to investigate the role of Pe-LPS in osteoblast proliferation and differentiation. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (10, 25, 50 mg/L) respectively. The relative growth rate (RGR) was detected by methyl thiazolyl tetrazolium (MTT) at different time point (12, 24, 48, 72 h). MC3T3-E1 cells were also stimulated with 10, 25 or 50 mg/L Pe-LPS for 6, 12, 24 and 48 h. The activity of ALP was detected by enzyme kinetics assay and the secretion of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). After the stimulation with 25 or 50 mg/L Pe-LPS for 72 h, the RGR of MC3T3-E1 cells descend to 87.46% and 71.12%. The ALP activities of MC3T3-E1 cells were inhibited obviously (P < 0.05) after the stimulation of different concentrations (10, 25, 50 mg/L) Pe-LPS for more than 24 hours. ELISA result showed that IL-6 increased to 32.21 ng/L treated with the 25 mg/L Pe-LPS for 6 h, 25 mg/L Pe-LPS gradually increased the expression of IL-6 from the ELISA results. Pe-LPS can induce the secretion of IL-6 in MC3T3-E1 and decrease the ALP activities of MC3T3-E1, the differentiation of osteoblasts was inhibited. with the long-time toxicity action of Pe-LPS, the proliferation rate of MC3T3-E1 also markedly decreased.

Curcumin induces apoptosis in pancreatic cancer cells through the induction of forkhead box O1 and inhibition of the PI3K/Akt pathway.

PubMed

Zhao, Zhiming; Li, Chenggang; Xi, Hao; Gao, Yuanxing; Xu, Dabin

2015-10-01

Previous population investigations have suggested that the application of curcumin may be associated with decreased incidence and improved prognosis in certain types of cancer. Forkhead box O1 (FOXO1) has been implicated in the regulation of several biological processes, including stress resistance, metabolism, DNA repair, cell cycle and apoptosis. The aims of the present study were to investigate the effects and molecular mechanisms of curcumin on the induction of anti‑proliferation, cell cycle arrest and apoptosis, by FOXO1, in pancreatic cancer cells. The MTT assay and ELISA‑Brdu assay were used to assess cell proliferation. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect the expression of PCNA, Ki‑67, B‑cell lymphoma‑2 (Bcl‑2), B‑cell‑associated X protein (Bax), cyclin D1, p21, p27 and FOXO1. Cell apoptosis was detected using a Cell Death ELISA detection kit. A Caspase‑3/9 Fluorescent Assay kit was used to detect caspase activity. The findings revealed that curcumin significantly decreased cell proliferation, which was associated with increased expression of the p21/CIP1 and p27/KIP1 cyclin‑dependent kinase inhibitors, and inhibited expression of cyclin D1. In addition, curcumin induced apoptosis by decreasing the Bcl‑2/Bax protein ratio and increasing caspase‑9/3 activation in the pancreatic cancer cells. Using siRNA against FOXO1, and Akt inhibitor and activator, the present study confirmed that curcumin induced the expression of FOXO1 by inhibition of phosphoinositide 3‑kinase/Akt signaling, leading to cell cycle arrest and apoptosis. In conclusion, these findings offer support for a mechanism that may underlie the anti‑neoplastic effects of curcumin and justify further investigation to examine the potential roles for activators of FOXO1 in the prevention and treatment of pancreatic cancer.

Coexpression of the genes for interleukin 6 and its receptor without apparent involvement in the proliferation of acute myeloid leukemia cells.

PubMed

Lopez, M; Maroc, N; Kerangueven, F; Bardin, F; Courcoul, M; Lavezzi, C; Birg, F; Mannoni, P

1991-09-01

Leukemic cells isolated from patients with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) were screened for their capacity to express the interleukin 6 (IL-6) and IL-6 receptor genes, both at the RNA and protein levels. Variable levels (10 to greater than 600 U/ml) of an IL-6 activity, inhibited by neutralizing anti-IL-6 antibodies, were detected in AML cell supernatants using the B9 cell bioassay. High levels (greater than 100 U/ml) were observed in differentiated (M4 and M5 stages) AML, as well as in less mature (M1 and M2 stages) AML. Detection of the IL-6 transcript correlated with the biological activity. In addition, both IL-6 activity and IL-6 mRNA were detected in "fresh" leukemic cells, indicating that the glycoprotein was actually synthesized in vivo. In contrast, the IL-6 gene was less frequently expressed in ALL. The IL-6 receptor gene was transcribed in both AML and ALL; binding experiments showed that the protein was present at the cell surface. The spontaneous in vitro proliferation of leukemic cells coexpressing the transcripts for IL-6 and its receptor was not significantly inhibited by a neutralizing anti-IL-6 antibody, suggesting that IL-6 is not primarily implicated in the proliferation of the leukemic clone via an autocrine loop. Synthesis of IL-6 could, however, confer on leukemic cells a selective growth advantage through activation of the cytokine cascade.

Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

PubMed

Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

2016-10-01

The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P < 0.05). Spectrophotometric assay showed that gem significantly increased caspase-3 and -9 activity in PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

Pirfenidone Inhibits Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the Wnt/β-Catenin Signaling Pathway.

PubMed

Zou, Wei-Jie; Huang, Zhi; Jiang, Tian-Peng; Shen, Ya-Ping; Zhao, An-Su; Zhou, Shi; Zhang, Shuai

2017-12-25

BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of β-catenin and p-β-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the β-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated β-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.

Developmental changes in cell proliferation and apoptosis in the normal duck bursa of Fabricius.

PubMed

Fang, Jing; Peng, Xi

2014-12-01

The aim of this work was to investigate developmental changes in cell proliferation and apoptosis in normal duck bursa of Fabricius using flow cytometry and immunohistochemistry. Studies were carried out on Tianfu ducks on days 24 and 27 of embryogenesis (E24 and E27) along with days 20, 70, and 200 of postnatal development (P20, P70, and P200). Results showed that the percentage of G0/G1 bursa cells significantly increased between E24 and P200 while the percentage of cells in the S phase or G2 + M phase as well as the proliferating index obviously decreased during the same period. Proliferation cell nuclear antigen was detected in lymphocyte and interfollicular epithelium. The proliferative lymphocyte density tended to decrease from E24 to P200. Apoptotic bodies in macrophages, free apoptotic bodies, or nuclei with condensed chromatin in lymphocytes in follicles were identified by transferase-mediated dUTP nick-end labeling. Both flow cytometry and microscopic analysis reveal that the proportion of apoptotic cells and apoptotic lymphocyte density increased from E24 to P20, fell on P70, then rose again on P200. Our foundings demonstrate that cell proliferation decreases and apoptosis increases with age. These changes may account for duck bursa development and involution.

The high dosage of earthworm (Eisenia andrei) extract decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

PubMed Central

Yan, Bing Chun; Yoo, Ki-Yeon; Park, Joon Ha; Lee, Choong Hyun; Choi, Jung Hoon

2011-01-01

Earthworm extract has shown anticancer characteristics. In the present study, we examined the effect of chronic treatment with a high dose of earthworm (Eisenia andrei) extract (EE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of 3-week-old mice using 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 immunohistochemistry for cell proliferation and doublecortin (DCX) immunohistochemistry for neuroblast differentiation, respectively. BrdU-, Ki-67-, and DCX-immunoreactive cells were easily detected in the subgranular zone of the DG in vehicle (saline)-treated mice. However, BrdU-, Ki-67-, and DCX-immunoreactive cells in the 500 mg/kg EE-treated mice decreased distinctively compared to those in the vehicle-treated mice. In addition, brain-derived neurotrophic factor (BDNF) immunoreactivity and its protein level decreased markedly in the DG of the EE-treated group compared to those in the vehicle-treated group. These results indicate that chronic treatment with high dose EE decreased cell proliferation and neuroblast differentiation, and that BDNF immunoreactivity decreased in the DG of EE-treated mice. PMID:22025974

Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer

PubMed Central

Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

2015-01-01

Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect ofAPCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P<0.05) and cervical cancer cell proliferation and invasive ability were decreased significantly (P<0.05). APOBEC-3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigationAPOBEC-3G effect in cervical cancer occurrence and development. PMID:26722417

Effect of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G in cervical cancer.

PubMed

Xu, Yanhua; Leng, Junhong; Xue, Fang; Dong, Ruiqian

2015-01-01

Cervical cancer is one of the most common gynecologic cancers. The role of apolipoprotein B mRNA-editing catalytic polypeptide-like protein-3G (APCBEC-3G) in cervical cancer has yet to be elucidated. This study intends to explore the effect of APCBEC-3G on cervical cancer cell proliferation and invasion. In vitro, the cervical cancer cell line Hela was transfected by APCBEC-3G plasmid. The mRNA and protein expression levels of APCBEC-3G were detected by Real-time PCR and Western blot, respectively. Cervical cancer cell proliferation was determined by MTT. Transwell assay was applied to measure the effect of APCBEC-3G on cell invasion. APCBEC-3G mRNA and protein increased significantly after transfection (P<0.05) and cervical cancer cell proliferation and invasive ability were decreased significantly (P<0.05). APOBEC-3G serves as a suppressor of cervical cancer cell proliferation and invasion. Our research provides theoretical basis for further investigation APOBEC-3G effect in cervical cancer occurrence and development.

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation.

PubMed

Du, Lingqian; Yang, Pishan; Ge, Shaohua

2012-03-01

The pivotal role of chemokine stromal cell-derived factor-1 (SDF-1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell-based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single-cell colonies by the limited-dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO-1. Differentiation potentials were assessed by alizarin-red staining and oil-red O staining. The expression of SDF-1 receptor CXCR4 was evaluated by real-time polymerase chain reaction (PCR) and immunocytochemical staining. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real-time PCR to determine the effect of SDF-1 on cells differentiation. Twenty percent of PDL single-cell colonies expressed STRO-1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF-1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF-1 exerted its effects on cells through CXCR4. SDF-1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. SDF-1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.

Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment.

PubMed

Chen, Miaojiao; Zhang, Jingjing; Hu, Fang; Liu, Shiping; Zhou, Zhiguang

2015-11-01

Accumulating evidence suggests an association between diabetes and cancer. Inflammation is a key event that underlies the pathological processes of the two diseases. Metformin displays anti-cancer effects, but the mechanism is not completely clear. This study investigated whether metformin regulated the microenvironment of macrophage polarization to affect the characteristics of HepG2 cells and the possible role of the Notch-signalling pathway. RAW264.7 macrophages were cultured alone or co-cultured with HepG2 cells and treated with metformin. We analysed classical (M1) and alternative (M2) gene expression in RAW264.7 cells using quantitative real-time polymerase chain reaction. Changes in mRNA and protein expressions of Notch signalling in both cell types were also detected using quantitative real-time polymerase chain reaction and Western-blotting analyses. The proliferation, apoptosis and migration of HepG2 cells were detected using Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Fitchburg, WI, USA), Annexin V-FITC/PI (7SeaPharmTech, Shanghai, China) and the cell scratch assay, respectively. Metformin induced single-cultured RAW264.7 macrophages with an M2 phenotype but attenuated the M2 macrophage differentiation and inhibited monocyte chemoattractant protein-1 (MCP-1) secretion in a co-culture system. The co-cultured group of metformin pretreatment activated Notch signalling in macrophages but repressed it inHepG2 cells. Co-culture also promoted the proliferation and migration of HepG2 cells. However, along with the enhanced apoptosis, the proliferation and the migration of HepG2 cells were remarkably inhibited in another co-culture system with metformin pretreatment. Metformin can skew RAW264.7 macrophages toward different phenotypes according to changes in the microenvironment, which may affect the inflammatory conditions mediated by macrophages, induce apoptosis and inhibit the proliferation and migration of HepG2 cells. Notch signalling pathway is a potentially important mechanism in the regulation of metformin on macrophage polarization and the subsequent change of hepatoma cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of gene silencing of CypB on gastric cancer cells.

PubMed

Guo, Feng; Zhang, Ying; Zhao, Chun-Na; Li, Lin; Guo, Yan-Jun

2015-04-01

To determine the effect of gene silencing of cyclophilin B (CypB) on growth and proliferation of gastric cancer cells. CypB siRNA lentivirus (LV-CypB-si) and control lentivirus (LV-si-con) were produced. CypB expression in gastric cancer cell lines was detected by Western blot. BGC823 and SGC7901 cells were chosen to be infected with LV-si-con and LV-CypB-si, and stable transfectants were isolated. The cell groups transfected with LV-CypB-siRNA, LV-siRNA-con and transfected no carrier were served as the experimental group, the implicit control group and the blank control group respectively. MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro. Cell cycle was analyzed with flow cytometry. The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot. Gene silencing of CypB can inhibit gastric cancer cell growth, proliferation, cell cycle progress and tumorigenesis. CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES. These two cell lines were infected with LV-si-con and LV-CypB-si respectively. MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si (P<0.05). Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G1 (P<0.05). These findings indicated that CypB could promote the G1-S transition of gastric cancer cell. In addition, the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group. Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis. These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

PubMed

Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

2013-08-01

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2

PubMed Central

XIN, JIA-XUAN; YUE, ZHEN; ZHANG, SHUAI; JIANG, ZHONG-HUA; WANG, PING-YU; LI, YOU-JIE; PANG, MIN; XIE, SHU-YANG

2013-01-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3′-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics. PMID:24137458

Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138.

PubMed

Yu, Dazhi; An, Fengmei; He, Xu; Cao, Xuecheng

2015-01-01

In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism. Affemitrix miRNA chip was used to detect the changes of miRNA expression profile in MG-63 cells before and after curcumin treatment, and screen different expression of miRNAs. The target gene of miRNA was analyzed by bioinformatics. The expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 were detected. MTT and Transwell Cell invasion assays were used to observe the effects of curcumin on MG-63 cells. Curcumin could significantly inhibit the proliferation of MG-63 cells and the expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 in MG-63 cells (P<0.05); overexpression of hsa-miR-138 down-regulated the expression levels of Smad4, NFκB p65 and cyclin D3 compared with the treatment of curcumin, while inhibition of hsa-miR-138 up-regulated the expression levels of Smad4, NFκB p65 and cyclin D3. Curcumin could increase the expression of hsa-miR-138, hsa-miR-138 inhibited cell proliferation and invasive ability by inhibition of its target genes.

SVCT2 vitamin C transporter expression in progenitor cells of the postnatal neurogenic niche

PubMed Central

Pastor, Patricia; Cisternas, Pedro; Salazar, Katterine; Silva-Alvarez, Carmen; Oyarce, Karina; Jara, Nery; Espinoza, Francisca; Martínez, Agustín D.; Nualart, Francisco

2013-01-01

Known as a critical antioxidant, recent studies suggest that vitamin C plays an important role in stem cell generation, proliferation and differentiation. Vitamin C also enhances neural differentiation during cerebral development, a function that has not been studied in brain precursor cells. We observed that the rat neurogenic niche is structurally organized at day 15 of postnatal development, and proliferation and neural differentiation increase at day 21. In the human brain, a similar subventricular niche was observed at 1-month of postnatal development. Using immunohistochemistry, sodium-vitamin C cotransporter 2 (SVCT2) expression was detected in the subventricular zone (SVZ) and rostral migratory stream (RMS). Low co-distribution of SVCT2 and βIII-tubulin in neuroblasts or type-A cells was detected, and minimal co-localization of SVCT2 and GFAP in type-B or precursor cells was observed. Similar results were obtained in the human neurogenic niche. However, BrdU-positive cells also expressed SVCT2, suggesting a role of vitamin C in neural progenitor proliferation. Primary neurospheres prepared from rat brain and the P19 teratocarcinoma cell line, which forms neurospheres in vitro, were used to analyze the effect of vitamin C in neural stem cells. Both cell types expressed functional SVCT2 in vitro, and ascorbic acid (AA) induced their neural differentiation, increased βIII-tubulin and SVCT2 expression, and amplified vitamin C uptake. PMID:23964197

The roles of RUNX3 in cervical cancer cells in vitro.

PubMed

Li, Zhen; Fan, Pan; Deng, Min; Zeng, Chao

2018-06-01

RUNX3 serves an important role in development of various types of human cancer. The purpose of the present study was to investigate the potential biological function of RUNX3 in cervical cancer cells. In the present study, a RUNX3 overexpressed model was constructed in Hec1 cells by PCDNA3.1-RUNX3 transfection. Western blot analysis was used to measure RUNX3 expression in cervical cancer cells. Immunofluorescence analysis was performed to examine subcellular localization of RUNX3 in cervical cancer cells. Effects of RUNX3 expression on proliferation, migration and invasion of cervical cancer cells were detected by colony formation assay, wound healing assay and Transwell assay, respectively. Immunofluorescence confirmed the nuclear location of RUNX3 in cervical cancer cell. Result sindicated that upregulation of RUNX3 expression inhibited proliferation, migration and invasion of cervical cancer cells. However, knockdown of RUNX3 expression promoted the proliferation, migration and invasion of cervical cancer cells. Hence, RUNX3 may serve as a tumor suppressor gene in cervical cancer.

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

PubMed Central

Inchley, C J

1987-01-01

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed. Images Figure 2 Figure 5 PMID:3493207

[Over-expression of miR-151a-3p inhibits proliferation and migration of PC-3 prostate cancer cells].

PubMed

Zhang, Yi; Hao, Tongtong; Zhang, Han; Wei, Pengtao; Li, Xiaohui

2018-03-01

Objective To observe the effect of microRNA-151a-3p (miR-151a-3p) up-regulation on the proliferation and migration of prostate cancer cells and explore the possible molecular mechanism. Methods The expression of miR-151a-3p in PC-3M, C4-2B, 22RV1, DU-145, PC-3, LNCap human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time fluorescence quantitative PCR. PC-3 cells with the lowest expression of miR-151a-3p were used for subsequent experiments. Bioinformatics and dual-luciferase reporter assay were performed to predict and test potential target genes of miR-151a-3p. The miR-151a-3p mimics or negative control microRNAs (miR-NCs) were transfected into PC-3 cells. Real-time fluorescence quantitative PCR was used to detect the expression of miR-151a-3p and potential target gene mRNA. The protein expressions of target genes and downstream signaling pathway proteins were analyzed by Western blotting. The proliferation of PC-3 cells was examined by MTT assay, and the migration of PC-3 cells was detected by Transwell TM assay. Results The expression level of miR-151a-3p in the prostate cancer cells was significantly lower than that in RWPE-1 normal human prostate epithelial cells. PC-3 cells had the lowest expression level of miR-151a-3p. The bioinformatics and dual-luciferase reporter assay showed that NEK2 was the potential target gene for miR-151a-3p. After transfection with miR-151a-3p mimics, the expression of miR-151a-3p in PC-3 cells significantly increased and the expression of NEK2 mRNA significantly decreased. The protein expressions of PI3K-AKT-mTOR signaling pathway were also reduced. Up-regulation of miR-151a-3p significantly inhibited the proliferation and migration of PC-3 cells. Conclusion The expression of miR-151a-3p is reduced in prostate cancer cells. Up-regulation of miR-151a-3p can inhibit the proliferation and migration of P-3 in prostate cancer by decreasing the expression of NEK2 and PI3K-AKT-mTOR signaling pathway proteins.

Direct effect of curcumin on porcine ovarian cell functions.

PubMed

Kádasi, Attila; Maruniaková, Nora; Štochmaľová, Aneta; Bauer, Miroslav; Grossmann, Roland; Harrath, Abdel Halim; Kolesárová, Adriana; Sirotkin, Alexander V

2017-07-01

Curcuma longa Linn (L.) is a plant widely used in cooking (in curry powder a.o.) and in folk medicine, but its action on reproductive processes and its possible mechanisms of action remain to be investigated. The objective of this study was to examine the direct effects of curcumin, the major Curcuma longa L. molecule, on basic ovarian cell functions such as proliferation, apoptosis, viability and steroidogenesis. Porcine ovarian granulosa cells were cultured with and without curcumin (at doses of 0, 1, 10 and 100μg/ml of medium). Markers of proliferation (accumulation of PCNA) and apoptosis (accumulation of bax) were analyzed by immunocytochemistry. The expression of mRNA for PCNA and bax was detected by RT-PCR. Cell viability was detected by trypan blue exclusion test. Release of steroid hormones (progesterone and testosterone) was measured by enzyme immunoassay (EIA). It was observed that addition of curcumin reduced ovarian cell proliferation (expression of both PCNA and its mRNA), promoted apoptosis (accumulation of both bax and its mRNA), reduced cell viability, and stimulated both progesterone and testosterone release. These observations demonstrate the direct suppressive effect of Curcuma longa L./curcumin on female gonads via multiple mechanisms of action - suppression of ovarian cell proliferation and viability, promotion of their apoptosis (at the level of mRNA transcription and subsequent accumulation of promoters of genes regulating these activities) and release of anti-proliferative and pro-apoptotic progesterone and androgen. The potential anti-gonadal action of curcumin should be taken into account by consumers of Curcuma longa L.-containing products. Copyright © 2017 Elsevier B.V. All rights reserved.

[Expression of Id1 and Id3 in endometrial carcinoma and their roles in regulating biological behaviors of endometrial carcinoma cells in vitro].

PubMed

Sun, Lili; Li, Xuenong; Liu, Guobing

2013-06-01

To investigate the expression of inhibitor of DNA differentiation/DNA binding 1 (Id1) and Id3 in endometrial carcinoma and explore their roles in regulating the proliferation, invasion, migration and adhesion of endometrial carcinoma cells in vitro. Id1 and Id3 expression in 4 fresh endometrial cancer tissue specimens and matched adjacent tissues were detected using Western blotting. Two endometrial cancer cell lines, HEC-1-B and RL-952, were both divided into 4 groups, namely the untreated group, blank virus group, promoter group and Id1/Id3 double-knockdown group, and their expressions of MMP2, CXCR4 and P21 were detected by qRT-PCR and Western blotting. The proliferation, invasion, migration and adhesion of the cells were evaluated with MTT, Transwell, wound-healing, and adhesion assays. Endometrial carcinoma tissues showed significantly higher Id1 and Id3 expression than the adjacent tissues (P<0.05). In the two endometrial carcinoma cell lines, Id1/Id3 double-knockdown significantly decreased MMP2 and CXCR4 expression and increased P21 expression at both mRNA and protein levels (P<0.05), and resulted in suppressed cell proliferation, invasion, migration and adhesion. Id1 and Id3 expressions are up-regulated in endometrial carcinoma to promote the proliferation, invasion, migration and adhesion of the tumor cells by increasing MMP2 and CXCR4 expression and reducing P21 expression. Therapies targeting Id1/Id3 can be a novel strategy for treatment of endometrial carcinoma.

De Novo Glutamine Synthesis

PubMed Central

He, Qiao; Shi, Xinchong; Zhang, Linqi; Yi, Chang; Zhang, Xuezhen

2016-01-01

Purpose: The aim of this study was to investigate the role of de novo glutamine (Gln) synthesis in the proliferation of C6 glioma cells and its detection with 13N-ammonia. Methods: Chronic Gln-deprived C6 glioma (0.06C6) cells were established. The proliferation rates of C6 and 0.06C6 cells were measured under the conditions of Gln deprivation along with or without the addition of ammonia or glutamine synthetase (GS) inhibitor. 13N-ammonia uptake was assessed in C6 cells by gamma counting and in rats with C6 and 0.06C6 xenografts by micro–positron emission tomography (PET) scanning. The expression of GS in C6 cells and xenografts was assessed by Western blotting and immunohistochemistry, respectively. Results: The Gln-deprived C6 cells showed decreased proliferation ability but had a significant increase in GS expression. Furthermore, we found that low concentration of ammonia was sufficient to maintain the proliferation of Gln-deprived C6 cells, and 13N-ammonia uptake in C6 cells showed Gln-dependent decrease, whereas inhibition of GS markedly reduced the proliferation of C6 cells as well as the uptake of 13N-ammoina. Additionally, microPET/computed tomography exhibited that subcutaneous 0.06C6 xenografts had higher 13N-ammonia uptake and GS expression in contrast to C6 xenografts. Conclusion: De novo Gln synthesis through ammonia–glutamate reaction plays an important role in the proliferation of C6 cells. 13N-ammonia can be a potential metabolic PET tracer for Gln-dependent tumors. PMID:27118759

KAI1 overexpression promotes apoptosis and inhibits proliferation, cell cycle, migration, and invasion in nasopharyngeal carcinoma cells.

PubMed

Guo, Zheng; Wang, Yili; Yang, Jing; Zhong, Jinghua; Liu, Xia; Xu, Mingjun

The purpose of this study is to characterize the effect of KAI1 overexpression on the biological behavior of nasopharyngeal carcinoma (NPC) cells. Nasopharyngeal carcinoma is a highly malignant tumor with a high rate of incidence in China. Currently, there are no ideal therapeutic options for patients with NPC, but a targeted therapy would have great potential for treating it. Therefore, there is an urgent need for novel therapeutic targets to provide new options for treating NPC. The KAI1 gene was originally identified as a metastasis suppressor gene for advanced human cancer. In NPC cell lines and tissues, the expression of KAI1 decreased as the metastatic potential of cells increased, but its potential as a therapeutic target has not been elucidated. Non-transformed nasopharyngeal epithelium cell NP69 and NPC cell line C666-1 were cultured and KAI1 expression in these cells was detected by qRT-PCR and Western blot. After the transfection of KAI1-pCDNA3.1 to NP69 and C666-1, the KAI1 expression in these cells was detected by qRT-PCR and Western blot, the proliferation was performed by MTS, the cell cycle and apoptosis were performed by flow cytometry, the migration and invasion were examined by transwell. Our results showed that KAI1 was significantly upregulated in C666-1 cells compared to that in NP69 cells. In addition, KAI1 overexpression significantly inhibited the proliferation, cell cycle, migration, and invasion, and promoted apoptosis of C666-1 cells, but had no significant effect on NP69 cells. Our findings suggest that KAI1 overexpression promotes apoptosis and inhibits proliferation, cell cycle, migration, and invasion in NPC cells. We hypothesize that KAI1 overexpression could be a potential therapeutic target for NPC. Copyright © 2016 Elsevier Inc. All rights reserved.

Low doses of bisphenol A stimulate the proliferation of breast cancer cells via ERK1/2/ERRγ signals.

PubMed

Song, Haixing; Zhang, Tao; Yang, Ping; Li, Minhui; Yang, Yuhan; Wang, Yuanyuan; Du, Jun; Pan, Kejian; Zhang, Kun

2015-12-25

The effects and mechanisms of bisphenol A (BPA) on the development of breast cancer are still not well illustrated. The present study revealed that nanomolar BPA significantly promoted the proliferation of both estrogen receptor (ER) positive (MCF-7) and negative (SkBr3) breast cancer cells, which was confirmed by up regulation of proliferating cell nuclear antigen (PCNA) and Bcl-2. Neither ERα nor G-protein-coupled estrogen receptor (GPER) mediated this effect of BPA because their inhibitors had no effect on the BPA induced cell proliferation. However, silencing of estrogen related receptor gamma (ERRγ) by its specific siRNA significantly abolished BPA induced proliferation of breast cancer cells, while si-ERRα had no similar effect. Moreover, nanomolar BPA up regulated the mRNA and protein levels of ERRγ and triggered its nuclear translocation via a time dependent manner. Further studies revealed that 10(-8)M BPA obviously increased the phosphorylation of ERK1/2, while had no similar effect on the phosphorylation of JNK and p38 MAPK. Further, PD 98059, the inhibitor of ERK1/2, significantly abolished the BPA induced up regulation of ERRγ and proliferation of breast cancer cells. Collectively, our results revealed that nanomolar BPA can trigger the proliferation of breast cancer cells via ERK1/2/ERRγ signals. Given that nanomolar BPA has been widely detected in human tissues, the clinical relevance of BPA and breast cancer progression should be further investigated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways.

PubMed

Ma, Yiming; Fu, Shaoting; Lu, Lin; Wang, Xiaohui

2017-07-15

To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK. C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot. ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells. Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

PubMed

Pan, J-L; Yuan, D-Z; Zhao, Y-B; Nie, L; Lei, Y; Liu, M; Long, Y; Zhang, J-H; Blok, L J; Burger, C W; Yue, L-M

2017-03-01

This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G 1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

[Effects of selenium compounds on proliferation, migration and adhesion of HeLa cells].

PubMed

Sun, Licui; Lu, Jiaxi; Wang, Qin; Liu, Yiqun; Han, Feng; Yang, Yanhua; Zhang, Hongkun; Huang, Zhenwu

2015-03-01

To explore the effects of methylseleninic acid (MeSeA), selenomethionine (SeMet) and methylselenocysteine (MeSeCys) on proliferation, migration and adhesion of HeLa cells. HeLa cells were cultured and treated with MeSeA, SeMet and MeSeCys for 12 - 72 h respectively. MTT assay, healing assay and in vitro cell Matrigel adhesion assay were used to detect the proliferation, migration and adhesion of HeLa cells. Compared to the control group, the proliferation of HeLa cells was remarkably inhibited by MeSeA (P <0. 01). The migration of HeLa cells in MeSeA group was inhibited by 34% (P < 0. 05) and 26% (P < 0. 05) in 4 h and 8 h, respectively. However, the migration of HeLa cells with inhibitions of 18% and 13% was in SeMet group in 4 h and 8 h. The inhibitions of HeLa cell migration in MeSeCys group was 28% (P < 0.05) and 5% in 4 h and 8 h, respectively. In addition, the adhesive function of HeLa cells in the MeSeA group, the SeMet group as well as the MeSeCys group were inhibited by 36% (P < 0. 01), 25% and 49% (P < 0. 01). The proliferation and migration of HeLa cell were effectively inhibited by MeSeA, while the adhesive function of HeLa cell was remarkably inhibited by MeSeCys.

Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de; Stagno, Matias Julian; Yan, Jing

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an intrinsic or early-acquisition of resistance to chemo- and radiation therapy. Molecular determinants pivotal for RMS migration, metastatic invasion, cell proliferation, and survival are incompletely identified. Migration and cell proliferation were shown to correlate with cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}). Store-operated Ca{sup 2+}-entry (SOCE) that increases intracellular [Ca{sup 2+}] is accomplished by Orai1, a pore-forming ion channel unit, the expression of which is stimulated by the transcription factor NFκB. The present study explored the expression of Orai1 and its regulators STIM1 and NFκB in human rhabdomyosarcoma cell linesmore » and analyzed their impact on cell proliferation and migration. For the study human rhabdomyosarcoma cell lines RD (embryonal) and RH30 (alveolar) were analyzed for Orai1, STIM1, and NFκB transcription by RT-PCR and their corresponding proteins in Western blot. [Ca{sup 2+}]{sub i} was detected via Fura-2 fluorescence and SOCE – resulting from [Ca{sup 2+}]{sub i} increase following store depletion with extracellular Ca{sup 2+} removal and inhibition of the sarcoendoplasmatic reticular Ca{sup 2+} ATPase – detected with thapsigargin. Cell migration was analyzed in transwell and mitotic cell death with the clonogenic assay. In summary, Orai1, STIM1, and NFκB are expressed in embryonal (RD) and alveolar (RH30) rhabdomyosarcoma. SOCE inhibitor BTP2, Orai1 inhibitor 2-APB, or NFκB inhibitor wogonin virtually abrogated (BTP2, 2-APB) or significantly reduced (wogonin) SOCE. Moreover, SOCE inhibitors 2-APB and BTP2 and wogonin significantly inhibited migration and proliferation of both, RD and RH30 cells. These results suggest that Orai1 signaling is involved in SOCE into rhabdomyosarcoma cells thus contributing to migration, invasion and proliferation. - Highlights: • Orai1, STIM1, and NFκB are expressed in RD and RH30 rhabdomyosarcoma cell lines. • Orai1, STIM1, and NFκB are significantly upregulated in the RH30 cell line and leads to a significantly increased SOCE. • Orai1 signaling is involved in SOCE thus contributing to migration, invasion and proliferation.« less

[Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

PubMed

Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

2009-06-01

This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

Association of Wnt1-inducible signaling pathway protein-1 with the proliferation, migration and invasion in gastric cancer cells.

PubMed

Jia, Shuqin; Qu, Tingting; Feng, Mengmeng; Ji, Ke; Li, Ziyu; Jiang, Wenguo; Ji, Jiafu

2017-06-01

Wnt1-inducible signaling pathway protein-1 is a cysteine-rich protein that belongs to the CCN family, which has been implicated in mediating the occurrence and progression through distinct molecular mechanisms in several tumor types. However, the association of Wnt1-inducible signaling pathway protein-1 with gastric cancer and the related molecular mechanisms remain to be elucidated. Therefore, this study aimed to clarify the biological role of Wnt1-inducible signaling pathway protein-1 in the proliferation, migration, and invasion in gastric cancer cells and further investigated the associated molecular mechanism on these biological functions. We first detected the expression level of Wnt1-inducible signaling pathway protein-1 in gastric cancer, and the reverse transcription polymerase chain reaction have shown that Wnt1-inducible signaling pathway protein-1 expression levels were upregulated in gastric cancer tissues. The expression of Wnt1-inducible signaling pathway protein-1 in gastric cancer cell lines was also detected by quantitative real-time polymerase chain reaction and Western blotting. Furthermore, two gastric cancer cell lines with high expression of Wnt1-inducible signaling pathway protein-1 were selected to explore the biological function of Wnt1-inducible signaling pathway protein-1 in gastric cancer. Function assays indicated that knockdown of Wnt1-inducible signaling pathway protein-1 suppressed cell proliferation, migration, and invasion in BGC-823 and AGS gastric cancer cells. Further investigation of mechanisms suggested that cyclinD1 was identified as one of Wnt1-inducible signaling pathway protein-1 related genes to accelerate proliferation in gastric cancer cells. In addition, one pathway of Wnt1-inducible signaling pathway protein-1 induced migration and invasion was mainly through the enhancement of epithelial-to-mesenchymal transition progression. Taken together, our findings presented the first evidence that Wnt1-inducible signaling pathway protein-1 was upregulated in gastric cancer and acted as an oncogene by promoting proliferation, migration, and invasion in gastric cancer cells.

Purification of high-molecular-weight subfraction from porcine skin inhibiting proliferation of A431 human carcinoma epidermoid cells.

PubMed

Belova, O V; Sergienko, V I; Arion, V Ya; Lukanidina, T A; Moskvina, S N; Zimina, I V; Borisenko, G G; Lutsenko, G V; Grechikhina, M V; Kovaleva, E V; Klyuchnikova, Zh I

2014-07-01

Subfraction with a molecular weight >250 kDa isolated from porcine skin and inhibiting the proliferation of A431 human carcinoma epidermoid cells was purified by DEAE 32 anion exchange chromatography with NaCl concentration step-gradient. The effects of the initial subfraction and fractions obtained by separation in DEAE 32 on the proliferation of A431 human carcinoma epidermoid cells were studied in vitro in two tests (MTT and fluorescent test). The more sensitive fluorescent test showed the highest inhibitory activity of fraction No. 2 released from the column at 0.15 M NaCl. One major protein component and a series of minor protein components were detected in this fraction by vertical PAAG-SDS electrophoresis.

PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis.

PubMed

Zhao, Yan; Yan, Xinfeng; Li, Xia; Zheng, Yabing; Li, Shufeng; Chang, Xiaotian

2016-10-01

Some studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA. Synovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT(2) Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF. Both PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1β and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF. PGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

1950MHz Radio Frequency Electromagnetic Radiation Inhibits Testosterone Secretion of Mouse Leydig Cells

PubMed Central

Lin, Yan-Yun; Wu, Tao; Liu, Jun-Ye; Gao, Peng; Li, Kang-Chu; Guo, Qi-Yan; Yuan, Meng; Lang, Hai-Yang; Zeng, Li-Hua; Guo, Guo-Zhen

2017-01-01

More studies that are focused on the bioeffects of radio-frequency (RF) electromagnetic radiation that is generated from the communication devices, but there were few reports with confirmed results about the bioeffects of RF radiation on reproductive cells. To explore the effects of 1950 MHz RF electromagnetic radiation (EMR) on mouse Leydig (TM3) cells. TM3 cells were irradiated or sham-irradiated continuously for 24 h by the specific absorption rate (SAR) 3 W/kg radiation. At 0, 1, 2, 3, 4, and 5 days after irradiation, cell proliferation was detected by cell counting kit-8 (CCK-8) method, cell cycle distribution, percentage of apoptosis, and cellular reactive oxygen species (ROS) were examined by flow cytometry, Testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) assay, messenger ribonucleic acid (mRNA) expression level of steroidogenic acute regulatory protein (StAR) and P450scc in TM3 cells was detected by real-time polymerase chain reaction (PCR). After being irradiated for 24 h, cell proliferation obviously decreased and cell cycle distribution, secretion capacity of Testosterone, and P450scc mRNA level were reduced. While cell apoptosis, ROS, and StAR mRNA level did not change significantly. The current results indicated that 24 h of exposure at 1950 MHz 3 W/kg radiation could cause some adverse effects on TM3 cells proliferation and Testosterone secretion, further studies about the biological effects in the reproductive system that are induced by RF radiation are also needed. PMID:29295490

1950MHz Radio Frequency Electromagnetic Radiation Inhibits Testosterone Secretion of Mouse Leydig Cells.

PubMed

Lin, Yan-Yun; Wu, Tao; Liu, Jun-Ye; Gao, Peng; Li, Kang-Chu; Guo, Qi-Yan; Yuan, Meng; Lang, Hai-Yang; Zeng, Li-Hua; Guo, Guo-Zhen

2017-12-23

More studies that are focused on the bioeffects of radio-frequency (RF) electromagnetic radiation that is generated from the communication devices, but there were few reports with confirmed results about the bioeffects of RF radiation on reproductive cells. To explore the effects of 1950 MHz RF electromagnetic radiation (EMR) on mouse Leydig (TM3) cells. TM3 cells were irradiated or sham-irradiated continuously for 24 h by the specific absorption rate (SAR) 3 W/kg radiation. At 0, 1, 2, 3, 4, and 5 days after irradiation, cell proliferation was detected by cell counting kit-8 (CCK-8) method, cell cycle distribution, percentage of apoptosis, and cellular reactive oxygen species (ROS) were examined by flow cytometry, Testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) assay, messenger ribonucleic acid (mRNA) expression level of steroidogenic acute regulatory protein (StAR) and P450scc in TM3 cells was detected by real-time polymerase chain reaction (PCR). After being irradiated for 24 h, cell proliferation obviously decreased and cell cycle distribution, secretion capacity of Testosterone, and P450scc mRNA level were reduced. While cell apoptosis, ROS, and StAR mRNA level did not change significantly. The current results indicated that 24 h of exposure at 1950 MHz 3 W/kg radiation could cause some adverse effects on TM3 cells proliferation and Testosterone secretion, further studies about the biological effects in the reproductive system that are induced by RF radiation are also needed.

Dixdc1 targets CyclinD1 and p21 via PI3K pathway activation to promote Schwann cell proliferation after sciatic nerve crush

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Weijie; Liu, Qingqing; Liu, Yuxi

Dixdc1 (DIX domain containing-1), the mammalian homolog of Ccd1 (Coiled-coil-Dishevelled-Axin1), is a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain. As a novel component of the Wnt pathway, Dixdc1 has been reported to be able to promote neural progenitor proliferation and neuronal differentiation via Wnt/β-catenin signaling. But there still remains something unknown about Dixdc1 distribution and functions in the lesion and regeneration of the peripheral nervous system (PNS), so we tried to investigate dynamic changes of Dixdc1 expression in a rat sciatic nerve crush (SNC) model in this study. First of all, we detected SNC-induced increased levels ofmore » Dixdc1 in Schwann cells and interestingly identified parallel expression of PCNA (proliferation cell nuclear antigen) with Dixdc1. Besides, we observed up-regulated Dixdc1 during the process of TNF-α-induced Schwann cell proliferation. Also, we discovered that Dixdc1 could promote G1-S phase transition accompanied with the up-regulation of CyclinD1 and down-regulation of p21. More importantly, enhanced effects of Dixdc1 on cell proliferation were confirmed to be associated with PI3K activation. Not only blocking of the PI3K but Dixdc1 knockdown led to significantly decreased ability for proliferation, as well as down-regulation of CyclinD1 and up-regulation of p21. In summary, these data demonstrated that Dixdc1 might participate in Schwann cell proliferation by targeting CyclinD1 and p21 at least partially through the PI3K/AKT activation. - Highlights: • The dynamic changes and location of Dixdc1 after sciatic nerve crush. • Dixdc1 was associated with Schwann cell proliferation. • Dixdc1 promoted Schwann cell proliferation partly via PI3K/AKT pathway.« less

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

PubMed Central

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells. PMID:29391870

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation.

PubMed

Xu, Falin; Bai, Qiongdan; Zhou, Kai; Ma, Li; Duan, Jiajia; Zhuang, Fangli; Xie, Cuicui; Li, Wenli; Zou, Peng; Zhu, Changlian

2017-01-01

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.

Effects of aluminum on the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in the dentate gyrus of D-galactose-treated mice via increasing oxidative stress

PubMed Central

Nam, Sung Min; Kim, Jong Whi; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Choi, Jung Hoon; Hwang, In Koo; Seong, Je Kyung

2016-01-01

Aluminum (Al) accumulation increases with aging, and long-term exposure to Al is regarded as a risk factor for Alzheimer's disease. In this study, we investigated the effects of Al and/or D-galactose on neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons in the hippocampal dentate gyrus. AlCl3 (40 mg/kg/day) was intraperitoneally administered to C57BL/6J mice for 4 weeks. In addition, vehicle (physiological saline) or D-galactose (100 mg/kg) was subcutaneously injected to these mice immediately after AlCl3 treatment. Neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons were detected using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN, respectively, via immunohistochemistry. Subchronic (4 weeks) exposure to Al in mice reduced neural stem cells, proliferating cells, and differentiating neuroblasts without causing any changes to mature neurons. This Al-induced reduction effect was exacerbated in D-galactose-treated mice compared to vehicle-treated adult mice. Moreover, exposure to Al enhanced lipid peroxidation in the hippocampus and expression of antioxidants such as Cu, Zn- and Mn-superoxide dismutase in D-galactose-treated mice. These results suggest that Al accelerates the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in D-galactose-treated mice via oxidative stress, without inducing loss in mature neurons. PMID:26243606

Downregulation of Pygopus 2 inhibits vascular mimicry in glioma U251 cells by suppressing the canonical Wnt signaling pathway

PubMed Central

WANG, HAIDONG; FU, JIANHUA; XU, DIANSHUANG; XU, WEIWEI; WANG, SHIYONG; ZHANG, LIU; XIANG, YONGSHENG

2016-01-01

Gliomas are the most common type of malignant primary brain tumor, and the Wnt signaling pathway is associated with glioma malignancy. Pygopus protein plays an important role in developmental brain patterning, and has been identified to be a component of the Wnt signaling pathway. In the present study, the Pygopus 2 (Pygo2) protein was examined in 80 glioma tissue samples. Short hairpin (sh)RNA-Pygo2 was transfected into glioma U251 cells, and the cell proliferation, colony formation and bromodeoxyuridine (BrdU) incorporation were analyzed. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect the expression of Pygo2. A vascular mimicry assay was performed to examine the vascular mimicry of U251 cells. A luciferase reporter assay was used to detect the β-catenin/Wnt system. The cyclin D1 protein was also detected using western blot analysis. The results demonstrated that inhibition of the expression of Pygo2 significantly triggered the decrease of cell proliferation, colony formation and BrdU incorporation compared with the cells treated with scramble control shRNA (shRNA-Scr). shRNA-Pygo2 transfection was found to inhibit vascular-mimicry and block the Wnt signaling pathway compared to the cells transfected with shRNA-Scr. The transfection of shRNA-Pygo2 also decreased the expression of the Wnt target gene cyclin D1. In conclusion, shRNA-Pygo2 suppressed glioma cell proliferation effectively and inhibited vascular mimicry by inhibiting the expression of cyclin D1 in the canonical Wnt/β-catenin pathway in brain glioma cells. PMID:26870266

CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

PubMed

Wu, Yu-Jing; Chen, Heng-Shi; Chen, Wen-Sheng; Dong, Jin; Dong, Xiao-Jie; Dai, Xing; Huang, Qiong; Wei, Wei

2018-01-01

Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'- O -benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4 + T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4 + T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4 + T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr 394 ). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4 + T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr 394 ) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4 + T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

Hot water-extracted Lycium barbarum and Rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells

PubMed Central

Chao, Jane C-J; Chiang, Shih-Wen; Wang, Ching-Chiung; Tsai, Ya-Hui; Wu, Ming-Shun

2006-01-01

AIM: To investigate the effect of hot water-extracted Lycium barbarum (LBE) and Rehmannia glutinosa (RGE) on cell proliferation and apoptosis in rat and/or human hepatocellular carcinoma (HCC) cells. METHODS: Rat (H-4-II-E) and human HCC (HA22T/VGH) cell lines were incubated with various concentrations (0-10 g/L) of hot water-extracted LBE and RGE. After 6-24 h incubation, cell proliferation (n = 6) was measured by a colorimetric method. The apoptotic cells (n = 6) were detected by flow cytometry. The expression of p53 protein (n = 3) was determined by SDS-PAGE and Western blotting. RESULTS: Crude LBE (2-5 g/L) and RGE (2-10 g/L) dose-dependently inhibited proliferation of H-4-II-E cells by 11% (P < 0.05) to 85% (P < 0.01) after 6-24 h treatment. Crude LBE at a dose of 5 g/L suppressed cell proliferation of H-4-II-E cells more effectively than crude RGE after 6-24 h incubation (P < 0.01). Crude LBE (2-10 g/L) and RGE (2-5 g/L) also dose-dependently inhibited proliferation of HA22T/VGH cells by 14%-43% (P < 0.01) after 24 h. Crude LBE at a dose of 10 g/L inhibited the proliferation of HA22T/VGH cells more effectively than crude RGE (56.8% ± 1.6% vs 70.3% ± 3.1% of control, P = 0.0003 < 0.01). The apoptotic cells significantly increased in H-4-II-E cells after 24 h treatment with higher doses of crude LBE (2-5 g/L) and RGE (5-10 g/L) (P < 0.01). The expression of p53 protein in H-4-II-E cells was 119% and 143% of the control group compared with the LBE-treated (2, 5 g/L) groups, and 110% and 132% of the control group compared with the RGE -treated (5, 10 g/L) groups after 24 h. CONCLUSION: Hot water-extracted crude LBE (2-5 g/L) and RGE (5-10 g/L) inhibit proliferation and stimulate p53-mediated apoptosis in HCC cells. PMID:16874858

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

PubMed

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Detection of mitogen-induced lymphocyte proliferation by bromodeoxyuridine (BrdU) incorporation in the chicken.

PubMed

Motobu, Maki; El-Abasy, Moshira; Na, Ki-Jeong; Hirota, Yoshikazu

2002-04-01

3H-thymidine (3H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with 3H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with 3H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.

Cyclosporin A promotes mineralization by human cementoblastoma-derived cells in culture.

PubMed

Arzate, Higinio; Alvarez, Marco A; Narayanan, A Sampath

2005-06-01

The immunosuppressive drug cyclosporin A has been shown to induce cementum deposition in vivo in experimental animals. Using cementoblastoma-derived cells, we have studied whether this drug will be useful to study cementum mineralization and differentiation in vitro. Human cementoblastoma cells and gingival fibroblasts (controls) were cultured and treated with 0.5, 1.0 and 5.0 microg/ml of cyclosporin A. Cell proliferation was evaluated by MTT (tetrazolium) assay and cell number, and cell viability was assessed by trypan blue dye exclusion. Induction of mineralization was evaluated by alizarin red S staining to detect mineralized nodules and by reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of bone differentiation markers alkaline phosphatase, osteocalcin, bone sialoprotein and core-binding factor a1 (Cbfa1). Cyclosporin A at 5.0 microg/ml concentration reduced significantly the increase in the number of cementoblastoma cells. A dose-dependent increase in the number of mineralized nodules occurred in cultures of cementoblastoma-derived cells treated with cyclosporin A, and RT-PCR analyses showed significantly higher levels of expression of alkaline phosphatase, bone sialoprotein, type I collagen, matrix metalloproteinase-1, osteocalcin, osteopontin, and Cbfa1. Human gingival fibroblast proliferation and cell number were not affected. Mineralized nodules were not detected in gingival fibroblasts and bone specific proteins were not expressed. Presence of cyclosporin A during 14-day culture period appears to suppress the proliferation of cementoblastoma cells and induce the formation mineralized-like tissue by these cells.

The chemokine receptor CXCR6 and its ligand CXCL16 are expressed in carcinomas and inhibit proliferation.

PubMed

Meijer, Joost; Ogink, Janneke; Kreike, Bas; Nuyten, Dimitry; de Visser, Karin E; Roos, Ed

2008-06-15

The chemokine receptor CXCR6 and its ligand CXCL16 are involved in inflammation. Thus far, they were known to be expressed mainly by T cells and macrophages, respectively. However, we detected both in all of 170 human primary mammary carcinomas and at similar levels in all 8 human mammary carcinoma cell lines tested by microarray analysis. Expression was confirmed by reverse transcription-PCR and for the cell lines also by fluorescence-activated cell sorting analysis. CXCR6 and CXCL16 were also detected in several mouse and human mammary, colon, and pancreatic carcinoma cell lines. CXCL16 is a transmembrane protein from which the soluble chemokine can be cleaved off. The transmembrane form is present on the surface of the carcinoma cells. Surprisingly, suppression of either CXCR6 or CXCL16 led to greatly enhanced proliferation in vitro as well as in vivo, indicating that their interaction inhibits proliferation. This notion was verified using inhibitory antibodies and by introduction of CXCL16 into a rare CXCL16-negative cell line. The effect was mediated by the G protein-coupled receptor CXCR6 because it was blocked by the G(i) protein inhibitor pertussis toxin. In contrast, the soluble CXCL16 chemokine enhanced proliferation, and this was also mediated by CXCR6 but not via G(i) protein. It is remarkable that both CXCR6 and CXCL16 are expressed by all mammary carcinomas because cells that lose either acquire a growth advantage and should be selected during tumor progression. This suggests an unknown important role in tumor formation. Proteases, possibly macrophage derived, might convert inhibitory transmembrane CXCL16 into the stimulatory chemokine.

MicroRNA-137 Affects Proliferation and Migration of Placenta Trophoblast Cells in Preeclampsia by Targeting ERRα.

PubMed

Lu, Tan-Min; Lu, Wei; Zhao, Long-Jun

2016-06-06

To investigate the effects of microRNA-137 (miRNA-137) in proliferation and migration of placenta trophoblast cells of preeclampsia and the targeting gene of miRNA-137. A total of 134 cases of puerperants were divided into normal pregnancy (n = 50), mild preeclampsia (n = 38), and severe preeclampsia groups (n = 46). MiRNA-137, estrogen-related receptor α (ERRα), and wingless INT (WNT)11 messenger RNAs (mRNAs) were measured in placental tissue and trophoblast cells after transfection, and ERRα protein in placental tissues was detected by immunohistochemistry. The target genes of miRNA-137, trophoblast cell proliferation, migration, and invasion abilities were detected. Both ERRα and WNT11 proteins in the trophoblast cells were measured after transfection. Relative expressions of miRNA-137 were higher, and positive expression rates and relative expression levels of ERRα protein were lower in mild and severe preeclampsia and early- and late-onset preeclampsia than in normal pregnancy group (all P < .05). MiRNA-137 in the placental tissues was negatively correlated with ERRα protein (P < .05). Luciferase reporter gene assay analysis showed that ERRα was a direct target gene of miRNA-137. Absorbance values, relative scratch-covered areas, cell membrane permeable rate, ERRα, and WNT11 mRNA and protein relative expressions were significantly lower, while cells at G1/G0 phase were higher in miRNA-137 mimic group than those in the blank, negative control, and miRNA-137 inhibitor group. MiRNA-137 significantly reduced the proliferation and migration of placenta trophoblast cells of preeclampsia by targeting ERRα, which might be a potential target for gene therapy. © The Author(s) 2016.

Binding of galectin-1 to breast cancer cells MCF7 induces apoptosis and inhibition of proliferation in vitro in a 2D- and 3D- cell culture model.

PubMed

Geiger, Pamina; Mayer, Barbara; Wiest, Irmi; Schulze, Sandra; Jeschke, Udo; Weissenbacher, Tobias

2016-11-08

Galectin-1 (gal-1) belongs to the family of β-galactoside-binding proteins which primarily recognizes the Galβ1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. The lectin recognizes correspondent glycoepitopes on human breast cancer cells. Galectin-1 is expressed both in normal and malignant tissues. Lymphatic organs naturally possessing high rates of apoptotic cells, express high levels of Galectin-1. Furthermore galectin-1 can initiate T cell apoptosis. Binding of galectin-1 to trophoblast tumor cells presenting the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen inhibits tumor cell proliferation. In this study we examined the impact galectin-1 has in vitro on cell proliferation, apoptotic potential and metabolic activity of MCF-7 and T-47D breast cancer cells in dependence to their expression of the Thomsen-Friedenreich (TF) tumor antigen. For proliferation and apoptosis assays cells were grown in presence of 10, 30 and 60 μg gal-1/ml medium. Cell proliferation was determined by a BrdU uptake ELISA. Detection of apoptotic cells was done by M30 cyto death staining, in situ nick translation and by a nucleosome ELISA method. Furthermore we studied the impact galectin-1 has on the metabolic activity of MCF-7 and T-47D cells in a homotypic three-dimensional spheroid cell culture model mimicking a micro tumour environment. Gal-1 inhibited proliferation of MCF-7 cells (strong expression of the TF epitope) but did not significantly change proliferation of T-47D cells (weak expression of the TF epitope). The incubation of MCF-7 cells with gal-1 raised number of apoptotic cells significantly. Treating the spheroids with 30 μg/ml galectin-1 in addition to standard chemotherapeutic regimes (FEC, TAC) resulted in further suppression of the metabolic activity in MCF-7 cells whereas T-47D cells were not affected. Our results demonstrate that galectin-1 can inhibit proliferation und metabolic cell activity and induce apoptosis in breast tumor cell lines with high expression levels of the Thomsen-Friedenreich (TF) antigen in monolayer and spheroid cell culture models.

Zinc oxide nanoparticles inhibit murine photoreceptor-derived cell proliferation and migration via reducing TGF-β and MMP-9 expression in vitro.

PubMed

Guo, Da Dong; Li, Qing Ning; Li, Chun Min; Bi, Hong Sheng

2015-04-01

To investigate behaviour and expression of transforming growth factor-β (TGF-β) and matrix metalloproteinases (MMP-9) in murine photoreceptor-derived cells (661W) after incubation with zinc oxide (ZnO) nanoparticles. We explored effects of ZnO nanoparticles on 661W cells using a real-time cell electronic sensing system, flow cytometry, multiple function microplate reading, real-time quantitative PCR detection system and enzyme-linked immunosorbent assay respectively. Our results indicate that ZnO nanoparticles induced overload of calcium and reactive oxygen species within cells, causing formation of apoptotic bodies, disruption of cell cycle distribution, and reduction in expression of TGF-β and MMP-9, to suppress cell proliferation and migration. Our findings show that disruption of intracellular calcium homoeostasis and overproduction of reactive oxygen species were closely associated with reduction of TGF-β and MMP-9 in 661W cells under ZnO nanoparticle treatment. Results of our study indicate that ZnO nanoparticles suppressed cell proliferation and migration, and reduced production of TGF-β and MMP-9 at both gene and protein levels. Our findings contribute to the understanding of the molecular mechanisms that reduced TGF-β and MMP-9 levels inhibit cell proliferation and migration under ZnO nanoparticle influence. © 2015 John Wiley & Sons Ltd.

NOR1 promotes hepatocellular carcinoma cell proliferation and migration through modulating the Notch signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

You, Kun; Sun, Peisheng; Yue, Zhongyi

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Previous studies have reported that the oxidored-nitro domain containing protein 1 (NOR1) is a novel tumor suppressor in several tumors. Recent evidence suggests that NOR1 is strongly expressed in HCC cells. However, its role and mechanism in HCC are unclear. In the current study, Western blot and qPCR detected strong NOR1 mRNA and protein expression in HepG2 and Hep3B cells. After transfection with NOR1 siRNA or pcDNA3.1-myc-his-NOR1, the proliferation and migration of HepG2 and Hep3B cells were analyzed in vitro. HepG2 or Hep3B cells overexpressing NOR1 showed anmore » increased proliferation and migration, whereas siRNA-mediated silencing of NOR1 showed the opposite effect. Furthermore, NOR1 activated the Notch signaling pathway, indicated by increased levels of Notch1, NICD, Hes1, and Hey1 in protein. Importantly, the Notch inhibitor DAPT downregulated Notch activation and further enhanced siNOR1-induced reduction of cell proliferation and migration in HepG2 and Hep3B cells, whereas DAPT reversed the effect of NOR1 overexpression on cell proliferation and migration. In conclusion, these results indicate that NOR1 may be involved in the progression of HCC and thus may be a potential target for the treatment of liver cancer. - Highlights: • NOR1 expression is up-regulated in HCC cells. • NOR1 promotes the proliferation and migration of HCC cells. • NOR1 promotes the progression of HCC cells by activating Notch pathway.« less

Sam68 promotes Schwann cell proliferation by enhancing the PI3K/Akt pathway and acts on regeneration after sciatic nerve crush

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Weijie, E-mail: 459586768@qq.com; Liu, Yuxi, E-mail: 924013616@qq.com; Wang, Youhua, E-mail: wyouhua1516@163.com

Sam68 (Src-associated in mitosis of 68 kD), a KH domain RNA-binding protein, is not only important in signaling transduction cascades, but crucial in a variety of cellular processes. Sam68 is reported to be involved in the phospoinositide3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) signaling pathways, and it is closely associated with cell proliferation, RNA metabolism, and tumor progression. However, we know little about the role of Sam68 during peripheral nervous system injury and regeneration. In this study, we investigated the expression of Sam68 and its biological significances in sciatic nerve crush. Interestingly, we found Sam68 had a co-localization with S100 (Schwannmore » cell marker). Moreover, after crush, Sam68 had a spatiotemporal protein expression, which was in parallel with proliferation cell nuclear antigen (PCNA). In vitro, we also observed increased expression of Sam68 during the process of TNF-α-induced Schwann cell proliferation model. Besides, flow cytometry analyses, CCK-8, and EDU were all performed with the purpose of investigating the role of Sam68 in the regulation of Schwann cell proliferation. Even more importantly, we discovered that Sam68 could enhance the phosphorylation of Akt while LY294002 (a PI3K inhibitor) obviously reversed Sam68-induced cell proliferation. Finally, we detected the variance during regeneration progress through the rat walk footprint test. In summary, all these evidences demonstrated that Sam68 might participate in Schwann cell proliferation partially via PI3K/Akt pathway and also regulate regeneration after sciatic nerve crush. -- Highlights: •The dynamic changes and location of Sam68 after sciatic nerve crush. •Sam68 promoted Schwann cell proliferation via PI3K/Akt pathway. •Sam68 modulated functional recovery after sciatic nerve crush.« less

Lithium Causes G2 Arrest of Renal Principal Cells

PubMed Central

de Groot, Theun; Alsady, Mohammad; Jaklofsky, Marcel; Otte-Höller, Irene; Baumgarten, Ruben; Giles, Rachel H.

2014-01-01

Vasopressin-regulated expression and insertion of aquaporin-2 channels in the luminal membrane of renal principal cells is essential for urine concentration. Lithium affects urine concentrating ability, and approximately 20% of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder characterized by polyuria and polydipsia. Lithium-induced NDI is caused by aquaporin-2 downregulation and a reduced ratio of principal/intercalated cells, yet lithium induces principal cell proliferation. Here, we studied how lithium-induced principal cell proliferation can lead to a reduced ratio of principal/intercalated cells using two-dimensional and three-dimensional polarized cultures of mouse renal collecting duct cells and mice treated with clinically relevant lithium concentrations. DNA image cytometry and immunoblotting revealed that lithium initiated proliferation of mouse renal collecting duct cells but also increased the G2/S ratio, indicating G2/M phase arrest. In mice, treatment with lithium for 4, 7, 10, or 13 days led to features of NDI and an increase in the number of principal cells expressing PCNA in the papilla. Remarkably, 30%–40% of the PCNA-positive principal cells also expressed pHistone-H3, a late G2/M phase marker detected in approximately 20% of cells during undisturbed proliferation. Our data reveal that lithium treatment initiates proliferation of renal principal cells but that a significant percentage of these cells are arrested in the late G2 phase, which explains the reduced principal/intercalated cell ratio and may identify the molecular pathway underlying the development of lithium-induced renal fibrosis. PMID:24408872

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

PubMed

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the therapeutic use of IgG1 monoclonal antibodies.

Effect of DJ-1 overexpression on the proliferation, apoptosis, invasion and migration of laryngeal squamous cell carcinoma SNU-46 cells through PI3K/AKT/mTOR.

PubMed

Wang, Bin; Qin, Hao; Wang, Yuejian; Chen, Weixiong; Luo, Jie; Zhu, Xiaolin; Wen, Weiping; Lei, Wenbin

2014-09-01

The aim of the present study was to explore the effect of DJ-1-mediated PI3K/AKT/mTOR pathway on the proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous cell SNU-46, through stable transfection and overexpression of the DJ-1 gene. Retrovirus carrying DJ-1 gene was used to stabilize transfected human laryngeal squamous carcinoma SNU-46 cell line, and monoclonal cell line of stably overexpressed DJ-1 protein was screened out by G418. DJ-1 protein expression was determined by western blotting, and changes of p-AKT, p-mTOR and PTEN protein content were detected, followed by the detection of changes in proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous carcinoma cell line with stably transfected DJ-1 protein overexpression by flow cytometry, CCK-8 method and Transwell. We successfully constructed a laryngeal squamous carcinoma cell line of stably overexpressed DJ-1 protein and termed it SNU-46-DJ-1. After overexpression of DJ-1 protein, the levels of PTEN expression in laryngeal squamous cell SNU-46 decreased and p-AKT and p-mTOR protein expression levels increased. Compared to the untreated SNU-46 cells, the proliferation rate of SNU-46-DJ-1 cells increased (0.834±0.336 vs. 0.676±0.112; p<0.001); invasiveness was enhanced (165.7±13.6 vs. 100.0±17.4; p=0.001), the migration ability was enhanced (207.3±13.1 vs. 175.3±13.3; p=0.036), and the apoptosis rate decreased (3.533±5.167 vs. 16.397±5.447%; p=0.019). The overexpression of DJ-1 protein in laryngeal squamous carcinoma SNU-46 cells can accelerate proliferation rate, increase the invasion and migration capacity, and reduce apoptosis, by activating the PI3K/AKT/mTOR pathway.

FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

NASA Astrophysics Data System (ADS)

Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

2002-06-01

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

Overexpression of Lin28 inhibits the proliferation, migration and cell cycle progression and induces apoptosis of BGC-823 gastric cancer cells.

PubMed

Song, Hu; Xu, Wei; Song, Jun; Liang, Yong; Fu, Wei; Zhu, Xiao-Cheng; Li, Chao; Peng, Jun-Sheng; Zheng, Jun-Nian

2015-02-01

Lin28 plays important roles in the development, maintenance of pluripotency and progression of various types of cancers. Lin28 represses the biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Oncogenic regulation of let-7 microRNAs has been demonstrated in several human malignancies, yet their correlation with Lin28 has not yet been studied in gastric cancer. Therefore, in the present study, we explored the possible mechanisms involved in the effects by Lin28 on the proliferation, migration, cell cycle arrest and apoptosis in gastric cancer cells via alteration of let-7 miRNA. The expression levels of Lin28 and let-7 were detected by real-time PCR in gastric cancer cell lines in vitro. Lin28 was overexpressed in the BGC-823 cells via lentiviral transfection, and let-7 expression was assessed. Cell proliferation and migration capabilities were investigated by MTT and Transwell assays, while cell cycle distribution and the apoptosis rate were detected using flow cytometry. The expression of Lin28 was moderately expressed in the GES cells while underexpressed in the BGC-823, SGC-7901 and HGC-27 cells. Let-7a miRNA was highly expressed in the GES, BGC-823, SGC-7901 and HGC-27 cells. Overexpression of Lin28 was inversely correlated with the downregulated expression of let-7a, and markedly suppressed the proliferation, migration, cell cycle progression and induced apoptosis in the BGC-823 cells. These findings demonstrated that overexpression of Lin28 can suppress the biological behavior of gastric cancer in vitro, and let-7 miRNA may play an important role in the process. We suggest that Lin28 may be a candidate predictor or an anticancer therapeutic target for gastric cancer patients.

Effect of ionizing radiation on human skeletal muscle precursor cells

PubMed Central

Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

2013-01-01

Background Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Materials and methods Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Results Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Conclusions Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions. PMID:24294183

Low level light promotes the proliferation and differentiation of bone marrow derived mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Ahn, Jin-Chul; Rhee, Yun-Hee; Choi, Sun-Hyang; Kim, Dae Yu; Chung, Phil-Sang

2015-03-01

Low-level light irradiation (LLLI) reported to stimulate the proliferation or differentiation of a variety of cell types. However, very little is known about the effect of light therapy on stem cells. The aim of the present study was to evaluate the effect of LLLI on the molecular physiological change of human bone marrow derived stem cells (hBMSC) by wavelength (470, 630, 660, 740 and 850, 50mW). The laser diode was performed with different time interval (0, 7.5, 15, 30J/cm2, 50mW) on hBMSC. To determine the molecular physiological changes of cellular level of hBMSC, the clonogenic assay, ATP assay, reactive oxygen species (ROS) detection, mitochondria membrane potential (MMPΦ) staining and calcium efflux assay were assessed after irradiation. There was a difference between with and without irradiation on hBMSCs. An energy density up to 30 J/cm² improved the cell proliferation in comparison to the control group. Among these irradiated group, 630 and 660nm were significantly increased the cell proliferation. The cellular level of ATP and calcium influx was increased with energy dose-dependent in all LLLI groups. Meanwhile, ROS and MMPΦ were also increased after irradiation except 470nm. It can be concluded that LLLI using infrared light and an energy density up to 30 J/cm² has a positive stimulatory effect on the proliferation or differentiation of hBMSCs. Our results suggest that LLLI may influence to the mitochondrial membrane potential activity through ATP synthesis and increased cell metabolism which leads to cell proliferation and differentiation.

A Rapid Survival Assay to Measure Drug-Induced Cytotoxicity and Cell Cycle Effects

PubMed Central

Valiathan, Chandni; McFaline, Jose L.

2012-01-01

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of BrdU. Cells that synthesize DNA in the presence of bromodeoxyuridine (BrdU) exhibit quenched Hoechst fluorescence easily detected by flow cytometry; quenching is used to determine relative proliferation in treated versus untreated cells. Finally, the multi-well setup of this assay allows the simultaneous screening of multiple cell lines, multiple doses, or multiple drugs to accurately measure cell survival and cell cycle changes after drug treatment. PMID:22133811

Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain.

PubMed

Kosi, Nina; Alić, Ivan; Kolačević, Matea; Vrsaljko, Nina; Jovanov Milošević, Nataša; Sobol, Margarita; Philimonenko, Anatoly; Hozák, Pavel; Gajović, Srećko; Pochet, Roland; Mitrečić, Dinko

2015-02-09

The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Effect of aromatase inhibitor letrozole on the proliferation of spermatogonia by regulating the MAPK pathway.

PubMed

Wang, Shunde; Wang, Shuhong; Li, Hang; Li, Xiaoxia; Xie, Menglin; Wen, Jiayu; Li, Meicai; Long, Tengbo

2018-06-01

The molecular mechanism of the aromatase inhibitor letrozole was investigated. It promotes the proliferation of spermatogonia by regulating the mitogen-activated protein kinase (MAPK) pathway. Six different concentrations were selected for letrozole in order to incubate mouse spermatogonia [GC-1 spermatogonia (spg)] for 24, 48 and 72 h, respectively. Cell Counting Kit-8 (CCK-8) was used to observe the effect of letrozole on the proliferation of GC-1 spg cells, and the effect was further verified by cell plate clone formation assay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the effects of letrozole on MAPK signaling pathways [Ras/extracellular signal-regulated kinase 1 (ERK1)/c-Myc], proliferation indexes [Ki-67 and proliferating cell nuclear antigen (PCNA)]. Bromodeoxyuridine (BrdU) staining was used to study the effects of letrozole and MAPK signaling pathways on cell proliferation. The results of CCK-8 showed that the proliferation rate of GC-1 spg cells was improved. Study results also revealed a significant increase in letrozole concentration along with the time of action. The results of plate clone formation assay further indicated that letrozole could significantly promote the proliferation capacity of GC-1 spg cells (p<0.05). The results of RT-PCR and western blot analysis confirmed letrozole significantly activated the expression of Ras/ERK1/c-Myc in the classical MAPK pathway. A significant increase was noted in the protein levels of Ki-67 and PCNA (p<0.05). By contrast, inhibition of the MAPK pathway resulted in a significant decrease in the levels of the above indexes (p<0.05). The number of BrdU cells in the letrozole group was also higher than that of the control group, while the number of BrdU-stained cells in the letrozole + MAPK inhibition group showed a significant decrease in comparison to the letrozole group. In conclusion, letrozole activated the MAPK signaling pathway and promoted the proliferation of mouse spermatogonia GC-1 spg cells. The present study provides a theoretical basis for the clinical application of letrozole.

In vitro analysis of low-level laser irradiation on human osteoblast-like cells proliferation

NASA Astrophysics Data System (ADS)

Bloise, Nora; Saino, Enrica; Bragheri, Francesca; Minzioni, Paolo; Cristiani, Ilaria; Imbriani, Marcello; Visai, Livia

2011-07-01

The objective of this study was to examine the in vitro effect of a single or a multiple doses of low-level laser irradiation (LLLI) on proliferation of the human osteosarcoma cell line, SAOS-2. SAOS-2 cells were divided in five groups and exposed to LLLI (659 nm diode laser; 11 mW power output): group I as a control (dark), group II exposed to a single laser dose of 1 J/cm2, group III irradiated with a single dose of 3 J/cm2, and group IV and V exposed for three consecutive days to 1 or 3 J/cm², respectively. Cellular proliferation was assessed daily up to 7 days of culturing. The obtained results showed an increase in proliferative capacity of SAOS-2 cells during the first 96 h of culturing time in once-irradiated cells, as compared to control cells. Furthermore, a significantly higher proliferation in the group IV and V was detected if compared to a single dose or to control group after 96 h and 7 days. In conclusion, the effect of the single dose on cell proliferation was transitory and repeated irradiations were necessary to observe a strong enhancement of SAOS-2 growth. As a future perspective, we would like to determine the potential of LLLI as a new approach for promoting bone regeneration onto biomaterials.

Bradykinin Promotes Cell Proliferation, Migration, Invasion, and Tumor Growth of Gastric Cancer Through ERK Signaling Pathway.

PubMed

Wang, Guojun; Sun, Junfeng; Liu, Guanghui; Fu, Yang; Zhang, Xiefu

2017-12-01

Bradykinin (BK) has been reported to be involved in the progression of diverse types of cancer. In the present study, we investigated the possible role of BK in cell proliferation, migration, invasion, and tumor growth of gastric cancer (GC). Cell proliferation was evaluated by MTT assays. Cell migration and invasion were assessed by Transwell assays. Tumor growth of nude mice was detected by establishing subcutaneous xenograft tumor model. Silencing of bradykinin B1 receptor (B1R) and the bradykinin B2 receptor (B2R) was performed by transfecting cells with si-B1R and si-B2R, respectively. The protein expression levels of phospho-ERK1/2 (p-ERK1/2), matrix metalloproteinase (MMP)-2, MMP-9, and E-Cadherin were examined by Western blot. Data revealed that BK promoted cell proliferation, migration, invasion, and the in vivo tumor growth of GC cells SGC-7901 and HGC-27. Furthermore, BK elevated the protein levels of p-ERK1/2, MMP-2, and MMP-9, but reduced E-Cadherin. In addition, by repressing B2R using si-B2R or inhibiting ERK signaling pathway using PD98059, BK-mediated promotion of cell proliferation, migration, and invasion and upregulation of p-ERK1/2, MMP-2/9, as well as downregulation of E-Cadherin were attenuated. Taken together, the present study demonstrated that BK promoted cell proliferation, migration, invasion, and tumor growth by binding to B2R via ERK signaling pathway. Our findings may provide promising options for the further treatment of GC. J. Cell. Biochem. 118: 4444-4453, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Activity of insulin growth factors and shrimp neurosecretory organ extracts on a lepidopteran cell line.

PubMed

Hatt, P J; Liebon, C; Morinière, M; Oberlander, H; Porcheron, P

1997-01-01

Ecdysteroids, or molting hormones, have been proven to be key differentiation regulators for epidermal cells in the postembryonic development of arthropods. Regulators of cell proliferation, however, remain largely unknown. To date, no diffusible insect peptidic growth factors have been characterized. Molecules structurally related to insulin have been discovered in insects, as in other eucaryotes. We developed in vitro tests for the preliminary characterization of potential growth factors in arthropods by adapting the procedures designed to detect such factors in vertebrates to an insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth. We verified the ability of these tests to measure the proliferation of IAL-PID2 cells. We tested mammalian insulin and insulin-like growth factors (IGF-I, IGF-II). Following an arrest of cell proliferation by serum deprivation, IGF-I and IGF-II caused partial resumption of the cell cycle, evidenced by DNA synthesis. In contrast, the addition of 20-hydroxyecdysone arrested the proliferation of the IAL-PID2 cells. The cell line was then used in a test for functional characterization of potential growth factors originating from the penaeid shrimp, Penaeus vannamei. Crude extracts of neurosecretory and nervous tissues, eyestalks, and ventral neural chain compensated for serum deprivation and stimulated completion of mitosis. Arch.

Inhibitory effects of blockage of intermediate conductance Ca(2+)-activated K (+) channels on proliferation of hepatocellular carcinoma cells.

PubMed

Yang, Xiao-wei; Liu, Jin-wen; Zhang, Ru-chao; Yin, Qian; Shen, Wen-zhuang; Yi, Ji-lin

2013-02-01

The roles of intermediate conductance Ca(2+)-activated K(+) channel (IKCa1) in the pathogenesis of hepatocellular carcinoma (HCC) were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCa1 protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCa1 mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCa1 in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCa1, was used to intervene with the function of IKCa1. As compared with para-carcinoma tissue, an over-expression of IKCa1 protein was detected in HCC tissue samples (P<0.05). The mRNA expression level of IKCa1 in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 (0.5, 1.0, 2.0 and 4.0 μmol/L) in vitro (P<0.05). Our results suggested that IKCa1 may play a role in the proliferation of human HCC, and IKCa1 blockers may represent a potential therapeutic strategy for HCC.

MiR-598: A tumor suppressor with biomarker significance in osteosarcoma.

PubMed

Liu, Kai; Sun, Xiaolu; Zhang, Yingang; Liu, Liang; Yuan, Qiling

2017-11-01

Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents. Identifying specific and sensitive biomarkers is beneficial to early detection and improvement of life qualities and overall survival rates of osteosarcoma patients. Realtime PCR was used to detect the expression of miR-598. CCK-8 assay was employed to detect the proliferation of osteosarcoma cells, while transwell assays were used to examine the migration and invasion. Tumor xenograft experiments were performed to test the in vivo malignancy of osteosarcoma cells. Co-culture experiment was used to study the relationship between osteosarcoma cells and osteoblast. Realtime PCR, Western Blotting and luciferase report assays were conducted for the target genes analysis. Using a cohort of 20 cases of osteosarcoma and paired adjacent tissue samples, we found that miR-598 expression was decreased in osteosarcoma tissues and serum, as well as the osteosarcoma cell lines. Over expression of miR-598 suppressed the proliferation, migration, and invasion of osteosarcoma cells, while inhibition of miR-598 expression stimulated the proliferation, migration, and invasion. However, MiR-598 had no effect on osteosarcoma cell apoptosis. Data from nude mice further demonstrated the inhibitory role of miR-598 in osteosarcoma progression in vivo. Mechanically, miR-598 played its role by modulating osteoblastic differentiation in the microenvironment and targeting PDGFB and MET. Our findings enrich the knowledge of miR-598 in osteosarcoma progression, and reveal miR-598 as a promising diagnostic, prognostic, therapeutic biomarker for osteosarcoma. Copyright © 2017 Elsevier Inc. All rights reserved.

Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating wnt/β-catenin signaling pathway.

PubMed

Sun, Huidong; Gao, Yanchao; Lu, Kemei; Zhao, Guimei; Li, Xuehua; Li, Zhu; Chang, Hong

2015-10-24

Klotho is a discovered aging suppressor gene, and its overexpression in mice extends the life span of the animal. Recently, Klotho is also identified as a tumor suppressor gene in variety of tumors; however, the potential role and the antitumor mechanism remain unclarified in liver cancers. RT-PCR and western blotting analysis were used to detect the expression of Klotho, β-catenin, C-myc, and Cyclin D1. MTT assay was used to detect the survival rates of HepG2 and SMMC-7721 cells. Colony formation assay was used to test the proliferation ability in Klotho transfected cells. FACS was used to detect the cell apoptosis rate in different groups. The results showed that lower expression of Klotho were found in liver cancer cell lines than the immortalized liver cell L02. Also, MTT assay results found that overexpression or recombinant Klotho administration suppressed the proliferation of liver cancer cells HepG2 and SMMC-7721. Moreover, the colony formation assay results showed that the number of colonies was significantly lower in the cells with transfection with pCMV-Klotho than the controls. Thus, functional analysis demonstrated that Klotho expression inhibited the proliferation of liver cancer cells and Klotho worked as an important antitumor gene in tumor progression. Next, the mechanism was partly clarified that Klotho expression induced cell apoptosis in HepG2 and SMMC-7721 cells, and this phenomenon was mainly involved in the Wnt/β-catenin signaling pathway. The western blotting analysis revealed that overexpression or recombinant administration of Klotho obviously decreased the expression levels of β-catenin, C-myc, and Cyclin D1 in HepG2 cells. Most importantly, the antitumor mechanism for Klotho due to that overexpression of Klotho not only decreased the endogenous β-catenin levels but also inhibited the nuclear translocation of β-catenin to delay the cell cycle progression. Klotho was a tumor suppressor gene, and overexpression of Klotho suppressed the proliferation of liver cancer cells partly due to negative regulation of Wnt/β-catenin signaling pathway. So, Klotho might be used as a potential target, and the study will contribute to treatment for therapy of liver cancer patients.

Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Duanmin; Su, Cunjin; Jiang, Min

There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs,more » siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.« less

Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

PubMed Central

Kiraly, Orsolya; Gong, Guanyu; Olipitz, Werner; Muthupalani, Sureshkumar; Engelward, Bevin P.

2015-01-01

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence. PMID:25647331

Up-regulation of neogenin-1 increases cell proliferation and motility in gastric cancer

PubMed Central

Kim, Seok-Jun; Wang, Yuan-Guo; Lee, Hyun-Woo; Gu Kang, Hyeok; La, Sun-Hyuk; Ju Choi, Il; Irimura, Tatsuro; Ro, Jae Y.; Bresalier, Robert S.; Chun, Kyung-Hee

2014-01-01

Although elevated expression of neogenin-1 has been detected in human gastric cancer tissue, its role in gastric tumorigenesis remains unclear due to the lack of neogenin-1 studies in cancer. Therefore, we demonstrated here the function and regulatory mechanism of neogenin-1 in gastric cancer. Neogenin-1 ablation decreased proliferation and migration of gastric cancer cells, whereas its over-expression reversed these effects. Xenografted analyses using gastric cancer cells displayed statistically significant inhibition of tumor growth by neogenin-1 depletion. Interestingly, galectin-3 interacted with HSF-1 directly, which facilitated nuclear-localization and binding on neogenin-1 promoter to drive its transcription and gastric cancer cell motility. The galectin-3-increased gastric cancer cell motility was down-regulated by HSF-1 depletion. Moreover, the parallel expression patterns of galectin-3 and neogenin-1, as well as those of HSF-1 and neogenin-1, were detected in the malignant tissues of gastric cancer patients. Taken together, high-expression of neogenin-1 promotes gastric cancer proliferation and motility and its expression is regulated by HSF-1 and galectin-3 interaction. In addition, we propose further studies for neogenin-1 and its associated pathways to provide them as a proper target for gastric cancer therapy. PMID:24930499

Evidence that thyroid hormone induces olfactory cellular proliferation in salmon during a sensitive period for imprinting.

PubMed

Lema, Sean C; Nevitt, Gabrielle A

2004-09-01

Salmon have long been known to imprint and home to natal stream odors, yet the mechanisms driving olfactory imprinting remain obscure. The timing of imprinting is associated with elevations in plasma thyroid hormone levels, with possible effects on growth and proliferation of the peripheral olfactory system. Here, we begin to test this idea by determining whether experimentally elevated plasma levels of 3,5,3'-triiodothyronine (T(3)) influence cell proliferation as detected by the 5-bromo-2'-deoxyuridine (BrdU) cell birth-dating technique in the olfactory epithelium of juvenile coho salmon (Oncorhynchus kisutch). We also explore how natural fluctuations in thyroxine (T(4)) relate to proliferation in the epithelium during the parr-smolt transformation. In both studies, we found that BrdU labeled both single and clusters of mitotic cells. The total number of BrdU-labeled cells in the olfactory epithelium was significantly greater in fish with artificially elevated T(3) compared with placebo controls. This difference in proliferation was restricted to the basal region of the olfactory epithelium, where multipotent progenitor cells differentiate into olfactory receptor neurons. The distributions of mitotic cluster sizes differed significantly from a Poisson distribution for both T(3) and placebo treatments, suggesting that proliferation tends to be non-random. Over the course of the parr-smolt transformation, changes in the density of BrdU cells showed a positive relationship with natural fluctuations in plasma T(4). This relationship suggests that even small changes in thyroid activity can stimulate the proliferation of neural progenitor cells in the salmon epithelium. Taken together, our results establish a link between the thyroid hormone axis and measurable anatomical changes in the peripheral olfactory system.

Effect of miRNA-203 on cervical cancer cells and its underlying mechanism.

PubMed

Yin, X Z; Zhao, D M; Zhang, G X; Liu, L

2016-09-23

miRNA-203 is involved in the development and progression of various types of cancer. However, its role in cervical cancer remains unclear. The aim of this study was to investigate the effect of miRNA-203 on the proliferation and migration of HeLa cervical cancer cells, as well as survivin expression in these cells. A miRNA-203 primer probe was designed according to a sequence obtained from NCBI. The expression of miRNA-203 in cervical epithelial cells and cervical cancer cells was detected by quantitative reverse transcriptase-polymerase chain reaction. The miRNA-203 expression pattern was compared between these two cell lines. The cervical cancer cells were transfected with miRNA-203 mimic or inhibitor to determine their effects on proliferation and migration. The expression of the miRNA-203 target protein (survivin) was analyzed by western blot. Cervical cancer cells showed reduced miRNA-203 expression compared to cervical epithelial cells. Transfection of miRNA-203 mimic upregulated the expression of miRNA-203, suppressed cell proliferation and migration, and downregulated survivin expression (P < 0.05). However, downregulation of miRNA-203 expression did not affect proliferation, migration, and survivin expression in cervical cancer cells (P > 0.05). In conclusion, upregulation of miRNA-203 in cervical cancer cells inhibits the proliferative and migratory capacities of these cells by downregulating the expression of survivin.

Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.

PubMed

Bullwinkel, Jörn; Baron-Lühr, Bettina; Lüdemann, Anja; Wohlenberg, Claudia; Gerdes, Johannes; Scholzen, Thomas

2006-03-01

The nuclear Ki-67 protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis. Copyright 2005 Wiley-Liss, Inc.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products.

PubMed

Xu, Yu-Dong; Wang, Yu; Yin, Lei-Miao; Peng, Ling-Ling; Park, Gyoung-Hee; Yang, Yong-Qing

2017-06-21

Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 μg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

[Cytogenetic characteristics of the uterine cervical epithelium in inflammatory diseases].

PubMed

Aleksieienko, O I

2011-01-01

Functional status of epithelial cells at inflammatory cervical pathologies has been studied with the use of cytogenetic method of detection of chromosome nucleolar organizer regions. The highest level of rRNA proliferation and synthesis has been detected in cylindrical epithelial cells using the indexes of compact and transitional nucleolonemic types of nucleolar organizer regions, a higher level--in squamous cells of intermediate type, and the lowest one in squamous epithelium of superficial type.

Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138

PubMed Central

Yu, Dazhi; An, Fengmei; He, Xu; Cao, Xuecheng

2015-01-01

Objective: In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism. Methods: Affemitrix miRNA chip was used to detect the changes of miRNA expression profile in MG-63 cells before and after curcumin treatment, and screen different expression of miRNAs. The target gene of miRNA was analyzed by bioinformatics. The expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 were detected. MTT and Transwell Cell invasion assays were used to observe the effects of curcumin on MG-63 cells. Results: Curcumin could significantly inhibit the proliferation of MG-63 cells and the expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 in MG-63 cells (P<0.05); overexpression of hsa-miR-138 down-regulated the expression levels of Smad4, NFκB p65 and cyclin D3 compared with the treatment of curcumin, while inhibition of hsa-miR-138 up-regulated the expression levels of Smad4, NFκB p65 and cyclin D3. Conclusions: Curcumin could increase the expression of hsa-miR-138, hsa-miR-138 inhibited cell proliferation and invasive ability by inhibition of its target genes. PMID:26823826

p53, Bcl-2 and cox-2 are involved in berberine hydrochloride-induced apoptosis of HeLa229 cells.

PubMed

Wang, Hai-Yan; Yu, Hai-Zhong; Huang, Sheng-Mou; Zheng, Yu-Lan

2016-10-01

The present study aimed to investigate the effects of berberine hydrochloride on the proliferation and apoptosis of HeLa229 human cervical cancer cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine the cytotoxicity of berberine hydrochloride against HeLa229 cells. The effects of berberine hydrochloride on the apoptosis of HeLa229 cells was detected by immunofluorescence and flow cytometry, and the mRNA expression levels of p53, B‑cell lymphoma 2 (Bcl‑2) and cyclooxygenase‑2 (cox‑2) were analyzed by reverse transcription-quantitative polymerase chain reaction. Berberine hydrochloride inhibited the proliferation of HeLa229 cells in a dose‑dependent manner; minimum cell viability (3.61%) was detected following treatment with 215.164 µmol/l berberine hydrochloride and the half maximal inhibitory concentration value was 42.93 µmol/l following treatment for 72 h. In addition, berberine hydrochloride induced apoptosis in HeLa229 cells in a dose‑ and time‑dependent manner. Berberine hydrochloride upregulated the mRNA expression levels of p53, and downregulated mRNA expression levels of Bcl‑2 and cox‑2, in a dose‑dependent manner. In conclusion, berberine hydrochloride inhibited the proliferation and induced apoptosis of HeLa229 cells, potentially via the upregulation of p53 and the downregulation of Bcl‑2 and cox‑2 mRNA expression levels.

Stem cells distribution, cellular proliferation and migration in the adult Austrolebias charrua brain.

PubMed

Torres-Pérez, Maximiliano; Rosillo, Juan Carlos; Berrosteguieta, Ines; Olivera-Bravo, Silvia; Casanova, Gabriela; García-Verdugo, José Manuel; Fernández, Anabel Sonia

2017-10-15

Our previous studies demonstrated that Austrolebias charrua annual fish is an excellent model to study adult brain cell proliferation and neurogenesis due to the presence of active and fast neurogenesis in several regions during its short lifespan. Our main goal was to identify and localize the cells that compose the neurogenic areas throughout the Austrolebias brain. To do this, we used two thymidine halogenated analogs to detect cell proliferation at different survival times: 5-chloro-2'-deoxyuridine (CldU) at 1day and 5-iodo-2'-deoxyuridine (IdU) at 30days. Three types of proliferating cells were identified: I - transient amplifying or fast cycling cells that uptake CldU; II - stem cells or slow cycling cells, that were labeled with both CldU and IdU and did not migrate; and III - migrant cells that uptake IdU. Mapping and 3D-reconstruction of labeled nuclei showed that type I and type II cells were preferentially found close to ventricle walls. Type III cells appeared widespread and migrating in tangential and radial routes. Use of proliferation markers together with Vimentin or Nestin evidenced that type II cells are the putative stem cells that are located at the ventricular lumen. Double label cells with IdU+ and NeuN or HuC/D allowed us identify migrant neurons. Quantitation of labeled nuclei indicates that the proportion of putative stem cells is around 10% in all regions of the brain. This percentage of stem cells suggests the existence of a constant brain cell population in Austrolebias charrua that seems functional to the maintainance of adult neurogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Akt-mediated phosphorylation of Oct4 is associated with the proliferation of stem-like cancer cells

PubMed Central

ZHAO, QING-WEI; ZHOU, YAN-WEN; LI, WEN-XIN; KANG, BO; ZHANG, XIAO-QIAN; YANG, YING; CHENG, JIE; YIN, SHENG-YONG; TONG, YING; HE, JIAN-QIN; YAO, HANG-PING; ZHENG, MIN; WANG, YING-JIE

2015-01-01

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self-renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti-Oct4A monoclonal antibody. Moreover, an anti-Oct4-pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti-Oct4A bands which was decreasable by a specific shRNA. The Oct4-pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem-like cancer cells. The levels of Oct4-pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K-Akt pathway. Akti-1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4-pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt-mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt-mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem-like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer. PMID:25625591

High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans.

PubMed

Suzuki, Shugo; Takeshita, Kentaro; Asamoto, Makoto; Takahashi, Satoru; Kandori, Hitoshi; Tsujimura, Kazunari; Saito, Fumiyo; Masuko, Kazuo; Shirai, Tomoyuki

2009-01-31

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.

Ferulic acid inhibits proliferation and promotes apoptosis via blockage of PI3K/Akt pathway in osteosarcoma cell.

PubMed

Wang, Ting; Gong, Xia; Jiang, Rong; Li, Hongzhong; Du, Weimin; Kuang, Ge

2016-01-01

Ferulic acid, a ubiquitous phenolic acid abundant in corn, wheat and flax, has potent anti-tumor effect in various cancer cell lines. However, the anti-tumor effect of ferulic acid on osteosarcoma remains unclear. Therefore, we conduct current study to examine the effect of ferulic acid on osteosarcoma cells and explore the underlying mechanisms. In present study, ferulic acid inhibited proliferation and induced apoptosis in both 143B and MG63 osteosarcoma cells dose-dependently, indicated by MTT assay and Annexin V-FITC apoptosis detection. Additionally, ferulic acid induced G0/G1 phase arrest and down-regulated the expression of cell cycle-related protein, CDK 2, CDK 4, CDK 6, confirmed by flow cytometry assay and western blotting. Moreover, ferulic acid upregulated Bax, downregulated Bcl-2, and subsequently enhanced caspase-3 activity. More importantly, ferulic acid dose-dependently inhibited PI3K/Akt activation. Using adenoviruses expressing active Akt, the anti-proliferation and pro-apoptosis of ferulic acid were reverted. Our results demonstrated that ferulic acid might inhibit proliferation and induce apoptosis via inhibiting PI3K/Akt pathway in osteosarcoma cells. Ferulic acid is a novel therapeutic agent for osteosarcoma.

Ferulic acid inhibits proliferation and promotes apoptosis via blockage of PI3K/Akt pathway in osteosarcoma cell

PubMed Central

Wang, Ting; Gong, Xia; Jiang, Rong; Li, Hongzhong; Du, Weimin; Kuang, Ge

2016-01-01

Ferulic acid, a ubiquitous phenolic acid abundant in corn, wheat and flax, has potent anti-tumor effect in various cancer cell lines. However, the anti-tumor effect of ferulic acid on osteosarcoma remains unclear. Therefore, we conduct current study to examine the effect of ferulic acid on osteosarcoma cells and explore the underlying mechanisms. In present study, ferulic acid inhibited proliferation and induced apoptosis in both 143B and MG63 osteosarcoma cells dose-dependently, indicated by MTT assay and Annexin V-FITC apoptosis detection. Additionally, ferulic acid induced G0/G1 phase arrest and down-regulated the expression of cell cycle-related protein, CDK 2, CDK 4, CDK 6, confirmed by flow cytometry assay and western blotting. Moreover, ferulic acid upregulated Bax, downregulated Bcl-2, and subsequently enhanced caspase-3 activity. More importantly, ferulic acid dose-dependently inhibited PI3K/Akt activation. Using adenoviruses expressing active Akt, the anti-proliferation and pro-apoptosis of ferulic acid were reverted. Our results demonstrated that ferulic acid might inhibit proliferation and induce apoptosis via inhibiting PI3K/Akt pathway in osteosarcoma cells. Ferulic acid is a novel therapeutic agent for osteosarcoma. PMID:27158383

Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aube, Michel, E-mail: 4aubem@videotron.ca; Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca; Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca

2011-04-15

Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cellsmore » and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The proliferative effect of the mixture on CAMA-1 cells in the presence of sex steroids appears mostly due to the antiandrogenic properties of p,p'-DDE, a major constituent of the mixture. Other mixtures of contaminants that include emerging compounds of interest such as brominated flame retardants and perfluoroalkyl compounds should be tested for their capacity to induce breast cancer cell proliferation. - Research highlights: {yields} We studied effects of a complex organochlorine mixture on breast cancer cell growth. {yields} Weak xenoestrogens in the mixture stimulated the proliferation of MCF-7 cells. {yields} Antiandrogens increased the proliferation CAMA-1 cells grown with sex steroids. {yields} High concentrations of the mixture decreased the proliferation of all cell lines.« less

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

PubMed

Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

2014-02-01

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Formononetin, an isoflavone from Astragalus membranaceus inhibits proliferation and metastasis of ovarian cancer cells.

PubMed

Zhang, Jing; Liu, Likun; Wang, Jing; Ren, Baoyin; Zhang, Lin; Li, Weiling

2018-07-15

Astragalus membranaceus which was originally described in the Shennong's Classic of Materia Medica, the earliest complete Pharmacopoeia of China written from the Warring States Period to Han Dynasty, has been widely used in Chinese medicine for > 2000 years, especially in the prescription of curing cancer. A. membranaceus has various bioactivities, such as anti-tumor, anti-viral, anti-oxidant, anti-diabetes, anti-inflammation, anti-atherosclerosis, immunomodulation, hepatoprotection, hematopoiesis, neuroprotection and so on. As an important component of A. membranaceus, whether formononetin has a close relationship with its tumor-inhibiting effect on ovarian cancer cell has been investigated. The present study aimed to demonstrate the anti-proliferation, anti- migration and invasion effects of formononetin on ovarian cancer cells and further explore the underlying molecular mechanisms associated with apoptosis, migration and invasion. MTT assay was performed to detect the viability of ovarian cancer cells. DAPI staining, Annexin-V assay and assay for mitochondrial membrane potential detected the apoptosis of ovarian cancer cells treated by formononetin. The migration and invasion of ovarian cancer cells which exposed to formononetin were detected by scratch assay and transwell assay. Meanwhile, the protein-level changes of in ovarian cancer cells treated by formononetin were assessed by western blot analysis. MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with formononetin. DAPI staining, Annexin-V assay and assay for mitochondrial membrane potential suggested that formononetin suppressed cells proliferation by inducing apoptosis. We detected the expression of apoptosis-related proteins in ovarian cancer cells after treatment with formononetin and found the expression of caspase 3/9 proteins and the ratio of Bax/Bcl-2 were increased in a dose-dependent manner. In addition, wound healing and transwell chamber assays showed that formononetin suppressed the migration and invasion of ovarian cancer cells. And formononetin decreased expression of MMP-2/9 proteins and phosphorylation level of ERK. The present results demonstrated that formononetin have potential effects on induction of apoptosis and suppression of migration and invasion. Copyright © 2018 Elsevier B.V. All rights reserved.

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Lin; Department of Oncology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710038; Lu, Yuanyuan

2013-10-18

Highlights: •Overexpression of Ran in pancreatic cancer was correlated with histological grade. •Downregulation of Ran could induce cell apoptosis and inhibit cell proliferation. •The effects were mediated by cell cycle proteins, Survivin and cleaved Caspase-3. -- Abstract: Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and inductionmore » of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3.« less

Effect of TRPV2 cation channels on the proliferation, migration and invasion of 5637 bladder cancer cells.

PubMed

Liu, Quanliang; Wang, Xinghuan

2013-11-01

Transient receptor potential vanilloid 2 (TRPV2), a nonselective cation channel, has become an attractive target gene for tumor studies due to its wide range of physiological and pathological functions. However, its specific role in bladder cancer development and progression remains unclear. The aim of the present study was to investigate the effects of TRPV2 on the proliferation, migration and invasion of 5637 bladder cancer cells in vitro . Rat TRPV2 cDNA was transfected into 5637 bladder cancer cells and changes in the behavior of the cells were detected. It was observed that TRPV2 enhanced bladder cancer cell migration and invasion; however, it did not affect cell proliferation in vitro . TRPV2 activity, which may be mediated by direct matrix metalloproteinase 2 (MMP2) regulation, is important in bladder tumor development and progression. The results of this study suggest that TRPV2 channels are a potential therapeutic target for bladder carcinoma.

Effect of TRPV2 cation channels on the proliferation, migration and invasion of 5637 bladder cancer cells

PubMed Central

LIU, QUANLIANG; WANG, XINGHUAN

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2), a nonselective cation channel, has become an attractive target gene for tumor studies due to its wide range of physiological and pathological functions. However, its specific role in bladder cancer development and progression remains unclear. The aim of the present study was to investigate the effects of TRPV2 on the proliferation, migration and invasion of 5637 bladder cancer cells in vitro. Rat TRPV2 cDNA was transfected into 5637 bladder cancer cells and changes in the behavior of the cells were detected. It was observed that TRPV2 enhanced bladder cancer cell migration and invasion; however, it did not affect cell proliferation in vitro. TRPV2 activity, which may be mediated by direct matrix metalloproteinase 2 (MMP2) regulation, is important in bladder tumor development and progression. The results of this study suggest that TRPV2 channels are a potential therapeutic target for bladder carcinoma. PMID:24223658

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection.

PubMed

Lokensgard, James R; Mutnal, Manohar B; Prasad, Sujata; Sheng, Wen; Hu, Shuxian

2016-05-20

Chemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19(+) B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection. Flow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation. The chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19(+) cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact. CXCR3 is the primary chemokine receptor on CD19(+) B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

Proliferation zones in the axolotl brain and regeneration of the telencephalon

PubMed Central

2013-01-01

Background Although the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue. Results There is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over. Conclusion There is a continual generation of neuronal cells from neural progenitor cells located within the ventricular zone of the axolotl brain. Variable rates of proliferation were detected across brain regions. These neural progenitor cells appear to mediate telencephalic tissue regeneration through an injury-induced olfactory cue. Identification of this cue is our future goal. PMID:23327114

Proliferation zones in the axolotl brain and regeneration of the telencephalon.

PubMed

Maden, Malcolm; Manwell, Laurie A; Ormerod, Brandi K

2013-01-17

Although the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue. There is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over. There is a continual generation of neuronal cells from neural progenitor cells located within the ventricular zone of the axolotl brain. Variable rates of proliferation were detected across brain regions. These neural progenitor cells appear to mediate telencephalic tissue regeneration through an injury-induced olfactory cue. Identification of this cue is our future goal.

Ethanol exacerbates T cell dysfunction after thermal injury.

PubMed

Choudhry, M A; Messingham, K A; Namak, S; Colantoni, A; Fontanilla, C V; Duffner, L A; Sayeed, M M; Kovacs, E J

2000-07-01

To understand the mechanism of suppressed immunity following alcohol consumption and thermal injury, we analyzed T cell functions in a mouse model of acute alcohol exposure and burn injury. Mice with blood alcohol levels at approximately 100 mg/dl were given a 15% scald or sham injury. Mice were sacrificed 48 h after injury. Our data demonstrated a 20-25% decrease in Con A-mediated splenic T cell proliferation (p<0.01) and 45-50% decrease in interleukin-2 (IL-2) production (p<0.01) following burn injury compared to the T cells from sham animals. A further decrease in the proliferation (25-30%) and IL-2 production (40-45%) was detected in T cells derived from burned animals receiving alcohol as compared to burn alone. No significant change in the proliferation and IL-2 production was observed in splenic T cells derived from sham-injured mice regardless of alcohol exposure. Additionally, there was no demonstrable difference in splenocyte apoptosis in any treatment group. These results suggest that alcohol consumption prior to burn injury causes a greater decrease in T cell proliferation and IL-2 production compared to either burn or alcohol injury alone that may further attenuate the cell-mediated immunity and thus enhance susceptibility to infection.

[Culture of pancreatic progenitor cells in hanging drop and on floating filter].

PubMed

Ma, Feng-xia; Chen, Fang; Chi, Ying; Yang, Shao-guang; Lu, Shi-hong; Han, Zhong-chao

2013-06-01

To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method. Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR). One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose. In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.

Proliferation of Prostate Stromal Cell Induced by Benign Prostatic Hyperplasia Epithelial Cell Stimulated With Trichomonas vaginalis via Crosstalk With Mast Cell.

PubMed

Kim, Jung-Hyun; Kim, Sang-Su; Han, Ik-Hwan; Sim, Seobo; Ahn, Myoung-Hee; Ryu, Jae-Sook

2016-11-01

Chronic inflammation has a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. Mast cells have been detected in chronic inflammatory infiltrate of the prostate, and it is possible that the interaction between prostate epithelial cells and Trichomonas vaginalis influences the activity of mast cells in the prostate stroma. Activated mast cells might influence the biological functions of nearby tissues and cells. In this study, we investigated whether mast cells reacted with the culture supernatant of BPH epithelial cells infected with T. vaginalis may induce the proliferation of prostate stromal cells. To measure the proliferation of prostate stromal cells in response to chronic inflammation caused by the infection of BPH-1 cells with T. vaginalis, the CCK-8 assay and wound healing assay were used. ELISAs, quantitative real-time PCR, western blotting and immunofluorescence were used to measure the production and expression of inflammatory cytokine and cytokine receptor. BPH-1 cells incubated with live trichomonads produced increased levels of CCL2, IL-1β, IL-6, and CXCL8, and induced the migration of mast cells and monocytes. When the culture supernatant of BPH-1 cells stimulated with trichomonads (TCM) was added to mast cells, they became activated, as confirmed by release of β-hexosaminidase and CXCL8. Prostate stromal cells incubated with the culture supernatant of mast cells activated with TCM (M-TCM) proliferated and expressed increased levels of CXCL8, CCL2, and the cytokine receptors CXCR1 and CCR2. Blocking the chemokine receptors reduced the proliferation of stromal cells and also decreased the production of CXCL8 and CCL2. Moreover, the expression of FGF2, cyclin D1, and Bcl-2 was increased in the proliferated stromal cells stimulated with M-TCM. Additionally, the M-TCM-treated stromal cells were more invasive than control cells. The inflammatory mediators released by BPH epithelial cells in response to infection by trichomonads induce the migration and activation of mast cells. The activated mast cells induce the proliferation of prostate stromal cells via CXCL8-CXCR1 and CCL2-CCR2 signaling. Our results therefore show that the inflammatory response by BPH epithelial cells stimulated with T. vaginalis induce the proliferation of prostate stromal cells via crosstalk with mast cells. Prostate 76:1431-1444, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Apatinib promotes apoptosis of the SMMC-7721 hepatocellular carcinoma cell line via the PI3K/Akt pathway.

PubMed

Zhang, Hua; Cao, Yumei; Chen, Yuru; Li, Guangxi; Yu, Hanshu

2018-04-01

The present study investigated the inhibitory effects of apatinib on the proliferation of the SMMC-7721 hepatocellular carcinoma cell line to explore the possible mechanism. The MTT assay was used to detect the inhibitory effects of the different concentrations of apatinib on the proliferation of SMMC-7721 cells. Annexin V/PI double staining was performed to investigate the effects of apatinib on the apoptosis of SMMC-7721 cells. Expression of the apoptosis-related genes Bcl-2, Bax and caspase-9 after apatinib treatment was detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Expression of the PI3K, p-PI3K, Akt and p-Akt proteins after apatinib treatment was detected using western blot analysis. The MTT results showed that apatinib inhibited the in vitro proliferation of SMMC-7721 cells. Annexin V/PI double staining showed that apatinib induced the apoptosis of SMMC-7721 cells in a concentration-dependent manner. Results of RT-qPCR and western blot analysis showed that apatinib was able to induce the expression of pro-apoptotic genes Bax and caspase-9 and inhibited the expression of anti-apoptotic gene Bcl-2 . In addition, the western blot analysis revealed that p-PI3K and p-Akt was significantly decreased following apatinib treatment, while no significant differences were found in the total protein levels of PI3K and Akt. The results of the present show that apatinib is capable of promoting the apoptosis of SMMC-7721 cells by inhibiting the PI3K/Akt signal transduction pathway, upregulating the expression of pro-apoptotic genes Bax and caspase-9 , and downregulating the expression level of the anti-apoptotic gene Bcl-2 .

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

USGS Publications Warehouse

Work, Thierry M.; Balazs, George H.; Rameyer, Robert; Chang, S.P.; Berestecky, J.

2000-01-01

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund’s complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Bloch, S. R.; Fiacco, G. J.; Partridge, N. C.

1999-01-01

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast. Copyright 1999 Wiley-Liss, Inc.

PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling.

PubMed

Chen, Xiaoguang; Lv, Qiongxia; Ma, Jun; Liu, Yumei

2018-02-11

The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes. A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT-PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase-3, -8 and -9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor. Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p-p38 and p-JNK MAPKs significantly decreased, and PLCγ2-induced cell proliferation inhibition and cell apoptosis were obviously reversed. This study showed that PLCγ2 regulates hepatocyte growth through PKCD-dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo. © 2018 John Wiley & Sons Ltd.

Long non-coding RNA OIP5-AS1 promotes proliferation of lung cancer cells and leads to poor prognosis by targeting miR-378a-3p.

PubMed

Wang, Maolong; Sun, Xiao; Yang, Yuling; Jiao, Wenjie

2018-06-13

The antisense of the OIP5-AS1 gene is a long non-coding RNA (lncRNA) that is reported to be upregulated and promotes cell proliferation in multiple human cancers; however, its function in lung cancer is unknown. We investigated the regulatory function and underlying mechanisms of OIP5-AS1 in lung cancer. OIP5-AS1 and microRNA (miR)-378a-3p expression were assayed by quantitative real-time PCR, and proliferation-related protein expression was measured by Western blotting. Cell viability was detected using methyl thiazolyl tetrazolium assay. Luciferase reporter assay and RNA immunoprecipitation were used to detect the direct regulation of miR-378a-3p by OIP5-AS1. Nude mice were used to test the function of OIP5-AS1 in vivo. OIP5-AS1 was highly expressed in lung cancer tissues and was correlated with tumor size and tumor growth speed. OIP5-AS1 overexpression increased lung cancer cell proliferation in vitro. Further investigation revealed that OIP5-AS1 functions as a competing endogenous RNA of miR-378a-3p. MiR-378a-3p overexpression inhibited cell proliferation and caused proliferation-associated proteins CDK4 and CDK6 to decrease in A549 cells. Overexpression of wild type OIP5-AS1 led to strong CDK4 and CDK6 expression; however, these two proteins did not change when mutated OIP5-AS1 was upregulated. Finally, in vivo assay showed that the speed of tumor growth was increased and decreased when OIP5-AS1 was upregulated and downregulated, respectively. Our results revealed that OIP5-AS1 acts as a growth-promoting lncRNA in lung cancer by suppressing miR-378a-3p function. OIP5-AS1 and miR-378a-3p interaction may provide a potential target for lung cancer treatment. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zitta, Karina; Brandt, Berenice; Wuensch, Annegret

Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model ofmore » primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1{beta} as a key molecule guiding tissue remodelling events after myocardial infarction.« less

Dual‑sensitive HRE/Egr1 promoter regulates Smac overexpression and enhances radiation‑induced A549 human lung adenocarcinoma cell death under hypoxia.

PubMed

Li, Chang-Feng; Chen, Li-Bo; Li, Dan-Dan; Yang, Lei; Zhang, Bao-Gang; Jin, Jing-Peng; Zhang, Ying; Zhang, Bin

2014-08-01

The aim of this study was to construct an expression vector carrying the hypoxia/radiation dual‑sensitive chimeric hypoxia response element (HRE)/early growth response 1 (Egr‑1) promoter in order to overexpress the therapeutic second mitochondria‑derived activator of caspases (Smac). Using this expression vector, the present study aimed to explore the molecular mechanism underlying radiotherapy‑induced A549 human lung adenocarcinoma cell death and apoptosis under hypoxia. The plasmids, pcDNA3.1‑Egr1‑Smac (pE‑Smac) and pcDNA3.1‑HRE/Egr-1‑Smac (pH/E‑Smac), were constructed and transfected into A549 human lung adenocarcinoma cells using the liposome method. CoCl2 was used to chemically simulate hypoxia, followed by the administration of 2 Gy X‑ray irradiation. An MTT assay was performed to detect cell proliferation and an Annexin V‑fluorescein isothiocyanate apoptosis detection kit was used to detect apoptosis. Quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression, respectively. Infection with the pE‑Smac and pH/E‑Smac plasmids in combination with radiation and/or hypoxia was observed to enhance the expression of Smac. Furthermore, Smac overexpression was found to enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis. The cytochrome c/caspase‑9/caspase‑3 pathway was identified to be involved in this regulation of apoptosis. Plasmid infection in combination with X‑ray irradiation was found to markedly induce cell death under hypoxia. In conclusion, the hypoxia/radiation dual‑sensitive chimeric HRE/Egr‑1 promoter was observed to enhance the expression of the therapeutic Smac, as well as enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis under hypoxia. This apoptosis was found to involve the mitochondrial pathway.

[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].

PubMed

Yang, Li-Juan; Liu, Yu-Qin; Gu, Bei; Bian, Xiao-Cui; Feng, Hai-Liang; Yang, Zhen-Li; Liu, Yan-Yan

2010-12-01

To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma. E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat. E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma. Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.

Ferritin expression in rat hepatocytes and Kupffer cells after lead nitrate treatment.

PubMed

Fan, Yang; Yamada, Toshiyuki; Shimizu, Takeshi; Nanashima, Naoki; Akita, Miki; Suto, Kohji; Tsuchida, Shigeki

2009-02-01

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation and leukocyte recruitment in vivo.

PubMed

Pagel, Judith-Irina; Ziegelhoeffer, Tibor; Heil, Matthias; Fischer, Silvia; Fernández, Borja; Schaper, Wolfgang; Preissner, Klaus T; Deindl, Elisabeth

2012-03-01

Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PubMed

Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

2016-08-01

To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

An Advanced Deep Learning Approach for Ki-67 Stained Hotspot Detection and Proliferation Rate Scoring for Prognostic Evaluation of Breast Cancer.

PubMed

Saha, Monjoy; Chakraborty, Chandan; Arun, Indu; Ahmed, Rosina; Chatterjee, Sanjoy

2017-06-12

Being a non-histone protein, Ki-67 is one of the essential biomarkers for the immunohistochemical assessment of proliferation rate in breast cancer screening and grading. The Ki-67 signature is always sensitive to radiotherapy and chemotherapy. Due to random morphological, color and intensity variations of cell nuclei (immunopositive and immunonegative), manual/subjective assessment of Ki-67 scoring is error-prone and time-consuming. Hence, several machine learning approaches have been reported; nevertheless, none of them had worked on deep learning based hotspots detection and proliferation scoring. In this article, we suggest an advanced deep learning model for computerized recognition of candidate hotspots and subsequent proliferation rate scoring by quantifying Ki-67 appearance in breast cancer immunohistochemical images. Unlike existing Ki-67 scoring techniques, our methodology uses Gamma mixture model (GMM) with Expectation-Maximization for seed point detection and patch selection and deep learning, comprises with decision layer, for hotspots detection and proliferation scoring. Experimental results provide 93% precision, 0.88% recall and 0.91% F-score value. The model performance has also been compared with the pathologists' manual annotations and recently published articles. In future, the proposed deep learning framework will be highly reliable and beneficial to the junior and senior pathologists for fast and efficient Ki-67 scoring.

Chlorogenic acid induces apoptosis to inhibit inflammatory proliferation of IL-6-induced fibroblast-like synoviocytes through modulating the activation of JAK/STAT and NF-κB signaling pathways

PubMed Central

LOU, LIXIA; ZHOU, JINGWEI; LIU, YUJUN; WEI, YI; ZHAO, JIULI; DENG, JIAGANG; DONG, BIN; ZHU, LINGQUN; WU, AIMING; YANG, YINGXI; CHAI, LIMIN

2016-01-01

Chlorogenic acid (CGA) is the primary constituent of Caulis Lonicerae, a Chinese herb used for the treatment of rheumatoid arthritis (RA). The present study aimed to investigate whether CGA was able to inhibit the proliferation of the fibroblast-like synoviocyte cell line (RSC-364), stimulated by interleukin (IL)-6, through inducing apoptosis. Following incubation with IL-6 or IL-6 and CGA, the cellular proliferation of RSC-364 cells was detected by MTT assay. The ratio of apoptosed cells were detected by flow cytometry. Western blot analysis was performed to observe protein expression levels of key molecules involved in the Janus-activated kinase/signal transducer and activator of transcription 3 (JAK/STAT) signaling pathway [phosphorylated (p)-STAT3, JAK1 and gp130] and the nuclear factor κB (NF-κB) signaling pathway [phosphorylated (p)-inhibitor of κB kinase subunit α/β and NF-κB p50). It was revealed that CGA was able to inhibit the inflammatory proliferation of RSC-364 cells mediated by IL-6 through inducing apoptosis. CGA was also able to suppress the expression levels of key molecules in the JAK/STAT and NF-κB signaling pathways, and inhibit the activation of these signaling pathways in the inflammatory response through IL-6-mediated signaling, thereby resulting in the inhibition of the inflammatory proliferation of synoviocytes. The present results indicated that CGA may have potential as a novel therapeutic agent for inhibiting inflammatory hyperplasia of the synovium through inducing synoviocyte apoptosis in patients with RA. PMID:27168850

Inhibitory effect of geraniol in combination with gemcitabine on proliferation of BXPC-3 human pancreatic cancer cells.

PubMed

Jin, Xiaoxin; Sun, Jichun; Miao, Xiongyong; Liu, Guoli; Zhong, Dewu

2013-08-01

To investigate the inhibitory effect of geraniol alone, or in combination with gemcitabine, on the proliferation of BXPC-3 pancreatic cancer cells. BXPC-3 cells were treated under different conditions: with geraniol at 10, 20, 40, 80 and 160 µmol/l each for 24 h, 48 h or 72 h; with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h; with 20 µmol/l geraniol for 24 h, 48 h and 72 h following 20 µmol/l gemcitabine for 24 h; or with 20 µmol/l gemcitabine alone as a control. Cell proliferation was assessed and changes in cell morphology were assessed by light and fluorescence microscopy. Apoptosis was detected using flow cytometry. Geraniol inhibited BXPC-3 cell proliferation in a time- and dosa-dependent manner. Geraniol alone or combined with gemcitabine induced BXPC-3 cell apoptosis. BXPC-3 inhibition rates with combined treatment were 55.24%, 50.69%, 49.83%, 41.85% and 45.27% following treatment with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h, or 20 µmol/l geraniol for 24 h, 48 h and 72 h, following 20 µmol/l gemcitabine for 24 h, respectively. Geraniol inhibited the proliferation of BXPC-3 cells. Geraniol significantly increased the antiproliferative and apoptosis-inducing effects of gemcitabine on BXPC-3 cells. Maximum inhibition of BXPC-3 cells was achieved with geraniol treatment for 24 h before gemcitabine treatment.

Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells.

PubMed

Mohammed, Fatima H; Khajah, Maitham A; Yang, Ming; Brackenbury, William J; Luqmani, Yunus A

2016-01-01

Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

Effect of microRNA-135a on Cell Proliferation, Migration, Invasion, Apoptosis and Tumor Angiogenesis Through the IGF-1/PI3K/Akt Signaling Pathway in Non-Small Cell Lung Cancer.

PubMed

Zhou, Yufei; Li, Shaoxia; Li, Jiangtao; Wang, Dongfeng; Li, Quanxing

2017-01-01

This study explored the ability of microRNA-135a (miR-135a) to influence cell proliferation, migration, invasion, apoptosis and tumor angiogenesis through the IGF-1/PI3K/Akt signaling pathway in non-small cell lung cancer (NSCLC). NSCLC tissues and adjacent normal tissues were collected from 138 NSCLC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-135a and IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 mRNA; western blotting was used to determine the expression levels of IGF-1, PI3K and Akt protein; and enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression levels of VEGF, bFGF and IL-8 protein. Human NSCLC cell lines (A549, H460, and H1299) and the human bronchial epithelial cell line (HBE) were selected. A549 cells were assigned to blank, negative control (NC), miR-135a mimics, miR-135a inhibitors, IGF-1 siRNA and miR-135a inhibitors + IGF-1 siRNA groups. The following were performed: an MTT assay to assess cell proliferation, a scratch test to detect cell migration, a Transwell assay to measure cell invasion, and a flow cytometry to analyze cell apoptosis. The expression level of miR-135a was lower while those of IGF-1, PI3K and Akt mRNA were higher in NSCLC tissues than in the adjacent normal tissues. Dual-luciferase reporter assay indicated IGF-1 as a target of miR-135a. The in vitro results showed that compared with the blank group, cell proliferation, migration and invasion were suppressed, mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 were reduced, and cell apoptosis was enhanced in the miR-135a mimics and IGF-1 siRNA groups. Compared with the IGF-1 siRNA group, cells in the miR-135a inhibitors + IGF-1 siRNA group demonstrated increased cell proliferation, migration and invasion, elevated mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 and reduced cell apoptosis. These findings indicated that miR-135a promotes cell apoptosis and inhibits cell proliferation, migration, invasion and tumor angiogenesis by targeting IGF-1 gene through the IGF-1/PI3K/Akt signaling pathway in NSCLC. © 2017 The Author(s). Published by S. Karger AG, Basel.

Restoration of miR-1305 relieves the inhibitory effect of nicotine on periodontal ligament-derived stem cell proliferation, migration, and osteogenic differentiation.

PubMed

Chen, Zhuo; Liu, Hui-Li

2017-04-01

Nicotine hinders the regenerative potentials of human periodontal ligament-derived stem cells (PDLSCs) and delays the healing process of periodontal diseases, but the underlying mechanism remains unclear. miR-1305 upregulation and its potential target RUNX2 downregulation exist in the PDLSCs exposed to nicotine. In this study, we aimed to investigate whether nicotine inhibits PDLSC proliferation, migration, and osteogenic differentiation by increasing miR-1305 level and decreasing RUNX2 level. Quantitative real-time PCR (qRT-PCR) and Western blot assays were performed to detect the expression levels of miR-1305 and RUNX2 in the PDLSCs exposed to nicotine, respectively. PDLSCs with miR-1305 overexpression, low expression, or RUNX2 overexpression were constructed by lipofectin transfection. MTT, migration, and Western blot assays were applied to assess the effect of miR-1305 on PDLSC proliferation, migration, and osteogenic differentiation, respectively. Target prediction and luciferase reporter assays were performed to investigate the targets of miR-1305. Nicotine promoted miR-1305 expression and inhibited RUNX2 expression in PDLSCs. Cell proliferation, migration, and differentiation detection showed that nicotine suppressed proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieved the inhibitory effect of nicotine on PDLSCs. Moreover, we identified and validated that RUNX2 was a direct target of miR-1305, and upregulation of RUNX2 had similar effects with the downregulation of miR-1305 on relieving the inhibitory effect of nicotine on PDLSCs. Nicotine suppresses proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieves the inhibitory effect of nicotine on PDLSCs depending on its target RUNX2. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Antioxidant axis Nrf2-keap1-ARE in inhibition of alcoholic liver fibrosis by IL-22

PubMed Central

Ni, Ya-Hui; Huo, Li-Juan; Li, Ting-Ting

2017-01-01

AIM To explore the effect of interleukin (IL)-22 on in vitro model of alcoholic liver fibrosis hepatic stellate cells (HSCs), and whether this is related to regulation of Nrf2-keap1-ARE. METHODS HSC-T6 cells were incubated with 25, 50, 100, 200 and 400 μmol/L acetaldehyde. After 24 and 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect proliferation of HSCs to choose the best concentration and action time. We used the optimal concentration of acetaldehyde (200 μmol/L) to stimulate HSCs for 24 h, and treated the cells with a final concentration of 10, 20 or 50 ng/mL IL-22. The cell proliferation rate was detected by MTT assay. The cell cycle was analyzed by flow cytometry. The expression of nuclear factor-related factor (Nrf)2 and α-smooth muscle antigen was detected by western blotting and immunocytochemistry. The levels of malondialdehyde (MDA) and glutathione (GSH) were measured by spectrophotometry. RESULTS In the MTT assay, when HSCs were incubated with acetaldehyde, activity and proliferation were higher than in the control group, and were most obvious after 48 h treatment with 200 μmol/L acetaldehyde. The number of cells in G0/G1 phases was decreased and the number in S phase was increased in comparison with the control group. When treated with different concentrations of IL-22, HSC-T6 cell activity and proliferation rate were markedly decreased in a dose-dependent manner, and cell cycle progression was arrested from G1 to S phase. Western blotting and immunocytochemistry demonstrated that expression of Nrf2 total protein was not significantly affected. Expression of Nrf2 nuclear protein was low in the control group, increased slightly in the model group (or acetaldehyde-stimulated group), and increased more obviously in the IL-22 intervention groups. The levels of MDA and GSH in the model group were significantly enhanced in comparison with those in the control group. In cells treated with IL-22, the MDA level was attenuated but the GSH level was further increased. These changes were dose-dependent. CONCLUSION IL-22 inhibits acetaldehyde-induced HSC activation and proliferation, which may be related to nuclear translocation of Nrf2 and increased activity of the antioxidant axis Nrf2-keap1-ARE. PMID:28373766

Estradiol-17β-Induced Human Neural Progenitor Cell Proliferation Is Mediated by an Estrogen Receptor β-Phosphorylated Extracellularly Regulated Kinase Pathway

PubMed Central

Wang, Jun Ming; Liu, Lifei; Brinton, Roberta Diaz

2008-01-01

Estradiol-17β (E2) induces rodent hippocampal neural progenitor cell (NPC) proliferation in vitro, in vivo, and after brain injury. The purpose of the present investigation was to determine whether E2-induced proliferation observed in rodent model systems generalized to cells of human neural origin and the signaling pathway by which E2 promotes mitosis of human NPCs (hNPCs). Results of these analyses indicate that E2 induced a significant increase in hNPC proliferation in a time- and dose-dependent manner. E2-induced hNPC DNA replication was paralleled by elevated cell cycle protein expression and centrosome amplification, which was associated with augmentation of total cell number. To determine whether estrogen receptor (ER) and which ER subtype were required for E2-induced hNPC proliferation, ER expression was first determined by real-time RT-PCR, followed by Western blot analysis, and subsequently verified pharmacologically using ERα or β-selective ligands. Results of these analyses indicated that ERβ expression was predominant relative to ERα, which was barely detectable in hNPCs. Activation of ERβ by the ERβ-selective ligand, diarylpropionitrile, led to an increase in phosphorylated extracellular signal-regulated kinase, and subsequent centrosome amplification and hNPC proliferation, which were blocked by the MEKK antagonist, UO126, but not its inactive analog, UO124. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2-induced hNPC proliferation in vitro. Therapeutic implications of these findings relevant to hormone therapy and prevention of neurodegenerative disease are discussed. PMID:17962344

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

PubMed Central

2012-01-01

Background Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. Methods siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. Results The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. Conclusions In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines. PMID:22257483

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis.

PubMed

Kapp, Friedrich G; Sommer, Anette; Kiefer, Thomas; Dölken, Gottfried; Haendler, Bernard

2012-01-18

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines.

Use of a SQUID array to detect T-cells with magnetic nanoparticles in determining transplant rejection

NASA Astrophysics Data System (ADS)

Flynn, Edward R.; Bryant, H. C.; Bergemann, Christian; Larson, Richard S.; Lovato, Debbie; Sergatskov, Dmitri A.

2007-04-01

Acute rejection in organ transplant is signaled by the proliferation of T-cells that target and kill the donor cells requiring painful biopsies to detect rejection onset. An alternative non-invasive technique is proposed using a multi-channel superconducting quantum interference device (SQUID) magnetometer to detect T-cell lymphocytes in the transplanted organ labeled with magnetic nanoparticles conjugated to antibodies specifically attached to lymphocytic ligand receptors. After a magnetic field pulse, the T-cells produce a decaying magnetic signal with a characteristic time of the order of a second. The extreme sensitivity of this technique, 10 5 cells, can provide early warning of impending transplant rejection and monitor immune-suppressive chemotherapy.

Mechanisms of taste bud cell loss after head and neck irradiation.

PubMed

Nguyen, Ha M; Reyland, Mary E; Barlow, Linda A

2012-03-07

Taste loss in human patients following radiotherapy for head and neck cancer is a common and significant problem, but the cellular mechanisms underlying this loss are not understood. Taste stimuli are transduced by receptor cells within taste buds, and like epidermal cells, taste cells are regularly replaced throughout adult life. This renewal relies on progenitor cells adjacent to taste buds, which continually supply new cells to each bud. Here we treated adult mice with a single 8 Gy dose of x-ray irradiation to the head and neck, and analyzed taste epithelium at 1-21 d postirradiation (dpi). We found irradiation targets the taste progenitor cells, which undergo cell cycle arrest (1-3 dpi) and apoptosis (within 1 dpi). Taste progenitors resume proliferation at 5-7 dpi, with the proportion of cells in S and M phase exceeding control levels at 5-6 and 6 dpi, respectively, suggesting that proliferation is accelerated and/or synchronized following radiation damage. Using 5-bromo-2-deoxyuridine birthdating to identify newborn cells, we found that the decreased proliferation following irradiation reduces the influx of cells at 1-2 dpi, while the robust proliferation detected at 6 dpi accelerates entry of new cells into taste buds. In contrast, the number of differentiated taste cells was not significantly reduced until 7 dpi. These data suggest a model where continued natural taste cell death, paired with temporary interruption of cell replacement, underlies taste loss after irradiation.

Mechanisms of taste bud cell loss after head and neck irradiation

PubMed Central

Nguyen, Ha M.; Reyland, Mary E.; Barlow, Linda A.

2012-01-01

Taste loss in human patients following radiotherapy for head and neck cancer is a common and significant problem, but the cellular mechanisms underlying this loss are not understood. Taste stimuli are transduced by receptor cells within taste buds, and like epidermal cells, taste cells are regularly replaced throughout adult life. This renewal relies on a progenitor cells adjacent to taste buds, which continually supply new cells to each bud. Here we treated adult mice with a single 8 Gy dose of X-ray irradiation to the head and neck, and analyzed taste epithelium at 1–21 days post-irradiation (dpi). We found irradiation targets the taste progenitor cells, which undergo cell cycle arrest (1–3 dpi) and apoptosis (within 1 dpi). Taste progenitors resume proliferation at 5–7 dpi, with the proportion of cells in S and M phase exceeding control levels at 5–6 and 6 dpi, respectively, suggesting that proliferation is accelerated and/or synchronized following radiation damage. Using BrdU birthdating to identify newborn cells, we found that the decreased proliferation following irradiation reduces the influx of cells at 1–2 dpi, while the robust proliferation detected at 6 dpi accelerates entry of new cells into taste buds. By contrast, the number of differentiated taste cells was not significantly reduced until 7 dpi. These data suggest a model where continued natural taste cell death, paired with temporary interruption of cell replacement underlies taste loss after irradiation. PMID:22399770

Environmental Enrichment, Age, and PPARα Interact to Regulate Proliferation in Neurogenic Niches

PubMed Central

Pérez-Martín, Margarita; Rivera, Patricia; Blanco, Eduardo; Lorefice, Clara; Decara, Juan; Pavón, Francisco J.; Serrano, Antonia; Rodríguez de Fonseca, Fernando; Suárez, Juan

2016-01-01

Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been shown to modulate recovery after brain insults such as ischemia and irradiation by enhancing neurogenesis. In the present study, we investigated the effect of the genetic deletion of PPARα receptors on the proliferative rate of neural precursor cells (NPC) in the adult brain. The study was performed in aged Pparα−/− mice exposed to nutritional (treats) and environmental (games) enrichments for 20 days. We performed immunohistochemical analyses of cells containing the replicating cell DNA marker 5-bromo-2′-deoxyuridine (BrdU+) and the immature neuronal marker doublecortin (Dcx+) in the main neurogenic zones of the adult brain: subgranular zone of dentate gyrus (SGZ), subventricular zone of lateral ventricles (SVZ), and/or hypothalamus. Results indicated a reduction in the number of BrdU+ cells in the neurogenic zones analyzed as well as Dcx+ cells in the SGZ during aging (2, 6, and 18 months). Pparα deficiency alleviated the age-related reduction of NPC proliferation (BrdU+ cells) in the SVZ of the 18-months-old mice. While no genotype effect on NPC proliferation was detected in the SGZ during aging, an accentuated reduction in the number of Dcx+ cells was observed in the SGZ of the 6-months-old Pparα−/− mice. Exposing the 18-months-old mice to nutritional and environmental enrichments reversed the Pparα−/−-induced impairment of NPC proliferation in the neurogenic zones analyzed. The enriched environment did not modify the number of SGZ Dcx+ cells in the 18 months old Pparα−/− mice. These results identify PPARα receptors as a potential target to counteract the naturally observed decline in adult NPC proliferation associated with aging and impoverished environments. PMID:27013951

Environmental Enrichment, Age, and PPARα Interact to Regulate Proliferation in Neurogenic Niches.

PubMed

Pérez-Martín, Margarita; Rivera, Patricia; Blanco, Eduardo; Lorefice, Clara; Decara, Juan; Pavón, Francisco J; Serrano, Antonia; Rodríguez de Fonseca, Fernando; Suárez, Juan

2016-01-01

Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been shown to modulate recovery after brain insults such as ischemia and irradiation by enhancing neurogenesis. In the present study, we investigated the effect of the genetic deletion of PPARα receptors on the proliferative rate of neural precursor cells (NPC) in the adult brain. The study was performed in aged Pparα(-/-) mice exposed to nutritional (treats) and environmental (games) enrichments for 20 days. We performed immunohistochemical analyses of cells containing the replicating cell DNA marker 5-bromo-2'-deoxyuridine (BrdU+) and the immature neuronal marker doublecortin (Dcx+) in the main neurogenic zones of the adult brain: subgranular zone of dentate gyrus (SGZ), subventricular zone of lateral ventricles (SVZ), and/or hypothalamus. Results indicated a reduction in the number of BrdU+ cells in the neurogenic zones analyzed as well as Dcx+ cells in the SGZ during aging (2, 6, and 18 months). Pparα deficiency alleviated the age-related reduction of NPC proliferation (BrdU+ cells) in the SVZ of the 18-months-old mice. While no genotype effect on NPC proliferation was detected in the SGZ during aging, an accentuated reduction in the number of Dcx+ cells was observed in the SGZ of the 6-months-old Pparα(-/-) mice. Exposing the 18-months-old mice to nutritional and environmental enrichments reversed the Pparα(-/-)-induced impairment of NPC proliferation in the neurogenic zones analyzed. The enriched environment did not modify the number of SGZ Dcx+ cells in the 18 months old Pparα(-/-) mice. These results identify PPARα receptors as a potential target to counteract the naturally observed decline in adult NPC proliferation associated with aging and impoverished environments.

Lattice-based model of ductal carcinoma in situ suggests rules for breast cancer progression to an invasive state.

PubMed

Boghaert, Eline; Radisky, Derek C; Nelson, Celeste M

2014-12-01

Ductal carcinoma in situ (DCIS) is a heterogeneous group of non-invasive lesions of the breast that result from abnormal proliferation of mammary epithelial cells. Pathologists characterize DCIS by four tissue morphologies (micropapillary, cribriform, solid, and comedo), but the underlying mechanisms that distinguish the development and progression of these morphologies are not well understood. Here we explored the conditions leading to the emergence of the different morphologies of DCIS using a two-dimensional multi-cell lattice-based model that incorporates cell proliferation, apoptosis, necrosis, adhesion, and contractility. We found that the relative rates of cell proliferation and apoptosis governed which of the four morphologies emerged. High proliferation and low apoptosis favored the emergence of solid and comedo morphologies. In contrast, low proliferation and high apoptosis led to the micropapillary morphology, whereas high proliferation and high apoptosis led to the cribriform morphology. The natural progression between morphologies cannot be investigated in vivo since lesions are usually surgically removed upon detection; however, our model suggests probable transitions between these morphologies during breast cancer progression. Importantly, cribriform and comedo appear to be the ultimate morphologies of DCIS. Motivated by previous experimental studies demonstrating that tumor cells behave differently depending on where they are located within the mammary duct in vivo or in engineered tissues, we examined the effects of tissue geometry on the progression of DCIS. In agreement with our previous experimental work, we found that cells are more likely to invade from the end of ducts and that this preferential invasion is regulated by cell adhesion and contractility. This model provides additional insight into tumor cell behavior and allows the exploration of phenotypic transitions not easily monitored in vivo.

[Effects of thyroid hormone on macrophage dysfunction induced by oxidized low-density lipoprotein].

PubMed

Ning, Yu; Zhang, Ming; DU, Yun-Hui; Zhang, Hui-Na; Li, Lin-Yi; Qin, Yan-Wen; Wen, Wan-Wan; Zhao, Quan-Ming

2018-04-25

It has been recognized that patients with hypothyroidism have higher risks of atherosclerosis and coronary heart disease, however, the mechanisms are largely unknown. Considering that macrophage dysfunction plays an important role in the formation and development of atherosclerosis plaques, this study aimed to investigate the direct effects of thyroid hormone on macrophage functions and to provide new insight for the mechanism of hypothyroid atherosclerosis. RAW264.7 cells (mouse leukaemic monocyte macrophage cell line) were incubated with oxidized low-density lipoprotein (oxLDL) to establish macrophage foam cells model in vitro, and the protective effects of different concentration of thyroxine (T4) on the macrophage foam cells function were explored. The proliferation, migration and cell aging of macrophages were detected by MTT method, scratch test and β-galactosidase staining respectively. The ELISA method was used to detect the secretion of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-1β (IL-1β). Western blot analysis was applied to measure the phosphorylation of focal adhesion kinase (FAK), which was required for the process of proliferation and migration of macrophages. The results showed that oxLDL significantly inhibited the macrophage proliferation and migration, induced cell senescence, and promoted the secretion of TNF-α, MCP-1, and IL-1β; while T4 reversed those effects of oxLDL on macrophage in a concentration-dependent manner. Moreover, oxLDL increased the phosphorylation of FAK in macrophage, while T4 concentration-dependently reversed the effect. These results suggest that T4 modulates macrophage proliferation, migration, senescence, and secretion of inflammation factors in a concentration-dependent way.

Histamine H{sub 3} receptor antagonist OUP-186 attenuates the proliferation of cultured human breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Satoshi; Sakaguchi, Minoru; Yoneyama, Hiroki

Histamine is involved in various physiological functions, including its neurotransmitter actions in the central nervous system and its action as a causative agent of inflammation, allergic reactions, and gastric acid secretions. Histamine expression and biosynthesis have been detected in breast cancer cells. It was recently suggested that the histamine H{sub 3} receptor (H{sub 3}R) plays a role in the proliferation of breast cancer cells. We recently developed the non-imidazole H{sub 3}R antagonist OUP-186 which exhibited a potent and selective human H{sub 3}R antagonistic activity as well as no activity against the human histamine H{sub 4} receptor (H{sub 4}R). In thismore » study, we compared the effects of OUP-186 on the proliferation of estrogen receptor negative (ER−) breast cancer cells (MDA-MB-231) and ER+ breast cancer cells (MCF7) to the effects of clobenpropit (potent imidazole-containing H{sub 3}R antagonist). OUP-186 and clobenpropit suppressed the proliferation of breast cancer cells. The IC{sub 50} values at 48 h for OUP-186 and clobenpropit were approximately 10 μM and 50 μM, respectively. Furthermore, OUP-186 potently induced cell death by activating caspase-3/7, whereas cell death was only slightly induced by clobenpropit. In addition, OUP-186 treatment blocked the proliferation increase triggered by 100 μM (R)-(-)-α-methylhistamine (H{sub 3}R agonist). The use of 4-methylhistamine (H{sub 4}R agonist) and JNJ10191584 (selective H{sub 4}R antagonist) did not affect breast cancer proliferation. These results indicate that OUP-186 potently suppresses proliferation and induces caspase-dependent apoptotic death in both ER+ and ER-breast cancer cells. - Highlights: • OUP-186, a histamine H{sub 3} receptor antagonist, effects breast cancer cell growth. • OUP-186 potently suppressed proliferation and induced caspase-dependent apoptosis. • OUP-186 may be an effective drug against ER+ and ER− breast cancers.« less

Effect and mechanism of PAR-2 on the proliferation of esophageal cancer cells.

PubMed

Quanjun, D; Qingyu, Z; Qiliang, Z; Liqun, X; Jinmei, C; Ziquan, L; Shike, H

2016-11-01

Esophageal Cancer (EC) is a common malignant tumor occurred in the digestive tract. In this study, we investigated the mechanism of Protease Activated Receptor 2 (PAR-2) on the proliferation of esophageal cancer cell. Transfected esophageal cancer (EC) cell (PAR-2shRNA EC109) was established with low stable PAR-2 expression. EC109 cell was treated with PAR-2 agonist, PAR-2 anti-agonist and MAPK inhibitor respectively; Untreated EC109 cell (blank control) and PAR-2shRNA EC109 cell were used for analysis also. The mRNA expressions of PAR-2, ERK1, Cyclin D1, and c-fos in each group were detected by reverse transcript and polymerase chain reaction. Western blot was used to detect the protein expressions in each group. The cell growth curves were drawn to compare the cell growth. Compared with the blank control, the mRNA and protein expressions of PAR-2, Cyclin D1, and c-fos in PAR-2 agonist group increased significantly (p < 0.05), while decreased significantly in PAR-2shRNA EC109 cell and MAPK inhibitor group (p < 0.05). The mRNA expression of ERK1 and protein expression of p-ERK1 increased in PAR-2 agonist group, decreased in PAR-2shRNA EC109 cell and MAPK inhibitor group when compared with blank control (p < 0.05). The growth of cells was upward in PAR-2 agonist group at cell growth phase when compared with blank control, while decreased in PAR-2 shRNA EC109 cell and MAPK inhibitor group with statistical difference (p < 0.05). PAR-2 regulate cell proliferation through the MAPK pathway in esophageal carcinoma cell, and Cyclin D1, c-fos are involved in this process.

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

PubMed

Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

[GPER silence inhibits the stimulation of growth and inhibition of apoptosis induced by tamoxifen in breast cancer-associated fibroblasts].

PubMed

Yan, Yuzhao; Yu, Tenghua; Tu, Gang; Liu, Manran; Yuan, Jie; Yang, Guanglun

2015-09-01

To construct a lentiviral vector (Lenti-GPER-shRNA) targeting G-protein coupled estrogen receptor (GPER) and explore the role of GPER in the effect of tamoxifen on cell proliferation and apoptosis in breast cancer associated fibroblasts (BCAFs). The target sequence of GPER gene and negative control were cloned into lentiviral vectors. The recombinant lentivirus and control were extracted after HEK293T cells were transfected with the recombinant vector and helper vectors. After infection of BCAFs with the GPER lentiviral vector under the best interfering condition, GPER expression was detected by real-time quantitative PCR and Western blotting. BCAFs were divided into negative control group, GPER-RNAi group, negative control combined with tamoxifen (10(-8) mmol/L) group and GPER-RNAi combined with tamoxifen (10(-8) mmol/L) group. CCK-8 assay was used to detect the proliferation and annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) combined with flow cytometry was used to detect the apoptosis of BCAFs after the treatment of tamoxifen. Lenti-GPER-shRNA significantly interfered the expression of GPER in BCAFs. Tamoxifen promoted the growth of BCAFs, which could be attenuated by knockdown of GPER. Moreover, the apoptosis of BCAFs was reduced by tamoxifen, which was also reversed by knockdown of GPER. Lenti-GPER-shRNA could effectively silence the GPER expression in BCAFs. The ability of tamoxifen to accelerate cell proliferation and decrease cell apoptosis could be weakened by knockdown of GPER.

Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.

PubMed

Okumura, Naoki; Ueno, Morio; Koizumi, Noriko; Sakamoto, Yuji; Hirata, Kana; Hamuro, Junji; Kinoshita, Shigeru

2009-08-01

The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. MCECs of cynomolgus monkeys were cultured in a medium containing 10 microM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

Effects of Vitamin K3 and K5 on Daunorubicin-resistant Human T Lymphoblastoid Leukemia Cells.

PubMed

Nakaoka, Eri; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

2015-11-01

Anticancer efficacy of vitamin K derivatives on multidrug-resistant cancer cells has been scarcely investigated. The effects of vitamins K3 and K5 on proliferation of human leukemia MOLT-4 cells and on daunorubicin-resistant MOLT-4/DNR cells were estimated by a WST assay. Apoptotic cells were detected by Annexin V and propidium iodide staining, followed by flow cytometry. Vitamins K3 and K5 significantly inhibited proliferation of leukemic cells at 10 and 100 μM (p<0.05), and these effects were almost equally observed in both MOLT-4 and MOLT/DNR drug-resistant cells. Vitamin K3 induced cell apoptosis at 10 and 100 μM in both MOLT-4 and MOLT-4/DNR cells (p<0.05). Vitamin K5 also increased apoptotic cells, while rather inducing necrotic cell death. Vitamins K3 and K5 suppress MOLT-4 and MOLT-4/DNR cell-proliferation partially through induction of apoptosis, and these vitamin derivatives can overcome drug resistance due to P-glycoprotein expression. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

PubMed

Xu, Yan; Hadjiargyrou, M; Rafailovich, Miriam; Mironava, Tatsiana

2017-07-11

Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). Herein, we report that titanium dioxide (TiO 2 ) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO 2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

Expression of a Novel RNA-Splicing Factor, RA301/Tra2β, in Vascular Lesions and Its Role in Smooth Muscle Cell Proliferation

PubMed Central

Tsukamoto, Yoshitane; Matsuo, Noriyuki; Ozawa, Kentaro; Hori, Osamu; Higashi, Toshio; Nishizaki, Junya; Tohnai, Norimitsu; Nagata, Izumi; Kawano, Kiyoshi; Yutani, Chikao; Hirota, Seiichi; Kitamura, Yukihiko; Stern, David M.; Ogawa, Satoshi

2001-01-01

RA301/Tra2β, a sequence-specific RNA-binding protein, was first cloned as a stress molecule in re-oxygenated astrocytes. In human vascular tissues, we have found enhanced RA301/Tra2β expression in coronary artery with intimal thickening, and atherosclerotic aorta. Balloon injury to the rat carotid artery induced RA301/Tra2β transcripts followed by expression of the antigen, which was detected in medial and neointimal vascular smooth muscle cells (VSMCs). In cultured VSMCs, hypoxia/re-oxygenation caused induction of RA301/Tra2β and was accompanied by cell proliferation, both of which were blocked by the addition of either diphenyl iodonium, a NADPH oxidase inhibitor, PD98059, a mitogen-activated protein kinase kinase inhibitor, or antisense oligonucleotide for RA301/Tra2β. Consistent with a link between RA301/Tra2β and cell proliferation, platelet-derived growth factor also induced expression of RA301/Tra2β in cultured VSMCs. These data suggest a possible role for RA301/Tra2β in the regulation of VSMC proliferation, especially in the setting of hypoxia/re-oxygenation-induced cell stress. PMID:11337366

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

PubMed Central

CRUZ, PAMELA; TORRES, CRISTIAN; RAMÍREZ, MARÍA EUGENIA; EPUÑÁN, MARÍA JOSÉ; VALLADARES, LUIS EMILIO; SIERRALTA, WALTER DANIEL

2010-01-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E2) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E2, and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E2 in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E2-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels. PMID:22993572

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol.

PubMed

Cruz, Pamela; Torres, Cristian; Ramírez, María Eugenia; Epuñán, María José; Valladares, Luis Emilio; Sierralta, Walter Daniel

2010-05-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E(2)) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E(2), and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E(2) in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E(2)-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels.

Regulation of pancreatic stellate cell activation by Notch3.

PubMed

Song, Haiyan; Zhang, Yuxiang

2018-01-05

Activated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC. The expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student's t-test. Notch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells. Notch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC.

[Inhibitory effect of baicalein on the proliferation and invasion of osteosarcoma cells and mechanism].

PubMed

Tang, Zhibin; Li, Chun; Chen, Zhiwei

2015-03-01

To explore the effect of baicalein on the proliferation and invasion of osteosarcoma cells and its related mechanism. Osteosarcoma MG-63 cells that were cultured in vitro were respectively treated with 20 μL culture medium (control group), dehydrated alcohol (0 μmol/L baicalein group), 100 and 200 μmol/L baicalein solution for 48 hours. Cell proliferation was analyzed by MTT assay. The cell invasion ability was detected using Transwell(TM) invasion assay. The expression of ezrin mRNA was examined by real-time quantitative PCR. The expressions of ezrin protein and p-ezrin protein were measured using Western blotting. Apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The inhibitory rates of cell proliferation significantly increased in 100 and 200 μmol/L baicalein groups as compared with 0 μmol/L baicalein group. Moreover, that was higher in 200 μmol/L baicalein group than in 100 μmol/L baicalein group. In comparison with control and 0 μmol/L baicalein groups, the mean cell numbers of permeated membrane and levels of ezrin mRNA, ezrin protein and p-ezrin protein gradually decreased, but AI was gradually elevated with the increase of baicalein concentrations, whereas there was no significant difference in these indicators between 0 μmol/L baicalein group and control group. Baicalein can inhibit the proliferation and invasion of osteosarcoma MG-63 cells. The mechanism may be associated with the inhibited expression and activity of ezrin protein and the promoted tumor cell apoptosis.

Traditional Chinese medicine Astragalus polysaccharide enhanced antitumor effects of the angiogenesis inhibitor apatinib in pancreatic cancer cells on proliferation, invasiveness, and apoptosis

PubMed Central

Wu, Jun; Wang, Jing; Su, Qiang; Ding, Wei; Li, Teng; Yu, Junxian; Cao, Bangwei

2018-01-01

Background Traditional chemotherapy and molecular targeted therapy have shown modest effects on the survival of patients with pancreatic cancer. The current study aimed to investigate the antitumor effects of apatinib, Astragalus polysaccharide (APS), and the combination of both the drugs in pancreatic cancer cells and further explore the molecular mechanisms in vitro. Materials and methods Expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human pancreatic cancer cell lines ASPC-1, PANC-1, and SW1990 was detected by Western blotting. Cell proliferation was measured by MTS, and migration and invasion were detected by wound-healing and Transwell assays, respectively. Cell apoptosis rate was determined by flow cytometry and cellular autophagy level affected by apatinib, and APS was analyzed by Western blotting. Results Human pancreatic cancer cell lines ASPC-1 and PANC-1 expressed VEGFR-2, but VEGFR-2 was not detected in SW1990. Either apatinib or APS inhibited cell proliferation in a dose-dependent manner in ASPC-1 and PANC-1. APS in combination with apatinib showed enhanced inhibitory effects on cell migration and invasion compared with apatinib monotherapy in ASPC-1 and PANC-1. Meanwhile, APS combined with apatinib strongly increased cell apoptosis percentage. Western blotting showed that the combination of APS and apatinib significantly enhanced the downregulation of phosphorylated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) (p-AKT and p-ERK) as well as matrix metalloproteinases-9 (MMP-9) expression. In addition, both apatinib and APS induced cellular autophagy. However, the expression of autophagy-related proteins was not further elevated in the combination group. Conclusion The study first demonstrated that apatinib showed potentially inhibitory effects in pancreatic cancer cells and that APS enhanced the antitumor effects of apatinib through further downregulating the expression of phosphorylation of AKT and ERK as well as MMP-9. PMID:29785118

Traditional Chinese medicine Astragalus polysaccharide enhanced antitumor effects of the angiogenesis inhibitor apatinib in pancreatic cancer cells on proliferation, invasiveness, and apoptosis.

PubMed

Wu, Jun; Wang, Jing; Su, Qiang; Ding, Wei; Li, Teng; Yu, Junxian; Cao, Bangwei

2018-01-01

Traditional chemotherapy and molecular targeted therapy have shown modest effects on the survival of patients with pancreatic cancer. The current study aimed to investigate the antitumor effects of apatinib, Astragalus polysaccharide (APS), and the combination of both the drugs in pancreatic cancer cells and further explore the molecular mechanisms in vitro. Expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human pancreatic cancer cell lines ASPC-1, PANC-1, and SW1990 was detected by Western blotting. Cell proliferation was measured by MTS, and migration and invasion were detected by wound-healing and Transwell assays, respectively. Cell apoptosis rate was determined by flow cytometry and cellular autophagy level affected by apatinib, and APS was analyzed by Western blotting. Human pancreatic cancer cell lines ASPC-1 and PANC-1 expressed VEGFR-2, but VEGFR-2 was not detected in SW1990. Either apatinib or APS inhibited cell proliferation in a dose-dependent manner in ASPC-1 and PANC-1. APS in combination with apatinib showed enhanced inhibitory effects on cell migration and invasion compared with apatinib monotherapy in ASPC-1 and PANC-1. Meanwhile, APS combined with apatinib strongly increased cell apoptosis percentage. Western blotting showed that the combination of APS and apatinib significantly enhanced the downregulation of phosphorylated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) (p-AKT and p-ERK) as well as matrix metalloproteinases-9 (MMP-9) expression. In addition, both apatinib and APS induced cellular autophagy. However, the expression of autophagy-related proteins was not further elevated in the combination group. The study first demonstrated that apatinib showed potentially inhibitory effects in pancreatic cancer cells and that APS enhanced the antitumor effects of apatinib through further downregulating the expression of phosphorylation of AKT and ERK as well as MMP-9.

Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

PubMed

Shi, Lei; Song, Ruigao; Yao, Xiaolei; Ren, Youshe

2017-04-15

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 μmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 μmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 μmol/L group. Appropriate Se level (2.0 μmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3β-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3β-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 μmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3β-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of Slug in S100β-protein-positive cells of postnatal developing rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Yako, Hideji; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

2016-02-01

Among heterogeneous S100β-protein-positive (S100β-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100β-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100β-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100β-promoter has allowed us to observe living S100β-positive cells. In the present study, we first confirmed that living S100β-positive cells in tissue cultures of S100β-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100β-positive cells. Interestingly, we detected Slug expression in S100β-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100β-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100β-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

MC3T3-E1 Cell Response to Ti1-xAgx and Ag-TiNx Electrodes Deposited on Piezoelectric Poly(vinylidene fluoride) Substrates for Sensor Applications.

PubMed

Marques, S M; Rico, P; Carvalho, I; Gómez Ribelles, J L; Fialho, L; Lanceros-Méndez, S; Henriques, M; Carvalho, S

2016-02-17

In the sensors field, titanium based coatings are being used for the acquisition/application of electrical signals from/to piezoelectric materials. In this particular case, sensors are used to detect dynamic mechanical loads at early stages after intervention of problems associated with prostheses implantation. The aim of this work is to select an adequate electrode for sensor applications capable, in an initial stage to avoid bone cell adhesion, but at a long stage, permit osteointegration and osteoinduction. This work reports on the evaluation of osteoblast MC3T3-E1 cells behavior in terms of proliferation, adhesion and long-term differentiation of two different systems used as sensor electrodes: Ti1-xAgx and Ag-TiNx deposited by d.c. and pulsed magnetron sputtering at room temperature on poly(vinylidene fluoride) (PVDF). The results indicated an improved effect of Ag-TiNx electrodes compared with Ti1-xAgx and TiN, in terms of diminished cell adhesion and proliferation at an initial cell culture stage. Nevertheless, when cell culture time is longer, cells grown onto Ag-TiNx electrodes are capable to proliferate and also differentiate at proper rates, indicating the suitability of this coating for sensor application in prostheses devices. Thus, the Ag-TiNx system was considered the most promising electrode for tissue engineering applications in the design of sensors for prostheses to detect dynamic mechanical loads.

Effect of miR-146a-5p on proliferation and metastasis of triple-negative breast cancer via regulation of SOX5.

PubMed

Si, Chengshuai; Yu, Qiao; Yao, Yufeng

2018-05-01

MicroRNA (miR)-146a-5p functions as a tumor suppressor in various types of cancer. However, the role of miR-146a-5p in the development of triple-negative breast cancer (TNBC) is unclear. The present study aimed to investigate the role of miR-146a-5p in TNBC. The expression level of miR-146a-5p in TNBC tissues and cell lines was initially detected using reverse transcription-quantitative polymerase chain reaction. To predict the target gene of miR-146a-5p, TargetScan software was used and a dual luciferase assay was performed to verify the prediction. Furthermore, in order to explore the role of miR-146a-5p in TNBC, miR-146a-5p was overexpressed in TNBC cells using miR-146a-5p mimics. An MTT assay was performed to detect cell proliferation, and a Transwell assay was conducted to determine cell migration and invasion. Furthermore, western blotting was performed to measure associated protein expression. It was revealed that miR-146a-5p was downregulated in TNBC tissues and cell lines. SOX5 was indicated to be a target gene of miR-146a-5p and was upregulated in TNBC cells. Additionally, miR-146a-5p could inhibit TNBC cell proliferation, migration and invasion, repress the expression of mesenchymal markers (N-cadherin, vimentin and fibronectin) and increase epithelial marker (E-cadherin) expression. Furthermore, SOX5 overexpression eliminated the effects of miR-146a-5p mimics on TNBC cells. In conclusion, the data of the present study indicated that miR-146a-5p inhibits the proliferation and metastasis of TNBC cells by regulating SOX5.

FBI-1 promotes cell proliferation and enhances resistance to chemotherapy of hepatocellular carcinoma in vitro and in vivo.

PubMed

Fang, Feng; Yang, Lianyue; Tao, Yiming; Qin, Wei

2012-01-01

The so-called factor that binds to inducer of short transcripts-1 (FBI-1) purportedly plays an important role in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) remains unknown. The objective of this study was to investigate the expression level, clinical relevance, and biologic function of FBI-1 in HCC. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis, Western blot analysis, and immunohistochemical staining were used to detect expression levels of FBI-1 and to analyze its relation to clinicopathologic parameters and to the prognosis of patients with HCC. In addition, the biologic functions of FBI-1 in regulating cell proliferation, migration, and reaction to chemotherapy were detected by using HepG2 cells and SMMC-7721 cells; subsequently, the molecular mechanism of FBI-1 also was investigated. Finally, a xenograft mouse model was used to validate the observations obtained from in vitro studies. Expression levels of FBI-1 messenger RNA and protein were elevated significantly in HCC tissues compared with adjacent nontumorous liver tissues (ANLTs). Increased FBI-1 expression was correlated with multiple tumor nodes, Edmondson-Steiner grade, and a poor prognosis in patients with HCC (P < .05). In vitro studies revealed that FBI-1 was capable of promoting cell proliferation (but not cell migration) by regulating the cell cycle regulation proteins p53, p21, and p27. In addition, FBI-1 could inhibit cell death induced by 5-fluorouracil or doxorubicin through suppressing the activation of p53. Consistent with the in vitro data, FBI-1 was capable of promoting cell proliferation and enhancing chemotherapy resistance of HCC in vivo. The current findings indicated that FBI-1 plays an important role in HCC carcinogenesis and chemotherapy tolerance, and FBI-1 may served as a novel prognostic marker and therapeutic target for HCC. Copyright © 2011 American Cancer Society.

Melatonin inhibits proliferation and invasion via repression of miRNA-155 in glioma cells.

PubMed

Gu, Junyi; Lu, Zhongsheng; Ji, Chenghong; Chen, Yuchao; Liu, Yuzhao; Lei, Zhe; Wang, Longqiang; Zhang, Hong-Tao; Li, Xiangdong

2017-09-01

Melatonin, an indolamine mostly synthesized in the pineal gland, exerts the anti-cancer effect by various mechanisms in glioma cells. Our previous study showed that miR-155 promoted glioma cell proliferation and invasion. However, the question of whether melatonin may inhibit glioma by regulating miRNAs has not yet been addressed. In this study, we found that melatonin (100μM, 1μM and 1nM) significantly inhibited the expression of miR-155 in human glioma cell lines U87, U373 and U251. Especially, the lowest expression of miR-155 was detected in 1μM melatonin-treated glioma cells. Melatonin (1μM) inhibits cell proliferation of U87 by promoting cell apoptosis. Nevertheless, melatonin had no effect on cell cycle distribution of U87 cells. Moreover, U87 cells treated with 1μM melatonin presented significantly lower migration and invasion ability when compared with control cells. Importantly, melatonin inhibited c-MYB expression, and c-MYB knockdown reduced miR-155 expression and migration and invasion in U87 cells. Taken together, for the first time, our findings show that melatonin inhibits miR-155 expression and thereby represses glioma cell proliferation, migration and invasion, and suggest that melatonin may downregulate the expression of miR-155 via repression of c-MYB. This will provide a theoretical basis for revealing the anti-glioma mechanisms of melatonin. Copyright © 2017. Published by Elsevier Masson SAS.

Targeting CXCR4 reverts the suppressive activity of T-regulatory cells in renal cancer.

PubMed

Santagata, Sara; Napolitano, Maria; D'Alterio, Crescenzo; Desicato, Sonia; Maro, Salvatore Di; Marinelli, Luciana; Fragale, Alessandra; Buoncervello, Maria; Persico, Francesco; Gabriele, Lucia; Novellino, Ettore; Longo, Nicola; Pignata, Sandro; Perdonà, Sisto; Scala, Stefania

2017-09-29

With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4 + CD25 hi FOXP3 hi CD45RA - ). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs ( P <0,001). CXCR4 is highly expressed on Tregs, thus we wished to modulate Tregs function through CXCR4 inhibition. CXCR4 antagonism, elicited by a new peptidic antagonist, Peptide-R29, efficiently reversed Tregs suppression of Teff proliferation. Thus Tregs functional evaluation precisely reflects Tregs status and may be a reliable biomarker of tumoral immune response. In addition, treatment with CXCR4 antagonist, impairing Tregs function, could improve the anticancer immune response, in combination with conventional therapy and/or immunotherapy such as checkpoints inhibitors.

Targeting CXCR4 reverts the suppressive activity of T-regulatory cells in renal cancer

PubMed Central

Santagata, Sara; Napolitano, Maria; D'Alterio, Crescenzo; Desicato, Sonia; Maro, Salvatore Di; Marinelli, Luciana; Fragale, Alessandra; Buoncervello, Maria; Persico, Francesco; Gabriele, Lucia; Novellino, Ettore; Longo, Nicola; Pignata, Sandro; Perdonà, Sisto; Scala, Stefania

2017-01-01

With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4+CD25hiFOXP3hiCD45RA-). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs (P <0,001). CXCR4 is highly expressed on Tregs, thus we wished to modulate Tregs function through CXCR4 inhibition. CXCR4 antagonism, elicited by a new peptidic antagonist, Peptide-R29, efficiently reversed Tregs suppression of Teff proliferation. Thus Tregs functional evaluation precisely reflects Tregs status and may be a reliable biomarker of tumoral immune response. In addition, treatment with CXCR4 antagonist, impairing Tregs function, could improve the anticancer immune response, in combination with conventional therapy and/or immunotherapy such as checkpoints inhibitors. PMID:29100374

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophsmore » taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways. - Highlights: • In hyperplastic pituitaries, LPS triggered the lactotroph cell proliferation and IL-6 release. • Functional Toll-like receptor 4 (TLR4) is expressed at the plasma membrane of tumoral lactotrophs. • Increases in TLR4 and CD14 intracellular expression levels were detected after an LPS challenge. • The proliferative stimulation and IL-6 release involved the PI3K-Akt pathway and NF-κB activation. • 17β-estradiol attenuated the LPS-evoked tumoral lactotroph proliferation and IL-6 secretion.« less

The influence of TLR4 agonist lipopolysaccharides on hepatocellular carcinoma cells and the feasibility of its application in treating liver cancer.

PubMed

Gu, Junsheng; Sun, Ranran; Shen, Shen; Yu, Zujiang

2015-01-01

This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on liver cancer cell and the feasibility to perform liver cancer adjuvant therapy. Human liver cancer cell lines HepG2, H7402, and PLC/PRF/5 were taken as models, and the expression of TLRs mRNA was detected by real time-polymerase chain reaction method semiquantitatively. WST-1 method was used to detect the influence of LPS on the proliferation ability of liver cancer cells; propidium iodide (PI) single staining and Annexin V/PI double staining were used to test the influence of LPS on the cell cycle and apoptosis, respectively, on human liver cancer cell line H7402. Fluorescent quantitative polymerase chain reaction and Western blot method were used to determine the change of expression of Cyclin D1. The results demonstrated that most TLRs were expressed in liver cancer cells; stimulating TLR4 by LPS could upregulate TLR4 mRNA and the protein level, activate NF-κB signaling pathway downstream of TLR4, and mediate the generation of inflammatory factors IL-6, IL-8, and TNF-α; LPS was found to be able to strengthen the proliferation ability of liver cancer cells, especially H7402 cells; the expression of Cyclin D1 rose and H7402 cells were promoted to transit from G1 stage to S stage under the stimulation of LPS, but cell apoptosis was not affected. It was also found that LPS was able to activate signal transducer and activator of transcription -3 (STAT3) signaling pathway in H7402 cells and meanwhile significantly increase the initiation activity of STAT3; proliferation promoting effect of LPS to liver cancer cells remarkably lowered once STAT3 was blocked or inhibited. Thus, TLR4 agonist LPS is proved to be able to induce liver cancer cells to express inflammation factors and mediate liver cancer cell proliferation and generation of multidrug resistance by activating the cyclooxygenase-2/prostaglandin signal axis as well as the STAT3 pathway.

The effects of ROS in prostatic stromal cells under hypoxic environment.

PubMed

Ren, Hailin; Li, Xiaona; Cheng, Guojun; Li, Ning; Hou, Zhi; Suo, Jiming; Wang, Jian; Za, Xi

2015-06-01

The objective of this study is to explore the effects of reactive oxygen species (ROS) under hypoxic environment in prostatic stromal cells (PSC). To detect the expression of ROS in PSC and the tissues of benign prostatic hyperplasia (BPH) by flow cytometry; under hypoxic conditions, to observe the changes of cells growth and ROS in PSC; quantitative PCR was used to detect hypoxia inducible factor-1α (HIF-1α), androgen receptors (AR), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in PSC; After edaravone intervening, to examine the changes of cells growth, ROS, HIF-1α, AR, VEGF, and IL-8 under hypoxic conditions. The expression of ROS in tissues and cells which under hypoxic condition was significantly increased. 3% O2 promoted the proliferation. The HIF-1α, AR, VEGF, and IL-8 were upregulated under 3% O2. After edaravone intervening, ROS significantly decreased, HIF-1α and VEGF were downregulated, and cell proliferation declined. Hypoxia stimulates the generation of ROS, and the ROS may play a key role in BPH.

Combined changes in Wnt signaling response and contact inhibition induce altered proliferation in radiation-treated intestinal crypts

PubMed Central

Dunn, S.-J.; Osborne, J. M.; Appleton, P. L.; Näthke, I.

2016-01-01

Curative intervention is possible if colorectal cancer is identified early, underscoring the need to detect the earliest stages of malignant transformation. A candidate biomarker is the expanded proliferative zone observed in crypts before adenoma formation, also found in irradiated crypts. However, the underlying driving mechanism for this is not known. Wnt signaling is a key regulator of proliferation, and elevated Wnt signaling is implicated in cancer. Nonetheless, how cells differentiate Wnt signals of varying strengths is not understood. We use computational modeling to compare alternative hypotheses about how Wnt signaling and contact inhibition affect proliferation. Direct comparison of simulations with published experimental data revealed that the model that best reproduces proliferation patterns in normal crypts stipulates that proliferative fate and cell cycle duration are set by the Wnt stimulus experienced at birth. The model also showed that the broadened proliferation zone induced by tumorigenic radiation can be attributed to cells responding to lower Wnt concentrations and dividing at smaller volumes. Application of the model to data from irradiated crypts after an extended recovery period permitted deductions about the extent of the initial insult. Application of computational modeling to experimental data revealed how mechanisms that control cell dynamics are altered at the earliest stages of carcinogenesis. PMID:27053661

In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

PubMed

Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

2016-03-01

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

PubMed

Diao, Lingyun; Wang, Shengying; Sun, Zhiguang

2018-01-01

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. LncRNA GAPLINC has been reported to be increased in gastric cancer (GC) tissues. Real-time PCR assays were used to measure expressions of GAPLINC, miR-378, and MAPK1 mRNA. Western blot assays were employed to examine MAPK1 protein expression. Cell proliferation and cell cycle were measured by CCK-8 and propidium iodide-detection assays, respectively. The interaction between GAPLINC and miR-378 was confirmed by site-directed mutagenesis and luciferase assays. Luciferase assays were also used to study whether GAPLINC was able to act as a molecular sponge of miR-378 to modulate MAPK1 expression. The lncRNA GAPLINC expression was upregulated and positively correlated with MAPK1 expression in gastric cancer tissues and cells. Additionally, lncRNA GAPLINC promoted the expression of MAPK1 and the enhancement of GC cell proliferation and cell cycle progression by LncRNA GAPLINC was dependent on MAPK1 in vitro and in vivo. Consequently, we found that miR-378 expression was inversely correlated with GAPLINC expression in GC tissues and cells. miR-378 could directly bind to GAPLINC and decreased GAPLINC expression, thus reducing MAPK1 expression. Furthermore, overexpression of miR-378 inhibited MAPK1 expression, cell proliferation, and cell cycle progression of gastric cancer cells, while these effects were abrogated by upregulating lncRNA GAPLINC expression. Taken together, lncRNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells

PubMed Central

Sheng, Lingling; Mao, Xiyuan; Yu, Qingxiong; Yu, Dong

2017-01-01

Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor. PMID:28123468

Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms

PubMed Central

Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang

2014-01-01

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27Kip1, p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27Kip1 at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. PMID:25349217

Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms.

PubMed

Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang; Li, Rongshan

2015-07-01

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. © 2014 by the Society for Experimental Biology and Medicine.

Influence of Partial O₂ Pressure on the Adhesion, Proliferation, and Osteogenic Differentiation of Human Dental Pulp Stem Cells on β-Tricalcium Phosphate Scaffold.

PubMed

Viña-Almunia, Jose; Mas-Bargues, Cristina; Borras, Consuelo; Gambini, Juan; El Alami, Marya; Sanz-Ros, Jorge; Peñarrocha, Miguel; Vina, Jose

To analyze, in vitro, the influence of O₂ pressure on the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSC) on β-tricalcium phosphate (β-TCP) scaffold. DPSC, positive for the molecular markers CD133, Oct4, Nestin, Stro-1, and CD34, and negative for CD45, were isolated from extracted third molars. Experiments were started by seeding 200,000 cells on β-TCP cultured under 3% or 21% O₂ pressure. No osteogenic medium was used. Eight different cultures were performed at each time point under each O₂ pressure condition. Cell adhesion, proliferation, and differentiation over the biomaterial were evaluated at 7, 13, 18, and 23 days of culture. Cell adhesion was determined by light microscopy, proliferation by DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity analysis. DPSC adhered to β-TCP with both O₂ conditions. Cell proliferation was found from day 7 of culture. Higher values were recorded at 3% O₂ in each time point. Statistically significant differences were recorded at 23 days of culture (P = .033). ALP activity was not detectable at 7 days. There was, however, an increase in ALP activity over time in both groups. At 13, 18, and 23 days of culture, higher ALP activity was recorded under 3% O₂ pressure. Statistical differences were found at day 23 (P = .014). DPSC display capacity of adhering to β-TCP under 3% or 21% O₂ pressure conditions. Cell proliferation on β-TCP phosphate is significantly higher at 3% than at 21% O₂ pressure, the most frequently used O₂ tension. β-TCP can itself promote osteogenic differentiation of DPSC and is enhanced under 3% O₂ compared with 21%.

Branches of the NF-κB signaling pathway regulate proliferation of oval cells in rat liver regeneration.

PubMed

Zhao, W M; Qin, Y L; Niu, Z P; Chang, C F; Yang, J; Li, M H; Zhou, Y; Xu, C S

2016-03-24

The NF-kB (nuclear factor kB) pathway is involved in the proliferation of many cell types. To explore the mechanism of the NF-kB signaling pathway underlying the oval cell proliferation during rat liver regeneration, the Rat Genome 230 2.0 Array was used to detect expression changes of NF-kB signaling pathway-related genes in oval cells. The results revealed that the expression levels of many genes in the NF-kB pathway were significantly changed. This included 48 known genes and 16 homologous genes, as well as 370 genes and 85 homologous genes related to cell proliferation. To further understand the biological significance of these changes, an expression profile function was used to analyze the potential biological processes. The results showed that the NF-kB pathway promoted oval cell proliferation mainly through three signaling branches; the tumor necrosis factor alpha branch (TNF-a pathway), the growth factor branch, and the chemokine branch. An integrated statistics method was used to define the key genes in the NF-kB pathway. Seven genes were identified to play vital roles in the NF-kB pathway. To confirm these results, the protein content, including two key genes (TNF and FGF11) and two non-key genes (CCL2 and TNFRSF12A), were analyzed using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The results were generally consistent with those of the array data. To conclude, three branches and seven key genes were involved in the NF-kB signaling pathway that regulates oval cell proliferation during rat liver regeneration.

The impact of 27-hydroxycholesterol on endometrial cancer proliferation.

PubMed

Gibson, Douglas A; Collins, Frances; Cousins, Fiona L; Esnal Zufiaurre, Arantza; Saunders, Philippa T K

2018-04-01

Endometrial cancer (EC) is the most common gynaecological malignancy. Obesity is a major risk factor for EC and is associated with elevated cholesterol. 27-hydroxycholesterol (27HC) is a cholesterol metabolite that functions as an endogenous agonist for Liver X receptor (LXR) and a selective oestrogen receptor modulator (SERM). Exposure to oestrogenic ligands increases risk of developing EC; however, the impact of 27HC on EC is unknown. Samples of stage 1 EC ( n  = 126) were collected from postmenopausal women undergoing hysterectomy. Expression of LXRs ( NR1H3 , LXRα; NR1H2 , LXRβ) and enzymes required for the synthesis ( CYP27A1 ) or breakdown ( CYP7B1 ) of 27HC were detected in all grades of EC. Cell lines originating from well-, moderate- and poorly-differentiated ECs (Ishikawa, RL95, MFE 280 respectively) were used to assess the impact of 27HC or the LXR agonist GW3965 on proliferation or expression of a luciferase reporter gene under the control of LXR- or ER-dependent promoters (LXRE, ERE). Incubation with 27HC or GW3965 increased transcription via LXRE in Ishikawa, RL95 and MFE 280 cells ( P  < 0.01). 27HC selectively activated ER-dependent transcription ( P  < 0.001) in Ishikawa cells and promoted proliferation of both Ishikawa and RL95 cells ( P  < 0.001). In MFE 280 cells, 27HC did not alter proliferation but selective targeting of LXR with GW3965 significantly reduced cell proliferation ( P  < 0.0001). These novel results suggest that 27HC can contribute to risk of EC by promoting proliferation of endometrial cancer epithelial cells and highlight LXR as a potential therapeutic target in the treatment of advanced disease. © 2018 The authors.

Alterations in the proliferating compartment of gastric mucosa during Helicobacter pylori infection: the putative role of epithelial cells expressing p27(kip1).

PubMed

Sougioultzis, Stavros; Foukas, Periklis G; Tzivras, Michalis; Kourtessas, Dimitrios; Gorgoulis, Vassilis G; Davaris, Panayiotis; Archimandritis, Athanasios J

2003-11-01

The proliferating zone contains stem cells that give rise to all epithelial cells of the gastric mucosa. In the present study, we investigated the turnover of gastric epithelial cells in the proliferating zone of Helicobacter pylori-infected mucosa, with or without intestinal metaplasia, before and after eradication of the microorganism. In addition, we studied the topographical distribution of the cyclin dependent kinase inhibitor p27(Kip1), which plays a critical role in cell cycle progression and differentiation programs. Twenty-eight patients (22 male), aged 32-78 years and with dyspeptic symptoms, were endoscoped, and gastric biopsies were obtained from antrum and corpus for histopathological examination and the Campylobacter-like organisms test; eradication therapy was given to infected patients, and all patients were re-endoscoped after 105 +/- 33 days (mean +/- SD). The kinetics of gastric epithelial cells and p27(Kip1) status was assessed by means of immunohistochemistry and TUNEL (Tdt-mediated dUTP-biotin nick end labeling) assay. Twenty-one (21) of 28 patients were H. pylori positive, and 7 were found H. pylori negative and served as controls. In antrum, intestinal metaplasia was detected in 7/21 (33.3%). In H. pylori gastritis, Ki67 expression was found increased in the proliferating zone, compared with normal (P =.03); analogous results were obtained with the other proliferation markers, namely retinoblastoma protein and topoisomerase IIalpha. An inverse relationship between proliferation index and atrophy was disclosed (P =.02). A reduction in the proliferation index was observed after eradication, albeit not significant. Apoptotic epithelial cells were found significantly increased (P <.01) in H. pylori gastritis, and a significant reduction was observed after eradication (P <.01). In addition, apoptotic index was found to correlate with H. pylori density. The topographical study of p27(Kip1) revealed a p27(kip1)-positive epithelial cell population that resided deep in the proliferating zone; these cells were considered to be stem cells and were found significantly increased in areas with intestinal metaplasia (P <.05); in H. pylori gastritis, there was also an increase that did not reach statistical significance. H. pylori infection induces apoptosis and increases proliferation in the proliferating zone. The increased cellular turnover, together with the increased number of putative p27(Kip1)-positive stem cells in the context of intestinal metaplasia, provides further evidence for the role of H. pylori infection in gastric carcinogenesis.

[Effect of shikonin, a phytocompound from Lithospermum erythrorhizon, on rat vascular smooth muscle cells proliferation and apoptosis in vitro].

PubMed

Zhang, Zhuo-qi; Cao, Xi-chuan; Zhang, Ling; Zhu, Wen-ling

2005-06-08

To study the anti-proliferation, pro-apoptosis and cell cycle blocking effects of shikonin on rat vascular smooth muscle cell (VSMC) in vitro. VSMCs were primarily cultured by explant method from the thoracic aorta of male SD rats. Shikonin of different concentration, 4, 2, 1, 0.5, 0.25, and 0 micromol/L was added. The cell viability was detected by MTT method. Cell growth curve was drawn by trypan blue exclusion method. (3)H-thymidine incorporation was used to calculate the inhibition rate of DNA synthesis. Flow cytometry was used to detect the cell cycle. Cell apoptosis was observed by fluorescence microscopy. Western blotting was performed to detect the expression of different cell apoptosis and cell cycle regulatory proteins, such as cyclin D(1) and E, proliferating cell nuclear antigen (PCNA), p21(waf1/cip1), p27(kip1), and p53. Compared with control group, shikonin had no obvious cytotoxic effect on cell viability at the concentration of 0.25-1 micromol/L (P > 0.05). While it could inhibit, both time- and dose-dependently, the growth of VSMC, which was predominant of 1 micromol/L at 72 h (1.9 x 10(5)/well vs 5.8 x 10(5)/well, P < 0.05), and DNA synthesis was also significantly inhibited in a time- and dose-dependent manner with inhibition rate varied from 33 to 98% (P < 0.05 or P < 0.01). 1 micromol/L shikonin significantly blocked the cell cycle progression in proliferative VSMC, decreased S, G(2)/M phase (P < 0.05) and increased G(0)/G(1) phase (P < 0.05) to quiescent level with sub-G(1) apoptotic distribution at 48 h (10.9% +/- 0.3%). Shikohin at the concentration of 1-2 micromol/L significantly increased the percentage of apoptotic cells in a time- and dose-dependent manner compared with control group (2.8%-23.7% vs 0.2%-0.4%, P < 0.05), and typical apoptotic nuclear morphological changes were observed. 1 micromol/L shikonin significantly down-regulated cyclin D(1), E and PCNA expression, up-regulated p21(wif1/cip1) expression, and did not obviously influence the p27(kip1) and p53 expression. Shikonin inhibits the proliferation, promotes the apoptosis and blocks cell cycle progression of VSMC. These effects are associated with the expression changes of cell cycle regulatory proteins.

Trypanosoma congolense: tissue distribution of long-term T- and B-cell responses in cattle.

PubMed

Lutje, V; Taylor, K A; Boulangé, A; Authié, E

1995-11-01

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

T cell function in tuatara (Sphenodon punctatus).

PubMed

Burnham, D Kim; Keall, Susan N; Nelson, Nicola J; Daugherty, Charles H

2005-05-01

Tuatara are the sole survivors of an entire order of reptiles that thrived during the age of the dinosaurs. Therefore, knowledge of their physiology is critical to understanding the phylogeny of reptiles. Previous studies of the immune system of the tuatara did not assess T cell function. We analyzed T cell function among six captive tuatara by assessing concanavalin A (Con A), phytohemagglutinin (PHA) and mixed lymphocyte reaction (MLR) induced T cell proliferation. Peripheral blood mononuclear cells from six out of six and four out of four tuatara tested exhibited significant proliferative responses to Con A and PHA, respectively, as measured by an MTT reduction assay. A lower level of proliferation was detected in an MLR. However, Con A activated lymphocytes were not cytotoxic for a xenogeneic murine mastocytoma cell line (P815).

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

PubMed

Morita, Maresuke; Fujita, Naoki; Takahashi, Ayaka; Nam, Eun Ryel; Yui, Sho; Chung, Cheng Shu; Kawahara, Naoya; Lin, Hsing Yi; Tsuzuki, Keiko; Nakagawa, Takayuki; Nishimura, Ryohei

2015-01-01

To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively. © 2014 American College of Veterinary Ophthalmologists.

Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.

2006-09-10

Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis.more » Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.« less

SASH1 inhibits cervical cancer cell proliferation and invasion by suppressing the FAK pathway.

PubMed

Chen, Hui; Wang, Dongliang; Liu, Yuling

2016-04-01

SAM and SH3 domain containing 1 (SASH1), a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in several types of cancer. However, the role of SASH1 in cervical cancer remains to be elucidated. Therefore, in the present study, the role of SASH1 in cervical cancer and the underlying mechanism was investigated. Cell proliferation was detected by the MTT assay. Cell invasion was measured by Transwell assay. The mRNA expression levels of SASH1, matrix metalloproteinase (MMP)‑2 and MMP‑9 were determined by reverse transcription quantitative polymerase chain reaction. The protein expression levels of SASH1, MMP‑2, MMP‑9 and focal adhesion kinase (FAK) were determined by western blot analysis. The results demonstrated that SASH1 was downregulated in cervical cancer tissues and cell lines. Subsequently, a vector that overexpresses SASH1 was constructed. Overexpression of SASH1 was found to significantly inhibit cervical cancer cell proliferation and invasion, and also significantly reduce the expression of MMP‑2 and MMP‑9 in cancer cells. In addition, SASH1 modulated the FAK signaling pathway. Overexpression of SASH1 suppressed the expression of FAK in cancer cells. Taken together, the results suggested that SASH1 inhibits cervical cancer cell proliferation and invasion by suppressing the FAK pathway.

miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhi; Li, Youjun, E-mail: liyoujunn@126.com; Wang, Nan

miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and proteinmore » exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.« less

Mangiferin inhibition of proliferation and induction of apoptosis in human prostate cancer cells is correlated with downregulation of B-cell lymphoma-2 and upregulation of microRNA-182.

PubMed

Li, Minglin; Ma, Huili; Yang, Lixin; Li, Peng

2016-01-01

Mangiferin, a flavonoid extracted from the mango tree, possesses anti-inflammatory, antibacterial, anti-herpes simplex and antitumor activity, and is able to affect immune function. The present study investigated the anticancer effects of mangiferin treatment on PC3 human prostate cancer cells, and the potential underlying mechanisms. In the present study, an MTT assay was used to analyze the proliferation of PC3 cells. Subsequently, flow cytometry and colorimetric assay kits were utilized to measure the PC3 cell apoptotic rate. The expression levels of B-cell lymphoma-2 (Bcl-2) and microRNA-182 (miR-182) were detected using western blot analysis and quantitative reverse transcription-polymerase chain reaction, respectively. Finally, miR-182 and anti-miR-182 were transfected into PC3 cells, which were used to investigate the effects of mangiferin. Mangiferin treatment reduced the proliferation of PC3 human prostate cancer cells in a concentration- and time-dependent manner. In addition, mangiferin was able to promote apoptosis and induce the caspase-3 activity of PC3 human prostate cancer cells. Mangiferin treatment was also able to significantly reduce Bcl-2 expression levels and enhance miR-182 expression in PC3 cells. Finally, it was observed that mangiferin inhibited proliferation and induced apoptosis in PC3 human prostate cancer cells, and this effect was correlated with downregulation of Bcl-2 and upregulation of miR-182.

In vitro effects and mechanisms of lycopene in MCF-7 human breast cancer cells.

PubMed

Peng, S J; Li, J; Zhou, Y; Tuo, M; Qin, X X; Yu, Q; Cheng, H; Li, Y M

2017-04-13

Breast cancer adversely affects the health status of women; therefore, the prevention and treatment of breast cancer is of critical importance. Lycopene is known to possess several biological effects such as removal of free radicals, alleviation of biological oxidative injury, and inhibition of tumor growth. In this study, we aimed to illustrate the effect of lycopene on tumor cell proliferation and modulation of cancer progression as well as its possible underlying mechanisms in human breast carcinoma cell line MCF-7 in vitro. MCF-7 cells were treated with different lycopene concentrations for 24, 48, and 72 h. Light field microscopy was used to observe cell morphology. MTT assay was used to determine the effect of lycopene on MCF-7 proliferation. Flow cytometry was employed to evaluate cell apoptosis. Real-time quantitative polymerase chain reaction was performed to detect the expression of p53 and Bax. Under microscopic examination, the untreated MCF-7 cells appeared to have a diamond or polygonal shape. Lycopene treatment resulted in cell shrinkage and breakage, whose severity increased in a dose and duration dependent manner. In addition, reduced cell proliferation and increased apoptosis (P < 0.05) were observed using MTT assay and flow cytometry, respectively. Moreover, lycopene could also upregulate the expression of p53 and Bax mRNAs in MCF-7 cells. In conclusion, lycopene inhibits proliferation and facilitates apoptosis of MCF-7 cells in vitro, possibly by regulating the expression of p53 and Bax.

Effects of Blue Light Emitting Diode Irradiation On the Proliferation, Apoptosis and Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Yuan, Ye; Yan, Gege; Gong, Rui; Zhang, Lai; Liu, Tianyi; Feng, Chao; Du, Weijie; Wang, Ying; Yang, Fan; Li, Yuan; Guo, Shuyuan; Ding, Fengzhi; Ma, Wenya; Idiiatullina, Elina; Pavlov, Valentin; Han, Zhenbo; Cai, Benzhi; Yang, Lei

2017-01-01

Blue light emitting diodes (LEDs) have been proven to affect the growth of several types of cells. The effects of blue LEDs have not been tested on bone marrow-derived mesenchymal stem cells (BMSCs), which are important for cell-based therapy in various medical fields. Therefore, the aim of this study was to determine the effects of blue LED on the proliferation, apoptosis and osteogenic differentiation of BMSCs. BMSCs were irradiated with a blue LED light at 470 nm for 1 min, 5 min, 10 min, 30 min and 60 min or not irradiated. Cell proliferation was measured by performing cell counting and EdU staining assays. Cell apoptosis was detected by TUNEL staining. Osteogenic differentiation was evaluated by ALP and ARS staining. DCFH-DA staining and γ-H2A.X immunostaining were used to measure intracellular levels of ROS production and DNA damage. Both cell counting and EdU staining assays showed that cell proliferation of BMSCs was significantly reduced upon blue LED irradiation. Furthermore, treatment of BMSCs with LED irradiation was followed by a remarkable increase in apoptosis, indicating that blue LED light induced toxic effects on BMSCs. Likewise, BMSC osteogenic differentiation was inhibited after exposure to blue LED irradiation. Further, blue LED irradiation was followed by the accumulation of ROS production and DNA damage. Taken together, our study demonstrated that blue LED light inhibited cell proliferation, inhibited osteogenic differentiation, and induced apoptosis in BMSCs, which are associated with increased ROS production and DNA damage. These findings may provide important insights for the application of LEDs in future BMSC-based therapies. © 2017 The Author(s). Published by S. Karger AG, Basel.

MicroRNA-29c overexpression inhibits proliferation and promotes apoptosis and differentiation in P19 embryonal carcinoma cells.

PubMed

Liu, Ming; Chen, Yumei; Song, Guixian; Chen, Bin; Wang, Lihua; Li, Xing; Kong, Xiangqing; Shen, Yahui; Qian, Lingmei

2016-01-15

Compared to healthy controls, microRNA-29c (miR-29c) is highly expressed in the heart during progression towards ventricular septal defect. However, studies on miR-29c function in heart development are scarce. We investigated the role of miR-29c in P19 cell proliferation, apoptosis, and differentiation and the underlying mechanisms. We evaluated proliferation and cell cycle progression, detected morphological changes; apoptosis rate; BAX, BCL2, GATA binding protein 4 (GATA4), cardiac troponin T (cTnT), and myocyte enhancer factor 2C (MEF2C) expression; and caspase-3, -8, and -9 activity in miR-29c-overexpressing P19 cells, and investigated whether WNT4 was a miR-29c target. MiR-29c-overexpressing cells had decreased proliferation, increased G1 cells, and significantly higher apoptotic rate than the controls. Expression of the apoptosis-related BAX and BCL2 genes and caspase-3, -8, and -9 activity were significantly increased in miR-29c-overexpressing cells. Expression of the cardiac-specific markers GATA4, cTnT, and MEF2C revealed promoted differentiation in miR-29c-overexpressing cells compared to the controls. Luciferase assay confirmed that WNT4 is a miR-29c target. Wnt4 and β-catenin expression was decreased in miR-29c-overexpressing cells. MiR-29c inhibits P19 cell proliferation and promotes apoptosis and differentiation, possibly by suppressing Wnt4 signaling, whose deregulation contributes to congenital heart disease development. Copyright © 2015 Elsevier B.V. All rights reserved.

Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

PubMed

Martelly, I; Soulet, L; Bonnavaud, S; Cebrian, J; Gautron, J; Barritault, D

2000-11-01

In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.

Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

PubMed

Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

2011-01-01

Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

Experimental Study of Nasopharyngeal Carcinoma Radionuclide Imaging and Therapy Using Transferred Human Sodium/Iodide Symporter Gene

PubMed Central

Zhong, Xing; Shi, Changzheng; Gong, Jian; Guo, Bin; Li, Mingzhu; Xu, Hao

2015-01-01

Purpose The aim of this study was to design a method of radionuclide for imaging and therapy of nasopharyngeal carcinoma (NPC) using the transferred human sodium/iodide symporter (hNIS) gene. Methods A stable NPC cell line expressing hNIS was established (CNE-2-hNIS). After 131I treatment, we detected proliferation and apoptosis of NPC cells, both in vitro and vivo. In vivo, the radioactivity of different organs of nude mice was counted and 99mTc imaging using SPECT was performed. The apparent diffusion coefficient (ADC) value changes of tumor xenografts were observed by diffusion-weighted magnetic resonance imaging (DW-MRI) within 6–24 days of 131I treatment. The correlation of ADC changes with apoptosis and proliferation was investigated. Post-treatment expression levels of P53, Bax, Bcl-2, Caspase-3, and Survivin proteins were detected by western blotting. Results 131I uptake was higher in CNE-2-hNIS than in CNE-2 cells. The proliferation and apoptosis rate decreased and increased respectively both in vitro and vivo in the experimental group after 131I treatment. The experimental group tumors accumulated 99mTc in vivo, leading to a good visualization by SPECT. DW-MRI showed that ADC values increased in the experimental group 6 days after treatment, while ADC values were positively and negatively correlated with the apoptotic and Ki-67 proliferation indices, respectively. After treatment, CNE-2-hNIS cells up-regulated the expression of P53 and Survivin proteins and activated Caspase-3, and down-regulated the expression of Bcl-2 proteins. Conclusions The radionuclide imaging and therapy technique for NPC hNIS-transfected cell lines can provide a new therapy strategy for monitoring and treatment of NPC. PMID:25615643

[Protective effect and mechanism of compound Ginkgo biloba granules on oxidative stress injury of HUVEC].

PubMed

Li, Qi; Chen, Xi; Kan, Xiao-Xi; Li, Yu-Jie; Yang, Qing; Wang, Ya-Jie; Chen, Ying; Weng, Xiao-Gang; Cai, Wei-Yan; Huang, He-Fei; Zhu, Xiao-Xin

2016-02-01

To reveal the protective and anti-apoptosis effect of compound Ginkgo biloba granules on oxidative stress injury of human umbilical vein endothelial cells (HUVEC). Negative control group, H2O2 model group and 4 drug pretreatment groups (80, 160, 320, 640 mg• L⁻¹) were established. The cell proliferation, morphological changes in each group after oxidative stress injury was detected by MTT assay and through microscope observation respectively. The content of LDH, MDA, SOD and NO and SOD activity in supernatant were detected to judge the protection effect of the drugs on endothelial cells. The protective effect on HUVEC apoptosis was analyzed by Caspase-3 activity test and Annexin V-FITC/PI staining. Western blot was used to observe the expression of apoptosis-related proteins Bcl-2 and Bax. Results showed that 1 200 μmol• L⁻¹ H2O2 can induce oxidative stress injury in endothelial cells and reduce the cell survival rate; cell proliferation inhibition degree is positively correlated with the effect time of H2O2. Besides, 80, 160, 320 640 mg•L⁻¹ compound Ginkgo biloba granules can protect HUVEC from oxidative stress injury, recover the normal proliferation level of cells, improve their state, prohibit cell apoptosis, and can up-regulate and down-regulate the expression level of Bcl-2 and Bax respectively. In conclusion, compound G. biloba granules can protect HUVEC from the oxidative stress injury induced by H2O2, its mechanism may be correlated with inhibition of the mitochondrial apoptotic pathway in HUVEC. Copyright© by the Chinese Pharmaceutical Association.

Neurogenic effects of β-amyloid in the choroid plexus epithelial cells in Alzheimer's disease.

PubMed

Bolos, Marta; Spuch, Carlos; Ordoñez-Gutierrez, Lara; Wandosell, Francisco; Ferrer, Isidro; Carro, Eva

2013-08-01

β-amyloid (Aβ) can promote neurogenesis, both in vitro and in vivo, by inducing neural progenitor cells to differentiate into neurons. The choroid plexus in Alzheimer's disease (AD) is burdened with amyloid deposits and hosts neuronal progenitor cells. However, neurogenesis in this brain tissue is not firmly established. To investigate this issue further, we examined the effect of Aβ on the neuronal differentiation of choroid plexus epithelial cells in several experimental models of AD. Here we show that Aβ regulates neurogenesis in vitro in cultured choroid plexus epithelial cells as well as in vivo in the choroid plexus of APP/Ps1 mice. Treatment with oligomeric Aβ increased proliferation and differentiation of neuronal progenitor cells in cultured choroid plexus epithelial cells, but decreased survival of newly born neurons. These Aβ-induced neurogenic effects were also observed in choroid plexus of APP/PS1 mice, and detected also in autopsy tissue from AD patients. Analysis of signaling pathways revealed that pre-treating the choroid plexus epithelial cells with specific inhibitors of TyrK or MAPK diminished Aβ-induced neuronal proliferation. Taken together, our results support a role of Aβ in proliferation and differentiation in the choroid plexus epithelial cells in Alzheimer's disease.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

PubMed

Kalish, R S; Wood, J A; LaPorte, A

1994-05-01

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

PubMed Central

Kalish, R S; Wood, J A; LaPorte, A

1994-01-01

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis. Images PMID:7910172

Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

PubMed Central

Huang, Shengkai; Dong, Xin; Wang, Jia; Ding, Jie; Li, Yan; Li, Dongdong; Lin, Hong; Wang, Wenjie; Zhao, Mei

2018-01-01

Background Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells. PMID:29807370

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eto, Masumi, E-mail: masumi.eto@jefferson.edu; Kirkbride, Jason A.; Chugh, Rishika

2013-04-26

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentratedmore » in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.« less

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells.

PubMed

Crosswell, Hal E; Dasgupta, Anindya; Alvarado, Carlos S; Watt, Tanya; Christensen, James G; De, Pradip; Durden, Donald L; Findley, Harry W

2009-11-25

c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-gamma agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. c-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted.

Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimura, Hiroaki; Akiyama, Haruhiko; Nakamura, Takashi

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblasticmore » cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.« less

Naringin inhibits vascular endothelial cell apoptosis via endoplasmic reticulum stress- and mitochondrial-mediated pathways and promotes intraosseous angiogenesis in ovariectomized rats

PubMed Central

Li, Zhan-Chun; Tang, Lu-Min; Shao, Jiang; Li, He

2017-01-01

In this study, to investigate the effects of naringin on vascular endothelial cell (VEC) function, proliferation, apoptosis, and angiogenesis, rat VECs were cultured in vitro and randomly divided into four groups: control, serum-starved, low-concentration naringin treatment, and high-concentration naringin treatment. MTT assay was used to detect cell proliferation while Hoechst 33258 staining and flow cytometry were used to detect apoptosis. Changes in the expression of apoptosis-associated proteins [GRP78, CHOP, caspase-12, and cytochrome c (Cyt.c)] were detected using western blotting. JC-1 staining was employed to detect changes in mitochondrial membrane potential. Intracellular caspase-3, -8, and -9 activity was determined by spectrophotometry. ELISA was used to detect endothelin (ET), and a Griess assay was used to detect changes in the expression of nitric oxide (NO) in culture medium. The study further divided an ovariectomized (OVX) rat model of osteoporosis randomly into four groups: OVX, sham-operated, low-concentration naringin treatment (100 mg/kg), and high-concentration naringin treatment (200 mg/kg). After 3 months of treatment, changes in serum ET and NO expression, bone mineral density (BMD), and microvessel density of the distal femur (using CD34 labeling of VECs) were determined. At each concentration, naringin promoted VEC proliferation in a time- and dose-dependent manner. Naringin also significantly reduced serum starvation-induced apoptosis in endothelial cells, inhibited the expression of GRP78, CHOP, caspase-12, and Cyt.c proteins, and reduced mitochondrial membrane potential as well as reduced the activities of caspase-3 and -9. Furthermore, naringin suppressed ET in vitro and in vivo while enhancing NO synthesis. Distal femoral microvascular density assessment showed that the naringin treatment groups had a significantly higher number of microvessels than the OVX group, and that microvascular density was positively correlated with BMD. In summary, naringin inhibits apoptosis in VECs by blocking the endoplasmic reticulum (ER) stress- and mitochondrial-mediated pathways. Naringin also regulates endothelial cell function and promotes angiogenesis to exert its anti-osteoporotic effect. PMID:29039439

Naringin inhibits vascular endothelial cell apoptosis via endoplasmic reticulum stress‑ and mitochondrial‑mediated pathways and promotes intraosseous angiogenesis in ovariectomized rats.

PubMed

Shangguan, Wen-Ji; Zhang, Yue-Hui; Li, Zhan-Chun; Tang, Lu-Min; Shao, Jiang; Li, He

2017-12-01

In this study, to investigate the effects of naringin on vascular endothelial cell (VEC) function, proliferation, apoptosis, and angiogenesis, rat VECs were cultured in vitro and randomly divided into four groups: control, serum‑starved, low‑concentration naringin treatment, and high‑concentration naringin treatment. MTT assay was used to detect cell proliferation while Hoechst 33258 staining and flow cytometry were used to detect apoptosis. Changes in the expression of apoptosis‑associated proteins [GRP78, CHOP, caspase‑12, and cytochrome c (Cyt.c)] were detected using western blotting. JC‑1 staining was employed to detect changes in mitochondrial membrane potential. Intracellular caspase‑3, ‑8, and ‑9 activity was determined by spectrophotometry. ELISA was used to detect endothelin (ET), and a Griess assay was used to detect changes in the expression of nitric oxide (NO) in culture medium. The study further divided an ovariectomized (OVX) rat model of osteoporosis randomly into four groups: OVX, sham‑operated, low‑concentration naringin treatment (100 mg/kg), and high‑concentration naringin treatment (200 mg/kg). After 3 months of treatment, changes in serum ET and NO expression, bone mineral density (BMD), and microvessel density of the distal femur (using CD34 labeling of VECs) were determined. At each concentration, naringin promoted VEC proliferation in a time‑ and dose‑dependent manner. Naringin also significantly reduced serum starvation‑induced apoptosis in endothelial cells, inhibited the expression of GRP78, CHOP, caspase‑12, and Cyt.c proteins, and reduced mitochondrial membrane potential as well as reduced the activities of caspase‑3 and ‑9. Furthermore, naringin suppressed ET in vitro and in vivo while enhancing NO synthesis. Distal femoral microvascular density assessment showed that the naringin treatment groups had a significantly higher number of microvessels than the OVX group, and that microvascular density was positively correlated with BMD. In summary, naringin inhibits apoptosis in VECs by blocking the endoplasmic reticulum (ER) stress‑ and mitochondrial‑mediated pathways. Naringin also regulates endothelial cell function and promotes angiogenesis to exert its anti‑osteoporotic effect.

[Effect of Endomorphin-1 on Maturation and Expression of TLR4 in Peripheral Blood Dendritic Cells Induced by High Glucose].

PubMed

Liu, Chuan-Miao; Yang, Tian-Hua; Huang, Min; Zhou, Cheng; Li, Yong-Hai; Li, Zheng-Hong

2018-06-01

To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function. Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR. Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4. EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

PubMed Central

Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

[Notochord cells enhance proliferation and phenotype-keeping of intervertebral disc chondroid cells].

PubMed

Zhao, Xianfeng; Liu, Hao; Feng, Ganjun; Deng, Li; Li, Xiuqun; Liang, Tao

2008-08-01

To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to evaluate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell proliferation and phenotype. The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) and co-cultured with notochord cells (group B) (1:1), and cell proliferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 microm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 microm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P > 0.05). The co-cultured cells (group B) increased in cell proliferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell proliferation in group B was more than that in group A after 4-day culture (P < 0.05). The co-cultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. The notochord cells are conducive for the proliferation and phenotype-keeping of the chondroid cells and may play a key role in preventing degeneration of the disc.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-κB activation and promotes apoptosis in human leukaemia T-cell lines.

PubMed

Chen, Qian; Pang, Min-Hui; Ye, Xiao-Hong; Yang, Guang; Lin, Chen

2018-05-18

Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells. The Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting. Western blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively. Our data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Reciprocal Activation of Transcription Factors Underlies the Dichotomy between Proliferation and Invasion of Glioma Cells

PubMed Central

Dhruv, Harshil D.; McDonough Winslow, Wendy S.; Armstrong, Brock; Tuncali, Serdar; Eschbacher, Jenny; Kislin, Kerri; Loftus, Joseph C.; Tran, Nhan L.; Berens, Michael E.

2013-01-01

Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the “Go or Grow” hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the “Go or Grow” hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a “growing-to-going” phenotype. PMID:23967279

Effects of an imprinting procedure on cell proliferation in the chick brain.

PubMed

Komissarova, N V; Anokhin, K V

2008-03-01

We report here studies on the effects of an imprinting procedure on cell proliferation in neonatal chicks in brain structures known to undergo plastic changes in imprinting. Proliferating cells were detected immunohistochemically on brain sections by incorporation of pre-training doses of 5-bromodeoxyuridine (BrdU) into DNA; numbers of new cells were counted in the intermediate medial mesopallium, the intermediate arcopallium, the medial part of the mesopallium and the nidopallium, the dorsocaudal nidopallium, the hippocampus, and the parahippocampal region 24 h and seven days after training. The intermediate medial mesopallium showed an increase in the number of BrdU-positive cells 24 h after training. However, at seven days post-training, the number of BrdU-containing cells decreased in the medial nidopallium and mesopallium, in the dorsocaudal nidopallium, and the right intermediate medial mesopallium. Thus, the imprinting procedure had differently directed transient and long-term influences on the genesis of new cells in the chick brain, inducing the appearance of a large number of cells in the parenchyma of the brain one day after training and decreases in the numbers of cells at later time points. This double effect may be associated with the fact that the imprinting procedure simultaneously initiates two brain processes involving the control of cell proliferation - one related to maturation of a species-specific functional system for tracking individuals of the same species and one related to remembering the characteristics of the actual parent.

Transcription factor NF-kappaB participates in regulation of epithelial cell turnover in the colon.

PubMed

Inan, M S; Tolmacheva, V; Wang, Q S; Rosenberg, D W; Giardina, C

2000-12-01

The transcription factor nuclear factor (NF)-kappaB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-kappaB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-kappaB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-kappaB1 (p50) knockout mice were used to study the role of NF-kappaB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-kappaB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-kappaB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-alpha and nerve growth factor-alpha genes at elevated levels, with in situ hybridization localizing some of the TNF-alpha expression to epithelial cells. TNF-alpha is NF-kappaB regulated, and its upregulation in NF-kappaB1 knockouts may result from an alleviation of p50-p50 repression. NF-kappaB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.

Mifepristone sensitizing cisplatin for cervical adenocarcinoma HeLa cell sensitivity to chemotherapy and its mechanism.

PubMed

Li, Caihong; Ye, Hong

2013-01-01

The study was designed to investigate proliferation inhibition for cervical adenocarcinoma HeLa cell treated with cisplatin combined with mifepristone and access its possible mechanism. HeLa cell was processed by different concentrations of mifepristone, cisplatin, and their combination respectively. Cell's proliferation inhibition rate and induction apoptosis ability were detected by MTT assay, FCM; the expression of P53, survivin and HPV E6 protein were measured by Western Blot. The results showed that cisplatin inhibits proliferation of HeLa cells in different concentrations (p <0.01). Mifepristone had no effect on HeLa cell proliferation inhibition rate during 24 and 48 hours (p > 0.05). Mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can significantly enhance the inhibitory effect of cisplatin on HeLa cell line. Flow cytometry showed that mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can induce apparent apoptosis of HeLa cell line in concentration dependent manner. Western blotting demonstrated that the expression of P53 protein increased and the expression of HPV E6 survivin protein decreased in HeLa cells treated with MIF at low concentrations (< or = 10 micromol/l) combined with cisplatin. Mifepristone at low concentrations (< or = 10 micromol/1) can enhance chemosensitivity and capability of inducing apoptosis of cisplatin to HeLa cells. The strengthening effect of growth inhibition and chemosensitivity to cisplatin of mifepristone are associated with down-regulating HPV E6 survivin protein and upregulating p53 protein.

Clinicopathological and immunohistochemical characterization of papillary proliferation of the endometrium: A single institutional experience.

PubMed

Park, Cheol Keun; Yoon, Gun; Cho, Yoon Ah; Kim, Hyun-Soo

2016-06-28

Papillary proliferation of the endometrium is an unusual lesion that is composed of papillae with fibrovascular stromal cores covered with benign-appearing glandular epithelium. We studied the clinicopathological and immunohistochemical features of four cases of endometrial papillary proliferations. All patients were postmenopausal. Two lesions were incidental findings in hysterectomy specimens, and two lesions were detected in endometrial curettage specimens. Based on the degree of architectural complexity and extent of proliferation, we classified papillary proliferations histopathologically into "simple" or "complex" growth patterns. Three cases were classified as simple papillary proliferation, and one case was classified as complex papillary proliferation. Simple papillary proliferations were characterized by slender papillae with delicate stromal cores. In contrast, complex papillary proliferations had intracystic papillary projections and cellular clusters with frequent branching and occasional cytological atypia. All cases showed coexistent metaplastic epithelial changes, including mucinous metaplasia, eosinophilic cell change, and ciliated cell metaplasia. One patient with simple papillary proliferations had coexistent well-differentiated endometrioid carcinoma. One patient had subsequent hyperplasia without atypia, and another patient had subsequent atypical hyperplasia/endometrioid intraepithelial neoplasia; both patients underwent total hysterectomy within four months. Our observations are consistent with previous data demonstrating that endometrial papillary proliferations coexist with or develop into atypical hyperplasia/endometrioid intraepithelial neoplasia or endometrioid carcinoma. It is very important for pathologists to discriminate papillary proliferations from neoplastic lesions (including atypical hyperplasia/endometrioid intraepithelial neoplasia and well-differentiated endometrioid carcinoma) and benign mimickers (including papillary syncytial metaplasia).

Clinicopathological and immunohistochemical characterization of papillary proliferation of the endometrium: A single institutional experience

PubMed Central

Park, Cheol Keun; Yoon, Gun; Cho, Yoon Ah; Kim, Hyun-Soo

2016-01-01

Papillary proliferation of the endometrium is an unusual lesion that is composed of papillae with fibrovascular stromal cores covered with benign-appearing glandular epithelium. We studied the clinicopathological and immunohistochemical features of four cases of endometrial papillary proliferations. All patients were postmenopausal. Two lesions were incidental findings in hysterectomy specimens, and two lesions were detected in endometrial curettage specimens. Based on the degree of architectural complexity and extent of proliferation, we classified papillary proliferations histopathologically into “simple” or “complex” growth patterns. Three cases were classified as simple papillary proliferation, and one case was classified as complex papillary proliferation. Simple papillary proliferations were characterized by slender papillae with delicate stromal cores. In contrast, complex papillary proliferations had intracystic papillary projections and cellular clusters with frequent branching and occasional cytological atypia. All cases showed coexistent metaplastic epithelial changes, including mucinous metaplasia, eosinophilic cell change, and ciliated cell metaplasia. One patient with simple papillary proliferations had coexistent well-differentiated endometrioid carcinoma. One patient had subsequent hyperplasia without atypia, and another patient had subsequent atypical hyperplasia/endometrioid intraepithelial neoplasia; both patients underwent total hysterectomy within four months. Our observations are consistent with previous data demonstrating that endometrial papillary proliferations coexist with or develop into atypical hyperplasia/endometrioid intraepithelial neoplasia or endometrioid carcinoma. It is very important for pathologists to discriminate papillary proliferations from neoplastic lesions (including atypical hyperplasia/endometrioid intraepithelial neoplasia and well-differentiated endometrioid carcinoma) and benign mimickers (including papillary syncytial metaplasia). PMID:27322430

Long non-coding RNA GHET1 promotes human breast cancer cell proliferation, invasion and migration via affecting epithelial mesenchymal transition.

PubMed

Song, Rui; Zhang, Jia; Huang, Junhua; Hai, Tao

2018-05-11

Breast cancer is a common malignancy in women and long non-coding RNAs (lncRNAs) have been shown to play key roles in the development and progression of breast cancer. In the present study, we examined the biological role of lncRNA gastric carcinoma highly expressed transcript 1 (GHET1) in breast cancer. The expression of GHET1 was determined by qRT-PCR assay; CCK-8, colony formation, Transwell invasion and migration assays detected breast cancer cell proliferation, invasion and migration; cell apoptosis and cell cycle were determined by flow cytometry; protein levels were determined by western blot assay. GHET1 was up-regulated in breast cancer tissues and cell lines, and the up-regulation of GHET1 was positively correlated with larger tumor size, advanced clinical stage, lymph node metastasis and shorter overall survival. Knockdown of GHET1 suppressed cell proliferation, invasion and migration, and induced apoptosis and G0/G1 cell cycle arrest in MCF-cells. Knockdown of GHET1 also suppressed the protein levels of N-cadherin, vimentin, and decreased the protein level of E-cadherin in MCF-7 cells. On the other hand, overexpression of GHET1 promoted cell proliferation, invasion and migration, and inhibited cell apoptosis and increased cell population at S phase in BT-20 cells. Overexpression of GHET1 also promoted epithelial mesenchymal transition (EMT) in BT-20 cells. Furthermore, knockdown of GHET1 also suppressed in vivo tumor growth of MCF-7 cells, and also decreased the protein levels of N-cadherin and vimentin, and increased the protein levels of E-cadherin in the tumor tissues from the nude mice. Our results demonstrated that GHET1 was up-regulated in breast cancer tissues and cell lines, and promoted breast cancer cell proliferation, invasion and migration by affecting EMT. Our study for the first time revealed the biological functions of GHET1 in breast cancer.

Compounds from Cynomorium songaricum with Estrogenic and Androgenic Activities Suppress the Oestrogen/Androgen-Induced BPH Process.

PubMed

Wang, Xueni; Tao, Rui; Yang, Jing; Miao, Lin; Wang, Yu; Munyangaju, Jose Edouard; Wichai, Nuttapong; Wang, Hong; Zhu, Yan; Liu, Erwei; Chang, Yanxu; Gao, Xiumei

2017-01-01

To investigate the phytoestrogenic and phytoandrogenic activities of compounds isolated from CS and uncover the role of CS in prevention of oestrogen/androgen-induced BPH. Cells were treated with CS compounds, and immunofluorescence assay was performed to detect the nuclear translocation of ER α or AR in MCF-7 or LNCaP cells; luciferase reporter assay was performed to detect ERs or AR transcriptional activity in HeLa or AD293 cells; MTT assay was performed to detect the cell proliferation of MCF-7 or LNCaP cells. Oestrogen/androgen-induced BPH model was established in rat and the anti-BPH, anti-estrogenic, and anti-androgenic activities of CS in vivo were further investigated. The nuclear translocation of ER α was stimulated by nine CS compounds, three of which also stimulated AR translocation. The transcriptional activities of ER α and ER β were induced by five compounds, within which only ECG induced AR transcriptional activity as well. Besides, ECG stimulated the proliferation of both MCF-7 cells and LNCaP cells. CS extract suppressed oestrogen/androgen-induced BPH progress in vivo by downregulation of E2 and T level in serum and alteration of the expressions of ER α , ER β , and AR in the prostate. Our data demonstrates that compounds from CS exhibit phytoestrogenic and phytoandrogenic activities, which may contribute to inhibiting the oestrogen/androgen-induced BPH development.

Influence of tumor cell proliferation and sex-hormone receptors on effectiveness of radiation therapy for dogs with incompletely resected meningiomas.

PubMed

Théon, A P; Lecouteur, R A; Carr, E A; Griffey, S M

2000-03-01

To assess the influence of tumor cell proliferation and sex-hormone receptors on the efficacy of megavoltage irradiation for dogs with incompletely resected meningiomas. Longitudinal clinical trial. 20 dogs with incompletely resected intracranial meningiomas. Dogs were treated with 48 Gy of radiation administered 3 times per week on an alternate-day schedule of 4 Gy/fraction for 4 weeks, using bilateral parallel-opposed fields. Tumor proliferative fraction measured by immunohistochemical detection of proliferating cell nuclear antigen (PFPCNA index) ranged from 10 to 42% (median, 24%). Progesterone receptor immunoreactivity was detected in 70% of tumors. Estrogen receptor immunoreactivity was not detected. An inverse correlation was found between detection of progesterone receptors and the PFPCNA index. The overall 2-year progression-free survival (PFS) rate was 68%. The only prognostic factor that significantly affected PFS rate was the PFPCNA index. The 2-year PFS was 42% for tumors with a high PFPCNA index (value > or = 24%) and 91% for tumors with a low PFPCNA index (value < 24%). Tumors with a high PFPCNA index were 9.1 times as likely to recur as were tumors with a low PFPCNA index. This study confirms the value of irradiation for dogs with incompletely resected meningiomas. Prognostic value of the PFPCNA index suggests-that duration of treatment and interval from surgery to start of irradiation may affect outcome. Loss of progesterone receptors in some tumors may be responsible for an increase in PFPCNA index and may indirectly affect prognosis after radiation therapy.

Dihydroartemisinin increases temozolomide efficacy in glioma cells by inducing autophagy

PubMed Central

ZHANG, ZE-SHUN; WANG, JING; SHEN, YOU-BI; GUO, CHENG-CHENG; SAI, KE; CHEN, FU-RONG; MEI, XIN; HAN, FU; CHEN, ZHONG-PING

2015-01-01

Artemisinin, a powerful antimalarial medicine, is extracted from the Chinese herb, Artemisia annua L., and has the ability to inhibit the proliferation of cancer cells. Dihydroartemisinin (DHA), the major active metabolite of artemisinin, is able to inhibit the growth of a variety of types of human cancer. However, the effect of DHA on human glioma cells remains unclear. The aim of the present study was to investigate the effect of DHA on the proliferation of glioma cells, and whether DHA was able to enhance temozolomide (TMZ) sensitivity in vitro and in vivo. In total, 10 human glioma cell lines were used to analyze the growth inhibition ability of DHA by MTT assay. The typical autophagic vacuoles were monitored by the application of the autofluorescent agent, monodansylcadaverine. Western blotting was used to detect markers of apoptosis and autophagy, namely Caspase-3, Beclin-1 and LC3-B. The combination efficiency of DHA and TMZ was assessed in vitro and in vivo. The half maximal inhibitory concentration (IC50) of DHA differed among the ten human glioma cell lines. The number of autophagic vacuoles was higher in DHA-treated SKMG-4 cells; this was highest of all cell lines analyzed. The expression of autophagy molecular markers, Beclin-1 and LC3-B, was increased following DHA treatment, while no significant alteration was detected in the expression of apoptotic marker Caspase-3. When combined with DHA, the IC50 of TMZ decreased significantly in the four glioma cell lines analyzed. Furthermore, DHA enhanced the tumor inhibition ability of TMZ in tumor-burdened mice. The results of the present study demonstrated that DHA inhibited the proliferation of glioma cells and enhanced the tumor inhibition efficacy of TMZ in vitro and in vivo through the induction of autophagy. PMID:26171034

Adhesion and proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation.

PubMed

Rosso, Francesco; Marino, Gerardo; Muscariello, Livio; Cafiero, Gennaro; Favia, Pietro; D'Aloia, Erica; d'Agostino, Riccardo; Barbarisi, Alfonso

2006-06-01

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones. Copyright 2006 Wiley-Liss, Inc.

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

PubMed

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells through the Toll-like receptor 4/nuclear factor-κB pathway.

PubMed

Jiang, Ninghong; Xie, Feng; Guo, Qisang; Li, Ming-Qing; Xiao, Jingjing; Sui, Long

2017-06-01

Toll-like receptor 4 is overexpressed in various tumors, including cervical carcinoma. However, the role of Toll-like receptor 4 in cervical cancer remains controversial, and the underlying mechanisms are largely elusive. Therefore, Toll-like receptor 4 in cervical cancer and related mechanisms were investigated in this study. Quantitative reverse transcription polymerase chain reaction and western blot analyses were used to detect messenger RNA and protein levels in HeLa, Caski, and C33A cells with different treatments. Proliferation was quantified using Cell Counting Kit-8. Cell cycle distribution and apoptosis were assessed by flow cytometry. Higher levels of Toll-like receptor 4 expression were found in human papillomavirus-positive cells compared to human papillomavirus-negative cells. Proliferation of HeLa and Caski cells was promoted in lipopolysaccharide-stimulated groups but suppressed in short hairpin RNA-transfected groups. Apoptosis rates were lower in lipopolysaccharide-stimulated groups relative to short hairpin RNA-transfected groups. In addition, G2-phase distribution was enhanced when Toll-like receptor 4 was downregulated. Moreover, the pNF-κBp65 level was positively correlated with the Toll-like receptor 4 level in HeLa and Caski cells, though when an nuclear factor-κB inhibitor was applied to lipopolysaccharide-stimulated groups, the patterns of proliferation and apoptosis were opposite to those of the lipopolysaccharide-stimulated groups without inhibitor treatment. In conclusion, these data suggest that Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells at least in part through the Toll-like receptor 4/nuclear factor-κB pathway, which may be correlated with the occurrence and development of cervical carcinoma.

Insulin and IGF-I effects on the proliferation of an osteoblast primary culture from sea bream (Sparus aurata).

PubMed

Capilla, Encarnación; Teles-García, Agueda; Acerete, Laura; Navarro, Isabel; Gutiérrez, Joaquim

2011-05-15

Bone deformities in several fish species, like gilthead sea bream (Sparus aurata), are currently a major problem in aquaculture. To gain knowledge of fish skeletal development, a primary cell culture has been established from sea bream vertebra. The initial fibroblastic phenotype of the cells changed to a polygonal shape during the culture, and the addition of an osteogenic medium promoted the deposition of minerals in the extracellular matrix. Cell proliferation was analyzed using the MTT assay in control and mineralizing conditions at different culture days, up to day 20. The capacity of the cells to differentiate into osteoblasts was evaluated using Alizarin red stain. The cells showed slightly increased proliferation and differentiation in the presence of osteogenic medium. Furthermore, pluripotentiality of these cells was demonstrated by inducing them to differentiate into adipocytes, and the accumulation of lipids into the cells was detected with Oil Red O staining. Subsequently, the effects of insulin (1, 10, 100 and 1000 nM) and IGF-I (0.1, 1 and 10nM) on cell proliferation were evaluated with the MTT assay at day 3. Both peptides significantly stimulated the proliferation of the cells in a dose-dependent manner after either 24 or 48 h of incubation, with IGF-I apparently being more potent than insulin. In summary, a primary culture of sea bream osteoblasts has been characterized. This cellular system can be a good model to study the process of osteoblastogenesis in fish and its endocrine regulation, which may help to improve the quality of the product in aquaculture. Copyright © 2011 Elsevier Inc. All rights reserved.

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

PubMed

Coste, Alicia; Jager, Muriel; Chambon, Jean-Philippe; Manuel, Michaël

2016-01-01

The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

MiR-155 promotes cell proliferation and inhibits apoptosis by PTEN signaling pathway in the psoriasis.

PubMed

Xu, Longjiang; Leng, Hong; Shi, Xin; Ji, Jiang; Fu, Jinxiang; Leng, Hong

2017-06-01

MicroRNAs (miRNAs) have been demonstrated to contribute to malignant progression in psoriasis development. The purposes of the study was to evaluated the effects of miRNA-155 on cell proliferation, migration and apoptosis in psoriasis development via PTEN singaling pathway and identify its direct target protein. Quantitative real-time RT-PCR (qRT-PCR) was performed to examine the level of miR-155 in psoriasis cells, miR-155 was downregulated in a psoriasis cell line Hacat by transfected with small interfering RNA (siRNA), respectively. Cell survival was detected by the MTT assay and colony formation assay. Cell migration and invasion were measured via wound-healing assayand transwell assay. In addition, cell cycle and apoptosis about psoriasis cells was measured by flow cytometry. In this study, qRT-PCR assay showed that the expressions of miR-155 mRNA in psoriasis tissues were significantly higher than that in normal tissues. The assays about cell growth and proliferation showed that miR-155 knockdown led to a significant decrease in cell proliferation which was determined by MTT assay and colony formation assay compared to those of Lv-NC cells. Flow cytometry analysis showed that depletion of miR-155 could cause cell cycle change and the number of apoptotic cells was significantly increased in Lv-miR155 cells compared with control cells. In addition, the expression of several apoptosis-related factors were dramatically changed, such as PTEN, PIP 3 , AKT, p-AKT, Bax and Bcl-2. Our findings indicate that down-regulation of miR-155 significantly inhibits proliferation, migration, invasion and promotes apoptosis through PTEN singaling pathway in psoriasis cells. miR-155 might function as an oncogene miRNA in the progress of psoriasis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells.

PubMed

Cohn, Zachary J; Kim, Agnes; Huang, Liquan; Brand, Joseph; Wang, Hong

2010-06-10

The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS)-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues. Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6, in mouse circumvallate and foliate papillae. TNF-alpha and IFN-gamma immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds, the niche for taste progenitor cells. PCR array experiments showed that the expression of cyclin B2 and E2F1, two key cell cycle regulators, was markedly downregulated by LPS in the circumvallate and foliate epithelia. Our results show that LPS-induced inflammation inhibits taste progenitor cell proliferation and interferes with taste cell renewal. LPS accelerates cell turnover and modestly shortens the average life span of taste cells. These effects of inflammation may contribute to the development of taste disorders associated with infections.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-inducedmore » inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.« less

Formononetin induces the mitochondrial apoptosis pathway in prostate cancer cells via downregulation of the IGF-1/IGF-1R signaling pathway.

PubMed

Huang, Wen-Jun; Bi, Ling-Yun; Li, Zhen-Zhao; Zhang, Xing; Ye, Yu

2013-12-20

Abstract Context: Formononetin, an isoflavone, can inhibit the proliferation of cancer cells, including those of the prostate. However, its antitumor mechanism remains unclear. Aim: To investigate whether the insulin-like growth factor 1 (IGF-1)/insulin-like growth factor 1 receptor (IGF-1 R) signaling pathway mediates the formononetin antitumor effect on prostate cancer cells. Materials and methods: The viability of PC-3 cells was measured by MTT assay 48 h after formononetin treatment (25, 50 and 100 μM). Formononetin-induced cell apoptosis was measured by Hoechst 33258 staining and flow cytometry. Expression of Bax mRNA was detected by real-time PCR, and the expression levels of Bax and IGF-1 R proteins were detected by western blots. Results: At concentrations >12.5 μM, formononetin significantly inhibited the proliferation of human prostate cancer cells. Formononetin increased Bax mRNA and protein expression levels and decreased the expression levels of pIGF-1 R protein in a dose-dependent manner. Conclusion: High concentrations of formononetin-induced apoptosis in androgen-independent prostate cancer cells through inhibition of the IGF-1/IGF-1 R pathway.

Differential responses to high- and low-dose ultraviolet-B stress in tobacco Bright Yellow-2 cells

PubMed Central

Takahashi, Shinya; Kojo, Kei H.; Kutsuna, Natsumaro; Endo, Masaki; Toki, Seiichi; Isoda, Hiroko; Hasezawa, Seiichiro

2015-01-01

Ultraviolet (UV)-B irradiation leads to DNA damage, cell cycle arrest, growth inhibition, and cell death. To evaluate the UV-B stress–induced changes in plant cells, we developed a model system based on tobacco Bright Yellow-2 (BY-2) cells. Both low-dose UV-B (low UV-B: 740 J m−2) and high-dose UV-B (high UV-B: 2960 J m−2) inhibited cell proliferation and induced cell death; these effects were more pronounced at high UV-B. Flow cytometry showed cell cycle arrest within 1 day after UV-B irradiation; neither low- nor high-UV-B–irradiated cells entered mitosis within 12 h. Cell cycle progression was gradually restored in low-UV-B–irradiated cells but not in high-UV-B–irradiated cells. UV-A irradiation, which activates cyclobutane pyrimidine dimer (CPD) photolyase, reduced inhibition of cell proliferation by low but not high UV-B and suppressed high-UV-B–induced cell death. UV-B induced CPD formation in a dose-dependent manner. The amounts of CPDs decreased gradually within 3 days in low-UV-B–irradiated cells, but remained elevated after 3 days in high-UV-B–irradiated cells. Low UV-B slightly increased the number of DNA single-strand breaks detected by the comet assay at 1 day after irradiation, and then decreased at 2 and 3 days after irradiation. High UV-B increased DNA fragmentation detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay 1 and 3 days after irradiation. Caffeine, an inhibitor of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) checkpoint kinases, reduced the rate of cell death in high-UV-B–irradiated cells. Our data suggest that low-UV-B–induced CPDs and/or DNA strand-breaks inhibit DNA replication and proliferation of BY-2 cells, whereas larger contents of high-UV-B–induced CPDs and/or DNA strand-breaks lead to cell death. PMID:25954287

Non-small cell lung carcinoma therapy using mTOR-siRNA.

PubMed

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition.

Non-small cell lung carcinoma therapy using mTOR-siRNA

PubMed Central

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition. PMID:22400071

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

RSPO1/β-Catenin Signaling Pathway Regulates Oogonia Differentiation and Entry into Meiosis in the Mouse Fetal Ovary

PubMed Central

Chassot, Anne-Amandine; Gregoire, Elodie P.; Lavery, Rowena; Taketo, Makoto M.; de Rooij, Dirk G.; Adams, Ian R.; Chaboissier, Marie-Christine

2011-01-01

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1−/− gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm. PMID:21991325

Inhibition of proliferation and induction of apoptosis in soft tissue sarcoma cells by interferon-α and retinoids

PubMed Central

Brodowicz, T; Wiltschke, C; Kandioler-Eckersberger, D; Grunt, T W; Rudas, M; Schneider, S M; Hejna, M; Budinsky, A; Zielinski, C C

1999-01-01

Uncontrolled proliferation and a defect of apoptosis constitute crucial elements in the development and progression of tumours. Among many other biological response modifiers known to influence these mechanisms, the efficacy of retinoids and interferons in the treatment of various malignant entities is currently matter of discussion. In the present study, we have investigated the effects of 9-cis-retinoic acid (9cRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (tRA) and interferon-α on proliferation and apoptosis of human soft tissue sarcoma (STS) cell lines HTB-82 (rhabdomyosarcoma), HTB-91 (fibrosarcoma), HTB-92 (liposarcoma), HTB-93 (synovial sarcoma) and HTB-94 (chondrosarcoma) in relation to p53 genotype as well as p53 expression. HTB-91, HTB-92 and HTB-94 STS cells exhibited mutant p53, whereas wild-type p53 was found in HTB-93 STS cells, and a normal p53 status in HTB-82 STS cells, carrying a silent point mutation only. Interferon-α, irrespective of p53 status, inhibited the proliferation of all five cell lines dose- and time-dependently. Similarly, 9cRA, 13cRA and tRA decreased the proliferation of HTB-82 and HTB-93 STS cells, whereas the proliferation of p53-mutated HTB-91, HTB-92 and HTB-94 STS cells remained unchanged. Furthermore, only 9cRA and tRA were capable of inducing apoptosis in HTB-82 and HTB-93 STS cells, whereas HTB-91, HTB-92 and HTB-94 STS cells did not undergo apoptosis under the influence of 9cRA or tRA. Retinoic acid receptor (RAR)-α and RAR-β mRNA were not detectable by Northern blot analysis in the five STS cell lines, whereas mRNA for the universal retinoic acid receptor, RAR-γ, was expressed in all STS cell lines indicating that retinoid resistance was not associated with a lack of RAR expression. Apoptosis was not induced by interferon-α or 13cRA in any of the five STS cell lines tested. Our results indicate that within the panel of tested STS cell lines, inhibition of proliferation and induction of apoptosis result from different mechanisms which differ in their dependence upon the presence of intact p53. © 1999 Cancer Research Campaign PMID:10424735

[18β-glycyrrhetinic acid piperazine derivative A30 inhibits the proliferation of SMMC-7721 hepatoma cells].

PubMed

Zhong, Like

2017-09-01

Objective To investigate the mechanism of 18β-glycyrrhetinic acid (GA) piperazine derivative A30 on the antiproliferation of hepatocellular carcinoma SMMC-7721 cells in vitro. Methods The experiment included three groups: control group, 18β-GA group and A30 group. The proliferation activity was detected by MTT assay. Cell apoptosis and the change in the cycle of SMMC-7721 cells were evaluated by flow cytometry. Western blotting was used to observe the expressions of Bcl2 and caspase-8. Results The proliferation of SMMC-7721 cells was inhibited by A30 at the concentration of 2-128 μg/mL in a dose-dependent manner. 18β-GA and A30 could induce the apoptosis of SMMC-7721 cells, and the apoptosis rate of A30 group was significantly higher than that of the 18β-GA group. In the cell cycle analysis, the G2/M phase cells of 18β-GA and A30 groups increased remarkably as compared with the control group. A30 and 18β-GA could significantly enhance the expression of caspase-8, and decreased the expression of Bcl2. Conclusion The 18β-GA piperazine derivative A30 can inhibit the proliferation of SMMC-7721 cells in vitro, and the inhibitory effect is stronger than that of 18β-GA. The mechanism may be related to the inhibition of intracellular Bcl2 protein expression and the enhancement of caspase-8 expression.

PI3K-mediated proliferation of fibroblasts by Calendula officinalis tincture: implication in wound healing.

PubMed

Dinda, Manikarna; Dasgupta, Uma; Singh, Namrata; Bhattacharyya, Debasish; Karmakar, Parimal

2015-04-01

Calendula officinalis, a member of the Asteraceae family, is a flowering plant and has been used for its antibacterial, antifungal, antiviral, antiinflammatory, anticancer and wound healing activity. The mode of action of C. officinalis tincture on wound healing is poorly understood. Here, we investigated the role of C. officinalis tincture (CDOT) on cell viability and wound closure. C. officinalis tincture stimulated both proliferation and migration of fibroblasts in a statistically significant manner in a PI3K-dependent pathway. The increase in phosphorylation of FAK (Tyr 397) and Akt (Ser 473) was detected after treatment of CDOT. Inhibition of the PI3K pathway by wortmannin and LY294002 decreased both cell proliferation and cell migration. HPLC-ESI MS revealed the presence of flavonol glycosides as the major compounds of CDOT. Altogether, our results showed that CDOT potentiated wound healing by stimulating proliferation and migration of fibroblast in a PI3K-dependent pathway, and the identified compounds are likely to be responsible for wound healing activity. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of Cyclic Mechanical Stretch on the Proliferation of L6 Myoblasts and Its Mechanisms: PI3K/Akt and MAPK Signal Pathways Regulated by IGF-1 Receptor.

PubMed

Fu, Shaoting; Yin, Lijun; Lin, Xiaojing; Lu, Jianqiang; Wang, Xiaohui

2018-06-02

Myoblast proliferation is crucial to skeletal muscle hypertrophy and regeneration. Our previous study indicated that mechanical stretch altered the proliferation of C2C12 myoblasts, associated with insulin growth factor 1 (IGF-1)-mediated phosphoinositide 3-kinase (PI3K)/Akt (also known as protein kinase B) and mitogen-activated protein kinase (MAPK) pathways through IGF-1 receptor (IGF-1R). The purpose of this study was to explore the same stretches on the proliferation of L6 myoblasts and its association with IGF-1-regulated PI3K/Akt and MAPK activations. L6 myoblasts were divided into three groups: control, 15% stretch, and 20% stretch. Stretches were achieved using FlexCell Strain Unit. Cell proliferation and IGF-1 concentration were detected by CCK8 and ELISA, respectively. IGF-1R expression, and expressions and activities of PI3K, Akt, and MAPKs (including extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38) were determined by Western blot. We found that 15% stretch promoted, while 20% stretch inhibited L6 myoblast proliferation. A 15% stretch increased IGF-1R level, although had no effect on IGF-1 secretion of L6 myoblasts, and PI3K/Akt and ERK1/2 (not p38) inhibitors attenuated 15% stretch-induced pro-proliferation. Exogenous IGF-1 reversed 20% stretch-induced anti-proliferation, accompanied with increases in IGF-1R level as well as PI3K/Akt and MAPK (ERK1/2 and p38) activations. In conclusion, stretch regulated L6 myoblasts proliferation, which may be mediated by the changes in PI3K/Akt and MAPK activations regulated by IGF-1R, despite no detectable IGF-1 from stretched L6 myoblasts.

[In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

PubMed

Luo, Tingting; Yan, Aifen; Liu, Lian; Jiang, Hong; Feng, Cuilan; Liu, Guannan; Liu, Fang; Tang, Dongsheng; Zhou, Tianhong

2018-03-28

To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.
 Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay.
 Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P<0.01).
 Conclusion: The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.

Effects of corticosteroids and hyaluronic acid on torn rotator cuff tendons in vitro and in rats.

PubMed

Nakamura, Hidehiro; Gotoh, Masafumi; Kanazawa, Tomonoshin; Ohta, Keisuke; Nakamura, Keiichirou; Honda, Hirokazu; Ohzono, Hiroki; Shimokobe, Hisao; Mitsui, Yasuhiro; Shirachi, Isao; Okawa, Takahiro; Higuchi, Fujio; Shirahama, Masahiro; Shiba, Naoto; Matsueda, Satoko

2015-10-01

Corticosteroids (CS) or hyaluronic acid (HA) is used in subacromial injection for the conservative treatment of rotator cuff tears (RCT); this study addresses the question of how CS and HA affect the tendon tissue and fibroblasts in vitro and in rats. Cell proliferation assays were performed in human tendon fibroblasts from RCT. Rats underwent surgery to create RCT, and the surgical sites were injected with CS or HA. The rotator cuff tendons were subjected to biomechanical testing, microscopic and immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), and ultrastructural analysis. Cell proliferation was significantly decreased with CS in vitro (p < 0.05). Maximal load of CS-treated tendons was significantly decreased compared with that of HA-treated tendons (p < 0.05), as well as PCNA(+) cells at 2 weeks (p < 0.05). Ultrastructural observations of the CS-treated rats detected apoptosis of tendon fibroblasts 24 h after surgery. Histological and biomechanical data 4 weeks after surgery were not significant among the three groups. Unlike HA, CS caused cell death, and inhibition of the proliferation of tendon fibroblasts, leading to a delay of tendon healing involved and a subsequent decrease of biomechanical strength at the surgical site. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Hormesis effect of trace metals on cultured normal and immortal human mammary cells.

PubMed

Schmidt, Craig M; Cheng, Chun N; Marino, Angelo; Konsoula, Roula; Barile, Frank A

2004-06-01

An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDA-MB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green, and Celltiter blue fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine, did not alter the hormesis effect displayed by MCF-12A. Growth of these cells however was not maintained in the alternative medium. The study demonstrates that a hormesis effect from trace metals is detectable in cultured mammary cells; fluorescent indicators, however, are not as sensitive as cell proliferation or MTT in recognizing the subtle responses. Also, sensitivity of mammary cells to lower concentrations of Cu, a biologically important trace metal, may play an important role in controlling cellular processes and proliferation. The ability to detect this in vitro phenomenon implies that similar processes, occurring in vivo, may be responsible for the development, induction, or enhancement of human cancers.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

PubMed Central

Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wang, Hong; Fu, Hongyong; Zhou, Fan; Yao, Chencheng; Wang, Xiaobo; Li, Zheng; He, Zuping

2017-01-01

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine. PMID:28152522

Hyper-O-GlcNAcylation of YB-1 affects Ser102 phosphorylation and promotes cell proliferation in hepatocellular carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qingqing; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province; Tao, Tao

As an essential post-translational modification, O-GlcNAcylation has been thought to be able to modulate various nuclear and cytoplasmic proteins and is emerging as a key regulator of multiple biological processes, such as transcription, cell growth, signal transduction, and cell motility. Recently, authoritative glycomics analyses have reported extensive crosstalk between O-GlcNAcylation and phosphorylation, which always dynamically interplay with each other and regulate signaling, transcription, and other cellular processes. Also, plentiful studies have shown close correlation between YB-1 phosphorylation and tumorigenesis. Therefore, our study aimed to determine whether YB-1 was O-GlcNAc modified and whether such modification could interact with its phosphorylation duringmore » the process of HCC development. Western blot and immunohistochemistry were firstly conducted to reveal obvious up-regulation of YB-1, OGT and O-GlcNAc modification in HCC tissues. What is more, not only YB-1 was identified to be O-GlcNAcylated but hyper-O-GlcNAcylation was demonstrated to facilitate HCC cell proliferation in a YB-1 dependent manner. Moreover, we detected four specific O-GlcNAc sites and confirmed T126A to be the most effective mutant in HCC cell proliferation via close O-GlcNAcylation-phosphorylation interaction. Even more interestingly, we discovered that T126A-induced HCC cell retardation and subdued transcriptional activity of YB-1 could be partially reversed by T126A/S102E mutant. From all above, it is not difficult to find that glycosylated-YB-1 mainly enhanced cell proliferation through congenerous actions with YB-1 phosphorylation and thus played indispensable roles in fine-tuning cell proliferation and procession of HCC. - Highlights: • YB-1 and OGT are associated with HCC prognosis. • YB-1 is O-GlcNAc modified in HCC. • Hyper-O-GlcNAcylation promotes HCC cell proliferation in dependent of YB-1. • The proliferating role of O-GlcNAcylation is based on Ser102 phosphorylation of YB-1.« less

Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA

PubMed Central

2012-01-01

Background Proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening. Methods Rat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay. Results MTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery. Conclusions The STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation. PMID:22216901

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

PubMed

Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca 2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals. © 2017 American Heart Association, Inc.

Cholecystokinin-8-induced hypoplasia of the rat pancreas: influence of nitric oxide on cell proliferation and programmed cell death.

PubMed

Trulsson, Lena M; Gasslander, Thomas; Svanvik, Joar

2004-10-01

The background of cholecystokinin-8 (CCK-8)-induced hypoplasia in the pancreas is not known. In order to increase our understanding we studied the roles of nitric oxide and NF-kappaB in rats. CCK-8 was injected for 4 days, in a mode known to cause hypoplasia, and the nitric oxide formation was either decreased by means of N(omega)-nitro-L-arginine (L-NNA) or increased by S-nitroso-N-acetylpencillamine (SNAP). The activation of NF-kappaB was quantified by ELISA detection, apoptosis with caspase-3 and histone-associated DNA-fragmentation and mitotic activity in the acinar, centroacinar and ductal cells were visualized by the incorporation of [(3)H]-thymidine. Pancreatic histology and weight as well as protein- and DNA contents were also studied. Intermittent CCK injections reduced pancreatic weight, protein and DNA contents and increased apoptosis, acinar cell proliferation and nuclear factor kappaB (NF-kappaB) activation. It also caused vacuolisation of acinar cells. The inhibition of endogenous nitric oxide formation by L-NNA further increased apoptosis and NF-kappaB activation but blocked the increased proliferation and vacuolisation of acinar cells. The DNA content was not further reduced. SNAP given together with CCK-8 increased apoptosis and other pathways of cell death, raised proliferation of acinar cells and strongly reduced the DNA content in the pancreas. Histological examination showed no inflammation in any group. We conclude that during CCK-8-induced pancreatic hypoplasia, endogenously formed nitric oxide suppresses apoptosis but increases cell death along non-apoptotic pathways and stimulates regeneration of acinar cells. Exogenous nitric oxide enhances the acinar cell turnover by increasing both apoptotic and non-apoptotic cell death and cell renewal. In this situation NF-kappaB activation seems not to inhibit apoptosis nor promote cell proliferation.

Silencing of Urothelial Carcinoma Associated 1 Inhibits the Proliferation and Migration of Medulloblastoma Cells.

PubMed

Zhengyuan, Xie; Hu, Xiao; Qiang, Wang; Nanxiang, Li; Junbin, Cai; Wangming, Zhang

2017-09-16

BACKGROUND UCA1 is a long non-coding RNA that has been found to be aberrantly upregulated in various cancers. The aim of this study was to determine the expression level and function of UCA1 in medulloblastoma, the most common malignant brain tumor during childhood. MATERIAL AND METHODS Real-time PCR was used to detect the expression of UCA1 in medulloblastoma specimens and cell lines. Lentiviral-mediated expression of a short hairpin RNA (shRNA) targeting UCA1 or a negative control shRNA was also achieved with the medulloblastoma cell line, Daoy. Cell proliferation and cell cycle progression were subsequently characterized with cell counting kit (CCK)-8 and flow cytometry. Cell migration was examined in wound healing and Transwell migration assays. RESULTS Levels of UCA1 mRNA were higher in the medulloblastoma specimens (p<0.05) and cell lines (p<0.05) compared to the corresponding nontumor adjacent tissue specimens and a glioblastoma cell line, respectively. For the Daoy cells with silenced UCA1, their proliferation was reduced by 30% compared to the Daoy cells expressing a negative control shRNA (p=0.017). Cell cycle arrest in the G0/G1 phase, resulting in a decreased number of cells in the S phase, as well as reduced cell migration in both wound scratch healing (p=0.001) and Transwell migration assays (p=0.021) were also observed for the Daoy cells with silenced UCA1. CONCLUSIONS UCA1 was highly expressed in part of medulloblastoma specimens and cell lines examined. In addition, knockdown of UCA1 significantly inhibited the proliferation and migration of medulloblastoma cells in vitro.

[Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells].

PubMed

Zhang, Yuanyuan; Liu, Zhe; Zhang, Qianwen; Chao, Zhenhua; Zhang, Pei; Xia, Fei; Jiang, Chenchen; Liu, Hao; Jiang, Zhiwen

2013-09-01

To study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism. MTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay. MTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells. 3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

The effects of electromagnetic fields on B16-BL6 cells are dependent on their spatial and temporal character.

PubMed

Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

2017-04-01

Exposure to low intensity, low frequency electromagnetic fields (EMF) has effects on several biological systems. Spatiotemporal characteristics of these EMFs are critical. The effect of several complex EMF patterns on the proliferation of B16-BL6 mouse melanoma cells was tested. Exposure to one of these patterns, the Thomas-EMF, inhibited cell proliferation and promoted calcium uptake. The Thomas-EMF is coded from a digital-to-analog file comprised of 849 points, which provides power to solenoids and can be set to alter timing, intensity, and duration of variable EMF. Setting the point duration to 3 ms generated a time-varying EMF pattern which began at 25 Hz and slowed to 6 Hz over a 2.5 s repeat. Exposing B16-BL6 cells to Thomas-EMF set to 3 ms for 1 h/day inhibited cell proliferation by 40% after 5 days, while setting the point duration to 1, 2, 4, or 5 ms had no effect on cell proliferation. Similarly, exposing cells to Thomas-EMF set to 3 ms promoted a three-fold increase in calcium uptake after 1 h, while the other timings had no effect. Exposure to Thomas-EMF for as short as 15 min/day slowed cell proliferation, but exposure for 1 h/day was optimal. This corresponded to the effect on calcium uptake where uptake was detected after 15 min exposure and was maximal by 1 h of treatment. Studies show that the specific spatiotemporal character of EMF is critical in mediating their biological activities. Bioelectromagnetics. 38:165-174, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Effects of Electromagnetic Radiation from Smartphones on Learning Ability and Hippocampal Progenitor Cell Proliferation in Mice.

PubMed

Choi, Yu-Jin; Choi, Yun-Sik

2016-02-01

Nonionizing radiation is emitted from electronic devices, such as smartphones. In this study, we intended to elucidate the effect of electromagnetic radiation from smartphones on spatial working memory and progenitor cell proliferation in the hippocampus. Both male and female mice were randomly separated into two groups (radiated and control) and the radiated group was exposed to electromagnetic radiation for 9 weeks and 11 weeks for male and female mice, respectively. Spatial working memory was examined with a Y maze, and proliferation of hippocampal progenitor cells were examined by 5-bromo-2'-deoxyuridine administration and immunohistochemical detection. When spatial working memory on a Y maze was examined in the 9(th) week, there was no significant difference in the spontaneous alternation score on the Y maze between the two groups. In addition, there was no significant difference in hippocampal progenitor cell proliferation. However, immunoreactivity to glial fibrillary acidic protein was increased in exposed animals. Next, to test the effect of recovery following chronic radiation exposure, the remaining female mice were further exposed to electromagnetic radiation for 2 more weeks (total 11 weeks), and spontaneous alternation was tested 4 weeks later. In this experiment, although there was no significant difference in the spontaneous alternation scores, the number of arm entry was significantly increased. These data indicate that although chronic electromagnetic radiation does not affect spatial working memory and hippocampal progenitor cell proliferation it can mediate astrocyte activation in the hippocampus and delayed hyperactivity-like behavior.

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

PubMed

Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

2013-12-01

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

[Effect of P38MAPK signal transduction pathway on apoptosis of THP-1 induced by allicin].

PubMed

Liao, Yang; Chen, Jianbin; Tang, Weixue; Ge, Qunfang; Lu, Qianwei; Yang, Zesong

2009-06-01

The objective of this paper was to study the change of P38MAPK and Fas in the apoptosis of THP-1 cells induced by allicin. The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay. Apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry. The expression and distribution change of the phosphorylation p38MAPK (P-p38MAPK) were detected by immunohistochemical staining. The changes of P-p38 MAPK and Fas proteins were detected by Western blot. The proliferations of leukemia cell line THP-1 are inhibited by allicin. MTT assay showed that allicin can inhibit the proliferation of the THP-1 cell, and the inhibition was dependent on both dose and time. The IC50 of 72 hours was 12.8 mg x L(-1). Apoptosis rate detected by Annexin V-FITC/PI was proportional to the concentration of the allicin. After the immunohistochemical staining test, the P-p38MAPK was located in the cell nucleus and plasma, showing deep brown, when adding allicin to THP-1 cell. Western blot test showed that the P-p38MAPK proteins expression was proportional to the concentration of Allicin and was also dose dependent. The levels of P-p38MAPK in negative control group, 1/2 IC50 of 72 hours group and IC50 of 72 hours group were 0.259 8 +/- 0.013 2, 0.61 2 +/- 0.008 3 and 0.505 6 +/- 0.005 5 respectively, and the levels of Fas proteins were 0.287 4 +/- 0.008 9, 0.426 8 +/- 0.007 9 and 0.597 1 +/- 0.010 9 respectively. The difference was statistically significant when compared with the negative control group (P < 0.01). Allicin can significantly induce THP-1 cells apoptosis, and its mechanism may be related to the activation of P38MAPK/Fas.

Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

PubMed Central

Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

2012-01-01

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer. PMID:22745580

Evolutionarily Conserved Epitopes on Human Immunodeficiency Virus Type 1 (HIV-1) and Feline Immunodeficiency Virus Reverse Transcriptases Detected by HIV-1-Infected Subjects

PubMed Central

Sanou, Missa P.; Roff, Shannon R.; Mennella, Antony; Sleasman, John W.; Rathore, Mobeen H.; Levy, Jay A.

2013-01-01

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV+) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV+ subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8+ T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8+ T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV+ subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine. PMID:23824804

Molecular Pathways: Is AMPK a Friend or a Foe in Cancer?

PubMed Central

Hardie, D. Grahame

2015-01-01

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status expressed in essentially all eukaryotic cells. Once activated by energetic stress via a mechanism that detects increases in AMP:ATP and ADP:ATP ratios, AMPK acts to restore energy homeostasis by switching on catabolic pathways that generate ATP, while switching off ATP-consuming processes, including anabolic pathways required for cell growth and proliferation. AMPK activation promotes the glucose-sparing, oxidative metabolism utilized by most quiescent cells, rather than the rapid glucose uptake and glycolysis used by most proliferating cells. Numerous pharmacological activators of AMPK are known, including drugs in long use such as salicylate and metformin, and there is evidence that regular use of either of the latter provides protection against development of cancer. Tumor cells appear to be under selection pressure to down-regulate AMPK, thus limiting its restraining influence on cell growth and proliferation, and several interesting mechanisms by which this occurs are discussed. Paradoxically, however, a complete loss of AMPK function, which appears to be rare in human cancers, may be deleterious to survival of tumor cells. AMPK can therefore either be a friend and a foe in cancer, depending on the context. PMID:26152739

Pleiotrophin antagonizes Brd2 during neuronal differentiation

PubMed Central

Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J.; Garcia-Dominguez, Mario

2014-01-01

ABSTRACT Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system. PMID:24695857

Taspine downregulates VEGF expression and inhibits proliferation of vascular endothelial cells through PI3 kinase and MAP kinase signaling pathways.

PubMed

Zhao, Jing; Zhao, Le; Chen, Wei; He, Langchong; Li, Xu

2008-01-01

Taspine is an active component isolated from Radix et Rhizoma Leonticis with inhibiting tumor angiogenic properties. The molecular mechanism(s) of taspine on tumor angiogenic inhibition have not been well documented. The aim of this study was to elucidate in detail the effects of taspine on genetic expressions of VEGF in human umbilical vein endothelial cells, and on VEGFR2-mediated intracellular signaling of human umbilical vein endothelial cells. The genetic expression of vascular endothelial growth factor (VEGF) in the human umbilical vein endothelial cells (HUVECs) treated with taspine in vitro was measured by the ELISA and RT-PCR methods. The effects of taspine on cell proliferation of HUVECs and HUVECs induced by VEGF165 were considered by using MTT assay. And also, a western blot was used to detect Akt and Erk1/2 expressions and their phosphorylation levels in HUVECs treated with taspine. Our results show that VEGF protein and mRNA expressions in the cells treated with taspine were significantly decreased. Taspine also significantly inhibited cell proliferation of HUVECs induced by VEGF165. HUVECs treated with taspine showed decreased Akt and Erk1/2 activities.

MiR-217 promoted the proliferation and invasion of glioblastoma by repressing YWHAG.

PubMed

Wang, Hongbin; Zhi, Hua; Ma, Dongzhou; Li, Tao

2017-04-01

To study the effects of miR-217 on glioblastoma cell proliferation, migration and invasion and its regulation on YWHAG. QRT-PCR was used to detect the expression of related mRNAs and miRNA in both glioblastoma tissues and cells. Western blot was used to determine the protein expression of related genes. The transfection was performed using lipo2000. MTT assay, colony formation assay, wound healing assay, Transwell assay as well as flow cytometry were employed to determine the viability, proliferation, migration, invasion and mitosis of UG87 MG cell line. Besides, the dual luciferase reporter gene assay was used to determine the direct targeting relationship between miR-217 and YWHAG. Xenograft models were also constructed and the effect of miR-217 on tumor growth was studied in vivo. MiR-217 was up-regulated, whereas YWHAG was down-regulated in glioblastoma tissues and cells. The down-regulation of miR-217 or the up-regulation of YWHAG suppressed the viability, proliferation, migration, invasion and mitosis of U87 MG cells in vitro. In addition, MiR-217 directly targeted 3'UTR of YWHAG and suppressed the expression of YWHAG. Up-regulation of miR-217 could efficiently attenuate the inhibitory effects of YWHAG overexpression on the proliferation and metastasis of U87 MG cells. YWHAG was able to accelerate the phosphorylation of MDM4 and lead to the degradation of P53, which provides a potential mechanism for the tumor-promoting role of miR-217 in glioblastoma cells. By constructing xenograft models, it was also confirmed that miR-217 could promote tumor growth in vivo. MiR-217 could promote the viability, proliferation, migration, invasion and mitosis of glioblastoma cells both in vitro and in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of role of Notch3 signaling pathway in human lung cancer cells.

PubMed

Hassan, Wael Abdo; Yoshida, Ryoji; Kudoh, Shinji; Motooka, Yamato; Ito, Takaaki

2016-05-01

There is still a debate on the extent to which Notch3 signaling is involved in lung carcinogenesis and whether such function is dependent on cancer type or not. To evaluate Notch3 expression in different types of human lung cancer cells. Notch3 was detected in human lung cancer cell lines and in tissues. Then, small interfering RNA (siRNA) was used to down-regulate the expression of Notch3 in H69AR small cell lung carcinoma (SCLC) cells; two non-small cell lung carcinoma (NSCLC) cells; A549 adenocarcinoma (ADC); and H2170 squamous cell carcinoma (SCC). In addition, Notch3 intracellular domain (N3ICD) plasmid was transfected into H1688 human SCLC cells. We observed the effect of deregulating Notch3 signaling on the following cell properties: Notch-related proteins, cell morphology, adhesion, epithelial-mesenchymal transition (EMT), motility, proliferation and neuroendocrine (NE) features of SCLC. Notch3 is mainly expressed in NSCLC, and the expression of Notch1, Hes1 and Jagged1 is affected by Notch3. Notch3 has opposite functions in SCLC and NSCLC, being a tumor suppressor in the former and tumor promoting in the latter, in the context of cell adhesion, EMT and motility. Regarding cell proliferation, we found that inhibiting Notch3 in NSCLC decreases cell proliferation and induces apoptosis in NSCLC. Notch3 has no effect on cell proliferation or NE features of SCLC. Notch3 signaling in lung carcinoma is dependent on cell type. In SCLC, Notch3 behaves as a tumor suppressor pathway, while in NSCLC it acts as a tumor-promoting pathway.

Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience

PubMed Central

Kokovic, Ira; Novakovic, Barbara Jezersek; Cerkovnik, Petra; Novakovic, Srdjan

2014-01-01

Background Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. Materials and methods. With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. Results The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. Conclusions In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value. PMID:24991205

All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes.

PubMed

Schroeder, M; Zouboulis, C C

2007-02-01

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.

NFATC3-PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines.

PubMed

Jang, Jee-Eun; Kim, Hwang-Phill; Han, Sae-Won; Jang, Hoon; Lee, Si-Hyun; Song, Sang-Hyun; Bang, Duhee; Kim, Tae-You

2018-06-14

This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer lines. We performed paired-end RNA sequencing of 28 colorectal cancer (CRC) cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. 1,380 FT candidates were detected through bioinformatics filtering. We selected 6 candidate FTs, including 4 inter-chromosomal and 2 intra-chromosomal FTs and each FT was found in at least 1 of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in 2. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Local application of periodontal ligament stromal cells promotes soft tissue regeneration.

PubMed

Baik, H S; Park, J; Lee, K J; Chung, C

2014-09-01

To test the potential stimulatory effect of local application of periodontal ligament (PDL) stromal cells on soft tissue regeneration. Fluorescently labeled PDL cells outgrown from extracted human premolars or phosphate-buffered saline were locally injected to the cutaneous wounds created on mice. Soft tissue regeneration was evaluated for 14 days using photographs and histomorphometry. PDL cell engraftment was tracked with confocal microscopy. To detect the paracrine effect of the PDL cells on soft tissue regeneration, PDL cell-conditioned medium (CM) was evaluated for the concentration of secretory factors, transforming growth factor-beta 1 (TGFβ1). The effect of PDL CM on the proliferation and migration of dermal fibroblast and keratinocyte was tested using MTT assay and migration assay. The application of PDL cells significantly promoted soft tissue regeneration compared with the application of PBS. Self-replicating PDL cells were engrafted into the hair follicles of the host tissue. Dermal fibroblast proliferation and keratinocyte migration were significantly enhanced by the treatment with PDL CM. Physiologically significant amount of TGFβ1 was secreted from PDL cells into the CM. Local injection of PDL cells promoted soft tissue regeneration in part by the enhancement of fibroblast proliferation and keratinocyte migration through a paracrine mechanism. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

PubMed Central

Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

2013-01-01

Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

MiR-338-5p Promotes Inflammatory Response of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via Targeting SPRY1.

PubMed

Yang, Yan; Wang, Yanfeng; Liang, Qingwei; Yao, Lutian; Gu, Shizhong; Bai, Xizhuang

2017-08-01

Our purpose is to study the roles of microRNA-338-5p (miR-338-5p) on the proliferation, invasion, and inflammatory response of fibroblast-like synoviocytes (SFs) in rheumatoid arthritis patients by regulating SPRY1. The target relationship between miR-338-5p and SPRY1 was validated through luciferase reporter system. The expression of miR-338-5p and SPRY1 in synovial tissues and synovial cells were detected using RT-PCR and western blot. The mimics and inhibitors of miR-338-5p were transfected into SFs. MTT, Transwell, and ELISA assays were used to analyze cell proliferation, invasiveness, and the secreted extracellular pro-inflammatory cytokines (such as IL-1a, IL-6, COX2) levels of SFs. MiR-338-5p was highly expressed in rheumatoid arthritis tissues and cells, and directly down-regulated the expression of SPRY1 in the SFs of rheumatoid arthritis patients. Cell proliferation, invasiveness and the expression level of pro-inflammatory cytokines in synovial cells increased after the transfection of miR-338-5p mimics, while the proliferation, invasion and expression level of pro-inflammatory cytokines decreased after the transfection of miR-338-5p inhibitors. In conclusion,miR-338-5p promoted the proliferation, invasion and inflammatory reaction in SFs of rheumatoid arthritis by directly down-regulating SPRY1 expression. J. Cell. Biochem. 118: 2295-2301, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Store-Operated Ca2+ Entry Does Not Control Proliferation in Primary Cultures of Human Metastatic Renal Cellular Carcinoma

PubMed Central

Turin, Ilaria; Potenza, Duilio Michele; Bottino, Cinzia; Glasnov, Toma N.; Ferulli, Federica; Mosca, Alessandra; Guerra, Germano; Rosti, Vittorio; Luinetti, Ombretta; Porta, Camillo; Pedrazzoli, Paolo

2014-01-01

Store-operated Ca2+ entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca2+ levels within the endoplasmic reticulum (ER) Ca2+ reservoir, and a number of a Ca2+-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1–7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca2+ store, but not by InsP3-dependent Ca2+ release. Metastatic RCC cells express Stim1-2, Orai1–3, and TRPC1–7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd3+ and Pyr6, while it was inhibited by 100 µM Gd3+, 2-APB, and carboxyamidotriazole (CAI). Neither Gd3+ nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca2+ signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors. PMID:25126575

Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

PubMed

Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

2015-07-22

Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopalan, Vinod; Smith, Robert A.; Lam, Alfred K.-Y., E-mail: a.lam@griffith.edu.au

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expressionmore » by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA's effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas. - Highlights: • miR-498 is a non-coding RNA located in 19q13.41. • Colon cancer cell lines showed reduced expression of miR-498. • Mimic driven over expression of miR-498 in colon cancer cells resulted in lower cell proliferation. • miR-498 expression was down regulated in all colorectal adenocarcinoma tissues.« less

Long non-coding RNA Gm2199 rescues liver injury and promotes hepatocyte proliferation through the upregulation of ERK1/2.

PubMed

Gao, Qiang; Gu, Yunyan; Jiang, Yanan; Fan, Li; Wei, Zixiang; Jin, Haobin; Yang, Xirui; Wang, Lijuan; Li, Xuguang; Tai, Sheng; Yang, Baofeng; Liu, Yan

2018-05-22

Long non-coding RNAs (lncRNAs) are a new class of regulators of various human diseases. This study was designed to explore the potential role of lncRNAs in experimental hepatic damage. In vivo hepatic damage in mice and in vitro hepatocyte damage in AML12 and NCTC1469 cells were induced by carbon tetrachloride (CCl 4 ) treatments. Expression profiles of lncRNAs and mRNAs were analyzed by microarray. Bioinformatics analyses were conducted to predict the potential functions of differentially expressed lncRNAs with respect to hepatic damage. Overexpression of lncRNA Gm2199 was achieved by transfection of the pEGFP-N1-Gm2199 plasmid in vitro and adeno-associated virus-Gm2199 in vivo. Cell proliferation and viability was detected by cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assay. Protein and mRNA expressions of extracellular signal-regulated kinase-1/2 (ERK1/2) were detected by western blot and quantitative real-time reverse-transcription PCR (qRT-PCR). Microarray analysis identified 190 and 148 significantly differentially expressed lncRNAs and mRNAs, respectively. The analyses of lncRNA-mRNA co-expression and lncRNA-biological process networks unraveled potential roles of the differentially expressed lncRNAs including Gm2199 in the pathophysiological processes leading to hepatic damage. Gm2199 was downregulated in both damaged livers and hepatocyte lines. Overexpression of Gm2199 restored the reduced proliferation of damaged hepatocyte lines and increased the expression of ERK1/2. Overexpression of Gm2199 also promoted the proliferation and viability of normal hepatocyte lines and increased the level of p-ERK1/2. Overexpression of Gm2199 in vivo also protected mouse liver injury induced by CCl 4 , evidenced by more proliferating hepatocytes, less serum alanine aminotransferase, less serum aspartate aminotransferase, and decreased hepatic hydroxyproline. The ability of Gm2199 to maintain hepatic proliferation capacity indicates it as a novel anti-liver damage lncRNA.

Effects of C-myc gene silencing on interleukin-1β-induced rat chondrocyte cell proliferation, apoptosis and cytokine expression.

PubMed

Zou, Jian; Li, Xiao-Lin; Shi, Zhong-Min; Xue, Jian-Feng

2018-05-01

This study explores the effects of C-myc gene silencing on cell proliferation, apoptosis and cytokine expression in interleukin (IL)-1β-induced rat chondrocytes. Primary chondrocytes were obtained from 40 Sprague-Dawley rats. For in vitro C-myc3-shRNA transfection, chondrocytes were assigned to a blank 1, model 1, IL-1β + C-myc3-shRNA, C-myc3-shRNA, (IL-1β + C-myc3-shRNA) + C-myc overexpression, C-myc3-shRNA + C-myc overexpression or IL-1β + C-myc-Con group. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect C-myc, PCNA and cyclin D1 mRNA and protein expression. Cell proliferation was analyzed via CCK-8 assay and cell cycle while apoptosis was measured through flow cytometry. ELISA was utilized to assess the levels of metallopeptidase 13 (MMP-13), IL-6 and tumor necrosis factor-α (TNF-α). Both the qRT-PCR and Western blotting results demonstrated that C-myc3-shRNA transfection inhibits C-myc expression and promotes PCNA and cyclin D1 expression. In comparison to the model 1 group, all groups except the (IL-1β + C-myc3-shRNA) + C-myc overexpression and IL-1β + C-myc-Con groups showed increases in cell proliferation and S phase cell count and decreases in G 0 /G 1 phase cell count, cell apoptosis and MMP-13, IL-6 and TNF-α levels. The model 1, C-myc3-shRNA and C-myc3-shRNA + C-myc overexpression groups displayed higher cell proliferation and S phase cell count and reduced G 0 /G 1 phase cell count, cell apoptosis and MMP-13, IL-6 and TNF-α levels than the IL-1β + C-myc3-shRNA group. In comparison to the model 1 and C-myc3-shRNA + C-myc overexpression groups, the C-myc3-shRNA group promoted cell proliferation and S phase cell counts but suppressed G 0 /G 1 phase cell count, cell apoptosis and MMP-13, IL-6 and TNF-α levels. In conclusion, the study demonstrates that C-myc gene silencing can promote cell proliferation and inhibit cell apoptosis and cytokine expression in IL-1β-induced rat chondrocytes.

Interferon-γ regulates the function of mesenchymal stem cells from oral lichen planus via indoleamine 2,3-dioxygenase activity.

PubMed

Zhang, Zhihui; Han, Ying; Song, Jiangyuan; Luo, Ruxi; Jin, Xin; Mu, Dongdong; Su, Sha; Ji, Xiaoli; Ren, Yan-Fang; Liu, Hongwei

2015-01-01

Little is known about mesenchymal stem cells (MSCs) in normal or inflammatory oral mucosal tissues, such as in oral lichen planus (OLP). Our objectives were to identify, isolate, and characterize MSCs from normal human oral mucosa and OLP lesions, and to evaluate indoleamine 2,3 dioxygenase (IDO) activity in mediating immunomodulation of MSCs from these tissues. Expressions of MSCs-related markers were examined in isolated cells by flow cytometry. Self-renewal and multilineage differentiations were studied to characterize these MSCs. Interferon-γ (IFN-γ), IDO, and STRO-1 were assessed by immunofluorescence. MSCs from oral mucosa and OLP or IFN-γ-pretreated MSCs were co-cultured with allogeneic mixed lymphocyte reaction assays (MLR). Proliferation and apoptosis of MLR or MSCs were detected by CCK8 and the annexin V-FITC apoptosis detection kit, respectively. IDO expression and activity were measured by real-time PCR, Western blotting, and high-performance liquid chromatography. Isolated cells from oral mucosa and OLP expressed MSC-related markers STRO-1, CD105, and CD90 but were absent for hematopoietic stem cell markers CD34. Besides, they all showed self-renewal and multilineage differentiation capacities. MSCs in OLP presented STRO-1/IDO+ phenotype by immunofluorescence. MSCs and IFN-γ-pretreated MSCs could inhibit lymphocyte proliferation via IDO activity, but not via cell apoptosis. Long-term IFN-γ could also inhibit MSC proliferation via IDO activity. Mesenchymal stem cells can be isolated from human oral mucosa and OLP tissues. Besides self-renewal and multilineage differentiation properties, these cells may participate in immunomodulation mediated by IFN-γ via IDO activity in human OLP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multichannel series piezoelectric quartz crystal cell sensor for real time and quantitative monitoring of the living cell and assessment of cytotoxicity.

PubMed

Tong, Feifei; Lian, Yan; Zhou, Huang; Shi, Xiaohong; He, Fengjiao

2014-10-21

A new multichannel series piezoelectric quartz crystal (MSPQC) cell sensor for real time monitoring of living cells in vitro was reported in this paper. The constructed sensor was used successfully to monitor adhesion, spreading, proliferation, and apoptosis of MG63 osteosarcoma cells and investigate the effects of different concentrations of cobalt chloride on MG63 cells. Quantitative real time and dynamic cell analyses data were conducted using the MSPQC cell sensor. Compared with methods such as fluorescence staining and morphology observation by microscopy, the MSPQC cell sensor is noninvasive, label free, simple, cheap, and capable of online monitoring. It can automatically record the growth status of cells and quantitatively evaluate cell proliferation and the apoptotic response to drugs. It will be a valuable detection and analysis tool for the acquisition of cellular level information and is anticipated to have application in the field of cell biology research or cytotoxicity testing in the future.

Composition for detection of cell density signal molecule

DOEpatents

Schwarz, Richard I.

2001-01-01

Disclosed herein is a novel proteinaceous cell density signal molecule (CDS), which is secreted by fibroblastic cells in culture, preferably tendon cells, and which provides a means by which the cells self-regulate their proliferation and the expression of differentiated function. CDS, and the antibodies which recognize them, are important for the development of diagnostics and treatments for injuries and diseases involving connective tissues, particularly tendon. Also disclosed are methods of production and use.

Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

PubMed

Liu, Qicai; Li, Ermao; Huang, Long; Cheng, Minsheng; Li, Li

2018-01-01

Background: The limb-bud and heart gene ( LBH ) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M. Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay. Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells. Conclusions : Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might be a therapeutic target and potential diagnostic marker in PCa.

[A preliminary study for the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells mixture 3D bio-printing].

PubMed

Song, Y; Wang, X F; Wang, Y G; Dong, F; Lv, P J

2016-10-18

To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis. P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×10 6 /mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed. The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group. Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.

Daucosterol promotes the proliferation of neural stem cells.

PubMed

Jiang, Li-hua; Yang, Nian-yun; Yuan, Xiao-lin; Zou, Yi-jie; Zhao, Feng-ming; Chen, Jian-ping; Wang, Ming-yan; Lu, Da-xiang

2014-03-01

Neural stem cells (NSCs) are self-regenerating cells, but their regenerative capacity is limited. The present study was conducted to investigate the effect of daucosterol (a sterolin) on the promotion of NSC proliferation and determine the corresponding molecular mechanism. Results of cell counting kit-8 (CCK-8) assay showed that daucosterol significantly increased the quantity of viable cells and the effectiveness of daucosterol was similar to that of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Flow cytometry detection of CFSE-labeled (CFSE, carboxyfluorescein diacetate succinimidyl ester) NSCs showed that Div Index (or the average number of cell divisions) and % Divided (or the percentage of cells that divided at least once) of the cells were increased, indicating that daucosterol increased the percentage of NSCs re-entering the cell cycle. mRNA microarray analysis showed that 333 genes that are mostly involved in the mitotic cell cycle were up-regulated. By contrast, 627 genes that are mostly involved in differentiation were down-regulated. In particular, insulin-like growth factor I (IGF1) was considered as an important regulatory gene that functionally promoted NSC proliferation, and the increased expression of IGF1 protein was validated by ELISA. In addition, the phosphorylation of AKT was increased, indicating that the proliferation-enhancing activity of daucosterol may be involved in IGF1-AKT pathway. Our study provided information about daucosterol as an efficient and inexpensive growth factor alternative that could be used in clinical medicine and research applications. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain.

PubMed

Noonin, Chadanat; Lin, Xionghui; Jiravanichpaisal, Pikul; Söderhäll, Kenneth; Söderhäll, Irene

2012-11-20

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

PubMed Central

Li, Yue-Hui; Liu, Yan; Li, Yan-Dong; Liu, Yan-Hong; Li, Feng; Ju, Qiang; Xie, Ping-Li; Li, Guan-Cheng

2012-01-01

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang’s liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC. PMID:22690081

A novel mouse model for neurotrophic keratopathy: trigeminal nerve stereotactic electrolysis through the brain.

PubMed

Ferrari, Giulio; Chauhan, Sunil K; Ueno, Hiroki; Nallasamy, Nambi; Gandolfi, Stefano; Borges, Lawrence; Dana, Reza

2011-04-01

To develop a mouse model of neurotrophic keratopathy by approaching the trigeminal nerve through the brain and to evaluate changes in corneal cell apoptosis and proliferation. Six- to 8-week-old male C57BL/6 mice underwent trigeminal stereotactic electrolysis (TSE) to destroy the ophthalmic branch of the trigeminal nerve. Clinical follow-up using biomicroscopy of the cornea was performed at days 2, 4, 5, and 7. To confirm the effectiveness of the procedure, we examined the gross nerve pathology, blink reflex, and immunohistochemistry of the corneal nerves. TUNEL-positive apoptotic and Ki-67-positive proliferating corneal cells were evaluated to detect changes from the contralateral normal eye. TSE was confirmed by gross histology of the trigeminal nerve and was considered effective if the corneal blink reflex was completely abolished. TSE totally abolished the blink reflex in 70% of mice and significantly reduced it in the remaining 30%. Animals with absent blink reflex were used for subsequent experiments. In these mice, a progressive corneal degeneration developed, with thinning of the corneal epithelium and eventually perforation after 7 days. In all mice, 48 hours after TSE, corneal nerves were not recognizable histologically. Seven days after TSE, an increase in cellular apoptosis in all the corneal layers and a reduction in proliferation in basal epithelial cells were detected consistently in all mice. TSE was able, in most cases, to induce a disease state that reflected clinical neurotrophic keratitis without damaging the periocular structures. Moreover, corneal denervation led to increased apoptosis and reduced proliferation of epithelial cells, formally implicating intact nerve function in regulating epithelial survival and turnover.

A Novel Mouse Model for Neurotrophic Keratopathy: Trigeminal Nerve Stereotactic Electrolysis through the Brain

PubMed Central

Ferrari, Giulio; Chauhan, Sunil K.; Ueno, Hiroki; Nallasamy, Nambi; Gandolfi, Stefano; Borges, Lawrence

2011-01-01

Purpose. To develop a mouse model of neurotrophic keratopathy by approaching the trigeminal nerve through the brain and to evaluate changes in corneal cell apoptosis and proliferation. Methods. Six- to 8-week-old male C57BL/6 mice underwent trigeminal stereotactic electrolysis (TSE) to destroy the ophthalmic branch of the trigeminal nerve. Clinical follow-up using biomicroscopy of the cornea was performed at days 2, 4, 5, and 7. To confirm the effectiveness of the procedure, we examined the gross nerve pathology, blink reflex, and immunohistochemistry of the corneal nerves. TUNEL-positive apoptotic and Ki-67–positive proliferating corneal cells were evaluated to detect changes from the contralateral normal eye. Results. TSE was confirmed by gross histology of the trigeminal nerve and was considered effective if the corneal blink reflex was completely abolished. TSE totally abolished the blink reflex in 70% of mice and significantly reduced it in the remaining 30%. Animals with absent blink reflex were used for subsequent experiments. In these mice, a progressive corneal degeneration developed, with thinning of the corneal epithelium and eventually perforation after 7 days. In all mice, 48 hours after TSE, corneal nerves were not recognizable histologically. Seven days after TSE, an increase in cellular apoptosis in all the corneal layers and a reduction in proliferation in basal epithelial cells were detected consistently in all mice. Conclusions. TSE was able, in most cases, to induce a disease state that reflected clinical neurotrophic keratitis without damaging the periocular structures. Moreover, corneal denervation led to increased apoptosis and reduced proliferation of epithelial cells, formally implicating intact nerve function in regulating epithelial survival and turnover. PMID:21071731

Dynamic Analysis of the Expression of the TGFβ/SMAD2 Pathway and CCN2/CTGF during Early Steps of Tooth Development

PubMed Central

Pacheco, Marcos S.; Reis, Alice H.; Aguiar, Diego P.; Lyons, Karen M.; Abreu, José G.

2009-01-01

Background/Aims CCN2 is present during tooth development. However, the relationship between CCN2 and the transforming growth factor β (TGFβ)/SMAD2/3 signaling cascade during early stages of tooth development is unclear. Here, we compare the expression of CCN2 and TGFβ/SMAD2/3 components during tooth development, and analyze the functioning of TGFβ/SMAD2/3 in wild-type (WT) and Ccn2 null (Ccn2−/−) mice. Methods Coronal sections of mice on embryonic day (E)11.5, E12.5, E13.5, E14.5 and E18.5 from WT and Ccn2−/− were immunoreacted to detect CCN2 and components of the TGFβ signaling pathway and assayed for 5′-bromo-2′-deoxyuridine immunolabeling and proliferating cell nuclear antigen immunostaining. Results CCN2 and TGFβ signaling components such as TGFβ1, TGFβ receptor II, SMADs2/3 and SMAD4 were expressed in inducer tissues during early stages of tooth development. Proliferation analysis in these areas showed that epithelial cells proliferate less than mesenchymal cells from E11.5 to E13.5, while at E14.5 they proliferate more than mesenchymal cells. We did not find a correlation between functioning of the TGFβ1 cascade and CCN2 expression because Ccn2−/− mice showed neither a reduction in SMAD2 phosphorylation nor a difference in cell proliferation. Conclusion CCN2 and the TGFβ/SMAD2/3 signaling pathway are active in signaling centers of tooth development where proliferation is dynamic, but these mechanisms may act independently. PMID:18089935

Comparison of Analysis and Quantification of Cell Death in vivo and in vitro

DTIC Science & Technology

1985-05-01

mammalian somatic cells appear to have a finite life span that is genetically programmed ( Hayflick , 1977). Following the consummation of this program... limited situations it is possible to evaluate the proliferation kinetics of cell populations in tis- sues by autoradiographically detecting radiolabeled...are, therefore, virtually limited to the analysis of toxicity of directly active chemicals. Primary cultures of target cells retain the ability to

HOXC6 promotes gastric cancer cell invasion by upregulating the expression of MMP9.

PubMed

Chen, Shi-Wei; Zhang, Qing; Xu, Zhi-Feng; Wang, Hai-Ping; Shi, Yi; Xu, Feng; Zhang, Wen-Jian; Wang, Ping; Li, Yong

2016-10-01

Previous studies have demonstrated that the homoebox C6 (HOXC6) gene is highly expressed in gastric cancer tissues and is associated with the depth of tumor invasion, and is associated with poor prognosis of gastric cancer patients expressing HOXC6. The present study investigated the effect and underlying mechanism of HOXC6 on the proliferation and metastasis of gastric cancer cells in vitro. Reverse transcription‑quantitative polymerase chain (PCR) reaction was used to investigate the expression levels of HOXC6 in different gastric cancer cell lines and the effect of different levels of expression on the proliferation of gastric cancer cells was determined by cell growth curve and plate colony formation. The effect of HOXC6 on the anchorage‑independent proliferation of gastric cancer cells was determined by soft agar colony formation assay while the Transwell invasion assay was used to investigate the effect of different levels of HOXC6 expression on the invasive and metastatic abilities of gastric cancer cells. Semi‑quantitative PCR was used to detect the effect of different levels of HOXC6 expression on the expression of matrix metalloproteinase (MMP)2 and MMP9 in gastric cancer cells. Immunoblotting was used to assess MMP9 signaling in the gastric cancer cells. The HOXC6 gene is highly expressed in the majority of the gastric cancer cell lines. Overexpression of HOXC6 promoted gastric cancer cell proliferation and colony formation ability while HOXC6 downregulation inhibited cell proliferation and clone forming ability. HOXC6 overexpression also enhanced the soft agar colony formation ability of gastric cancer cells while HOXC6 downregulation decreased the colony formation ability. Upregulated HOXC6 increased the migration and invasion abilities of gastric cancer cells while interfering with HOXC6 expression inhibited the migration and invasion of the gastric cancer cells. The expression of MMP9 was enhanced with an upregulation of HOXC6 expression while HOXC6 downregulation lowered MMP9 gene expression levels. Increased expression of HOXC6 in gastric cancer cell lines significantly activated extracellular signal‑regulated kinase signaling and upregulated MMP9. The HOXC6 gene promotes the proliferation of gastric cancer cells while upregulation of MMP9 promotes migration and invasion of gastric cancer cells.

The effects of focal adhesion kinase on the motility, proliferation and apoptosis of Caco2 and SMMC-7721 cells.

PubMed

Gao, Ning; He, Huimin; Xiao, Li; Gao, Xiaojie; Shi, Hongyan; Wu, Qiong; Xu, Nan; Lei, Yuqing; Song, Xigui; Hou, Yingchun

2015-04-01

Focal adhesion kinase (FAK) plays important roles in cancer development. However, the significance of FAK expression in colorectal carcinoma and hepatocellular carcinoma has not been clarified. This study aims to explore the roles FAK played in the progression of colorectal carcinoma and hepatocellular carcinoma. RNAi method was used to inhibit the expression of FAK in Caco2 and SMMC-7721 cells. Reverse transcriptase polymerase chain reaction analysis and Western blot analysis were used to examine mRNA and protein expression of FAK. Then, the proliferation, motility and apoptosis of both types of cells were detected using MTT assay, wound healing/transwell assay and nuclear staining assay. The microstructure changes (F-actin, β-tubulin and lamin B1) of SMMC-7721 cells were visualized by immunofluorescence. FAK was overexpressed in both cell lines and down-regulation of FAK resulted in suppression of cell proliferation, inhibition of cell migration and invasion. The apoptosis of cells was increased significantly following the FAK expression inhibition. Moreover, actin polymerization, β-tubulin and lamin B1 expression of cells were significantly decreased. The results highlight the role of FAK in the progression of cancers. These findings suggest FAK serve as a potential therapeutic target for cancer therapy.

MiR-129-5p Inhibits Proliferation and Invasion of Chondrosarcoma Cells by Regulating SOX4/Wnt/β-Catenin Signaling Pathway.

PubMed

Zhang, Peng; Li, Jifeng; Song, Yuze; Wang, Xiao

2017-01-01

Recently, microRNAs (miRNA) have been identified as novel regulators in Chondrosarcoma (CHS). This study was aimed to identify the roles of miR-129-5p-5p in regulation of SOX4 and Wnt/β-catenin signaling pathway, as well as cell proliferation and apoptosis in chondrosarcomas. Tissue samples were obtained from chondrosarcoma patients. Immunohistochemistry, real-time quantitative RT-PCR (RT-qPCR) and western blot analysis were performed to detect the expressions of miR-129-5p and SOX4. Luciferase assay was conducted to confirm that miR-129-5p directly targeted SOX4 mRNA. Manipulations of miR-129-5p and SOX4 expression were achieved through cell transfection. Cell proliferation, migration and apoptosis were evaluated by CCK-8 assay, colony forming assay, wound healing assay and flow cytometry in vitro. For in vivo experiment, the tumor xenograft model was established to evaluate the effects of miR-129-5p and SOX4 on chondrosarcomas. The expression of miR-129-5p was significantly down-regulated in chondrosarcoma tissues as well as cells in comparison with normal ones, while SOX4 was over-activated. Further studies suggested that miR-129-5p suppressed cell proliferation, migration and promoted apoptosis by inhibiting SOX4 and Wnt/β-catenin pathway. MiR-129-5p inhibits the Wnt/β-catenin signaling pathway by targeting SOX4 and further suppresses cell proliferation, migration and promotes apoptosis in chondrosarcomas. © 2017 The Author(s). Published by S. Karger AG, Basel.

Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin.

PubMed Central

Terzi, F.; Maunoury, R.; Colucci-Guyon, E.; Babinet, C.; Federici, P.; Briand, P.; Friedlander, G.

1997-01-01

Proliferation and dedifferentiation of tubular cells are the hallmark of early regeneration after renal ischemic injury. Vimentin, a class III intermediate filament expressed only in mesenchymal cells of mature mammals, was shown to be transiently expressed in post-ischemic renal tubular epithelial cells. Vimentin re-expression was interpreted as a marker of cellular dedifferentiation, but its role in tubular regeneration after renal ischemia has also been hypothesized. This role was evaluated in mice bearing a null mutation of the vimentin gene. Expression of vimentin, proliferating cell nuclear antigen (a marker of cellular proliferation), and villin (a marker of differentiated brush-border membranes) was studied in wild-type (Vim+/+), heterozygous (Vim+/-), and homozygous (Vim-/-) mice subjected to transient ischemia of the left kidney. As expected, vimentin was detected by immunohistochemistry at the basal pole of proximal tubular cells from post-ischemic kidney in Vim+/+ and Vim+/- mice from day 2 to day 28. The expression of the reporter gene beta-galactosidase in Vim+/- and Vim-/- mice confirmed the tubular origin of vimentin. No compensatory expression of keratin could be demonstrated in Vim-/- mice. The intensity of proliferating cell nuclear antigen labeling and the pattern of villin expression were comparable in Vim-/-, Vim+/- and Vim+/+ mice at any time of the study. After 60 days, the structure of post-ischemic kidneys in Vim-/- mice was indistinguishable from that of normal non-operated kidneys in Vim+/+ mice. In conclusion, 1) the pattern of post-ischemic proximal tubular cell proliferation, differentiation, and tubular organization was not impaired in mice lacking vimentin and 2) these results suggest that the transient tubular expression of vimentin is not instrumental in tubular regeneration after renal ischemic injury. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 PMID:9094992

Reconstituted normal human breast in nude mice: effect of host pregnancy environment and human chorionic gonadotropin on proliferation.

PubMed

Popnikolov, N; Yang, J; Liu, A; Guzman, R; Nandi, S

2001-03-01

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although hCG receptor transcripts were detected in human breast epithelial cells, raising the possibility of a direct mitogenic action, the hCG effect observed in this study may have been mediated via the ovary by increased secretion of ovarian steroids. In summary, using our in vivo nude mice system, the proliferation of normal human breast epithelial cells could be stimulated by host mouse pregnancy and by administration of hCG.

A cancer cell-specific fluorescent probe for imaging Cu2 + in living cancer cells

NASA Astrophysics Data System (ADS)

Wang, Chao; Dong, Baoli; Kong, Xiuqi; Song, Xuezhen; Zhang, Nan; Lin, Weiying

2017-07-01

Monitoring copper level in cancer cells is important for the further understanding of its roles in the cell proliferation, and also could afford novel copper-based strategy for the cancer therapy. Herein, we have developed a novel cancer cell-specific fluorescent probe for the detecting Cu2 + in living cancer cells. The probe employed biotin as the cancer cell-specific group. Before the treatment of Cu2 +, the probe showed nearly no fluorescence. However, the probe can display strong fluorescence at 581 nm in response to Cu2 +. The probe exhibited excellent sensitivity and high selectivity for Cu2 + over the other relative species. Under the guidance of biotin group, could be successfully used for detecting Cu2 + in living cancer cells. We expect that this design strategy could be further applied for detection of the other important biomolecules in living cancer cells.

ERα36 gene silencing promotes tau protein phosphorylation, inhibits cell proliferation, and induces apoptosis in human neuroblastoma SH-SY5Y cells.

PubMed

Wang, Hong-Bin; Li, Tao; Ma, Dong-Zhou; Zhi, Hua

2018-06-22

Neuroblastoma is the most common cancer in infants and the third most common cancer in children after leukemia and brain cancer. The purpose of our study was to investigate the effects of estrogen receptor (ER)-α36 gene silencing on tau protein phosphorylation, cell proliferation, and cell apoptosis in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with estrogen or left untreated, to investigate the effects of estrogen stimulation on ERα36 and the ERK/protein B kinase (AKT) signaling pathway. ERα36 mRNA expressions were detected by quantitative RT-PCR. A phosphatase kit was used to test protein phosphatase (PP)-2A activity before and after treatment. Western blot analysis was conducted to detect protein expression of ERα36; tau protein; phosphorylated- tau (p-tau) at site Thr231 [p-tau (Thr231)]; glycogen synthase kinase (GSK)3β and its specificity sites (Tyr216 and Ser9); Cyclin Dl; proliferating cell nuclear antigen (PCNA); B-cell lymphoma (Bcl)-2; and Bcl-2-associated X protein (Bax). A cell-counting kit (CCK)-8 assay was used to determine cell viability. Cell apoptosis and rate of tumor growth and volume were determined by Annexin V-FITC/PI staining and a xenotransplanted tumor model in nude mice. Results show that without estrogen stimulation, ERα36 was inactivated. When stimulated by estrogen, expression of ERα36, PP2A, p-GSK3β (Ser9)/total protein ( t)-GSK3β, Cyclin Dl, PCNA, and Bcl-2 were up-regulated, and p-GSK3β (Tyr216)/ t-GSK3β expression was down-regulated, as was p-tau (Thr231) and Bax expression. The expression of p-ERK/ERK, p-AKT/AKT, p-methyl ethyl ketone (MEK)/MEK, and p-mammalian target of rapamycin (mTOR)/mTOR expression was up-regulated, suggesting that the ERK/AKT signaling pathway is activated. Cell proliferation was also accelerated, whereas apoptosis was inhibited with stimulation by estrogen. However, we found that the effects of silencing ERα36 on the expression of related intracellular factors had no association with estrogen. Our study demonstrates that ERα36 gene silencing can inhibit the activation of the ERK/AKT signaling pathway, increase tau protein phosphorylation, decrease cell vitality and tumorigenicity, and promote apoptosis of human neuroblastoma SH-SY5Y cells.-Wang, H.-B., Li, T., Ma, D.-Z., Zhi, H. ERα36 gene silencing promotes tau protein phosphorylation, inhibits cell proliferation, and induces apoptosis in human neuroblastoma SH-SY5Y cells.

Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice.

PubMed

Igarashi, Miyuki; Setoguchi, Mayumi; Takada, Sanae; Itoh, Satoru; Furuhama, Kazuhisa

2007-08-15

To ascertain an optimum condition for detecting micronuclei in the liver caused by numerical aberration inducers, either carbendazim (125-1000mg/kg, p.o.), colchicine (0.375-1.5mg/kg, i.v.), cytochalasin B (2.5-20mg/kg, i.v.), diazepam (3.13-25mg/kg, i.v.), noscapine (7.8-62.5mg/kg, i.v.), paclitaxel (1-100mg/kg, i.v.) or trichlorfon (18.75-150mg/kg, i.v.) was administered once to male Slc:ddY mice 1 day before or after partial hepatectomy (PH, Day 1). Five days after PH (on Day 6), hepatic micronuclei were determined in conjunction with classifications of the main nuclei and relative liver weights as a proliferative indicator or a dysfunction marker of cell division. Additionally, hepatocyte proliferation index (HPI) was calculated by using mono-, bi- and multinucleated cell counts. Treatment of mice with six compounds, except for colchicine, after PH showed higher incidence of micronucleated hepatocytes (MNH) than that before PH, and also increases in binucleated and multinucleated cells. Especially for carbendazim, diazepam, noscapine and trichlorfon, the dosing after PH was essential for the detecting numerical aberration. Colchicine evidently increased HPI and decreased relative liver weights without MNH induction on Day 6. On Day 8 when HPI and relative liver weights almost returned to the basal range, a significant increase in MNH was noted. This implied that the strong inhibition of colchicine on hepatocyte proliferation may obstruct the induction of MNH on Day 6. In conclusion, to detect the potential numerical aberration, exposure of mice to test chemicals should be performed 1 day after PH, during which enhanced proliferation of hepatocytes was seen, and it would be better to analyze the liver specimens on Day 6 or more post-PH.

Balance between fibroblast growth factor 10 and secreted frizzled-relate protein-1 controls the development of hair follicle by competitively regulating β-catenin signaling.

PubMed

Zhang, Haihua; Nan, Weixiao; Wang, Shiyong; Si, Huazhe; Li, Guangyu

2018-07-01

Growth of hairs depends on the regular development of hair follicles which are hypothesized to be regulated by fibroblast growth factor 10 (FGF10) and secreted frizzled-relate protein-1 (sFRP1). In the current study, the effect of FGF10 or sFRP1 on hair follicle cells was assessed and the possible mechanism mediating the interaction between FGF10 and sFRP1 in hair follicle cells was explored. Out root sheath (ORS) and dermal papilla (DP) cells were isolated from mink skin tissues and subjected to administrations of FGF10 (50 ng/ml) or sFRP1 (10 ng/ml). Then proliferation, cell cycle distribution, and migration potentials of both cell types were detected. Moreover, the nuclear translocation of β-catenin was determined. The results showed that the administration of FGF10 increased cell proliferation and migration potential in both cell types, which was associated with the up-regulated nuclear level of β-catenin. To the contrary, the administration of sFRP1 decreased cell proliferation and migration potentials while induced the G1 cell cycle arrest in both cell types by inhibiting nuclear translocation of β-catenin. Compared with the sole administrations, the co-treatment of FGF10 and sFRP1 had a medium effect on cell proliferation, cell cycle distribution, cell migration, and nuclear β-catenin level, representing an antagonistic interaction between the two factors, which was exerted by competitively regulating β-catenin pathway. Conclusively, the cycle of hair follicles was promoted by FGF10 while blocked by sFRP1 and the interplay between the two factors controlled the development of hair follicles by competitively regulating β-catenin signaling. Copyright © 2018. Published by Elsevier Masson SAS.

Immunohistochemical and ultrastructural evidence of functional organization along the Corydoras paleatus intestine.

PubMed

Plaul, Silvia E; Pastor, Raquel; Díaz, Alcira O; Barbeito, Claudio G

2016-03-01

The Neotropical catfish, Corydoras paleatus (Callichthyidae) is a facultative air-breathing teleost that makes use of the caudal portion of the intestine as an accessory air-breathing organ. This portion is highly modified, being well vascularized with capillaries between epithelial cells, which makes it well suited for gas exchange. Instead, the cranial portion is a digestion and absorption site, as it has a typical intestinal epithelium with columnar cells arranged in a single row, villi and less vascularized tunica mucosa. Therefore, the intestine was studied by light and electron microscopy to assess differences between the cranial, middle and caudal portions. To characterize the potential for cell proliferation of this organ, we used anti-proliferating cell nuclear antigen antibody and anti-Na(+) K(+) -ATPase monoclonal antibody to detect the presence of Na(+) /K(+) pump. In C. paleatus it was observed that cell dynamics showed a decreasing gradient of proliferation in cranio-caudal direction. Also, the intestine of this catfish is an important organ in ionoregulation: the basolateral Na(+) /K(+) pump may have an active role, transporting Na(+) out of the cell while helping to maintain the repose potential and to regulate cellular volume. © 2016 Wiley Periodicals, Inc.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

PubMed

Ginani, Fernanda; Soares, Diego Moura; de Oliveira Rocha, Hugo Alexandre; de Souza, Lélia Batista; Barboza, Carlos Augusto Galvão

2018-01-01

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm 2 -16 s; 1.0 J/cm 2 -33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm 2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm 2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm 2 ) promote the proliferation of SHEDs and the maintenance of cell viability.

Overexpression of PHRF1 attenuates the proliferation and tumorigenicity of non-small cell lung cancer cells.

PubMed

Wang, Yadong; Wang, Haiyu; Pan, Teng; Li, Li; Li, Jiangmin; Yang, Haiyan

2016-09-27

The aim of this study was to investigate the potential role of PHRF1 in lung tumorigenesis. Western blot analysis was used to detect the expression of proteins. Quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, soft agar assay and tumor formation assay in nude mice were applied. Cell cycle distribution was analyzed by flow cytometry. The lower level of PHRF1 mRNA was observed in human lung cancer tissues than that in paracancerous tissues. The decreased expression of PHRF1 protein was observed in H1299 and H1650 cell lines than that in 16HBE and BEAS-2B cell lines. The decreased expression of PHRF1 protein was observed in malignant 16HBE cells compared to control cells. The reduced expression of PHRF1 protein was observed in mice lung tissues treated with BaP than that in control group. Overexpression of PHRF1 inhibited H1299 cell proliferation, colony formation in vitro and growth of tumor xenograft in vivo, and arrested cell cycle in G1 phase. The decreased expression of TGIF and c-Myc proteins and the increased expression of p21 protein were observed in H1299-PHRF1 cells compared with H1299-pvoid cells. In conclusion, our findings suggest that overexpression of PHRF1 attenuated the proliferation and tumorigenicity of non-small cell lung cancer cell line of H1299.

Comparative analysis of cell proliferation ratio in plaque and erosive oral lichen planus: An immunohistochemical study.

PubMed

Redder, C Pramod; Pandit, Siddharth; Desai, Dinkar; Kandagal, V Suresh; Ingaleshwar, Pramod S; Shetty, Sharan J; Vibhute, Nupura

2014-05-01

Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S-phase of the cell cycle. Detection of this protein represents a useful marker of the proliferation status of lesions. This study has been carried out to evaluate the cell proliferation rate in oral lichen planus (OLP) and comparison between plaque and erosive lichen planus, which indicates the potential for malignant transformation. This study was comprised of 64 cases of histologically proven lichen planus, out of which 32 cases of plaque and erosive each was taken. Two sections were taken from each, one for H and E staining to verify histological diagnosis according to Eisenberg criteria, other sections were stained according to super sensitive polymer horse radish peroxidise method for identifying immunohistochemical expression of PCNA. Data were statistically analyzed by Tukey high-range statistical domain test. Statistically significant P value was considered <0.05. In two types of lichen planus, erosive type (66.86%) showed higher expression of PCNA followed by plaque (17.07%). Overall, P value was <0.001, which was statistically significant. It indicates that proliferation activity is more in erosive lichen planus followed by plaque type, which ultimately results in increased rate of malignant transformation. PCNA is a good nuclear protein marker to evaluate the proliferation status of OLP. Out of the two types of lichen planus, erosive type possesses more proliferative ratio and chances of malignant change is more in this type. It emphasizes the importance of long-term follow-up with erosive type when compared with plaque type.

Diadenosine polyphosphates Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of vascular smooth muscle cells.

PubMed

Bobbert, Peter; Schlüter, Hartmut; Schultheiss, Heinz Peter; Reusch, Hans Peter

2008-05-15

Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways. Proliferation of VSMCs was measured by the cell count and [(3)H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca(2+)](i) measurements were done to determine the influence of [Ca(2+)](i) on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of G alpha(12). Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCbeta and the increase of [Ca(2+)](i). In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.

Order-of-magnitude estimates of latency (time to appearance) and refill time of a cancer from a single cancer 'stem' cell compared by an exponential and a logistic equation.

PubMed

Anderson, Ken M; Rubenstein, Marvin; Guinan, Patrick; Patel, Minu

2012-01-01

The time required before a mass of cancer cells considered to have originated from a single malignantly transformed cancer 'stem' cell reaches a certain number has not been studied. Applications might include determination of the time the cell mass reaches a size that can be detected by X-rays or physical examination or modeling growth rates in vitro in order to compare with other models or established data. We employed a simple logarithmic equation and a common logistic equation incorporating 'feedback' for unknown variables of cell birth, growth, division, and death that can be used to model cell proliferation. It can be used in association with free or commercial statistical software. Results with these two equations, varying the proliferation rate, nominally reduced by generational cell loss, are presented in two tables. The resulting equation, instructions, examples, and necessary mathematical software are available in the online appendix, where several parameters of interest can be modified by the reader www.uic.edu/nursing/publicationsupplements/tobillion_Anderson_Rubenstein_Guinan_Patel1.pdf. Reducing the proliferation rate by whatever alterations employed, markedly increases the time to reach 10(9) cells originating from an initial progenitor. In thinking about multistep oncogenesis, it is useful to consider the profound effect that variations in the effective proliferation rate may have during cancer development. This can be approached with the proposed equation, which is easy to use and available to further peer fine-tuning to be used in future modeling of cell growth.

Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells.

PubMed

Singer, Bernhard B; Scheffrahn, Inka; Kammerer, Robert; Suttorp, Norbert; Ergun, Suleyman; Slevogt, Hortense

2010-01-18

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

Proliferating cell nuclear antigen promotes cell proliferation and tumorigenesis by up-regulating STAT3 in non-small cell lung cancer.

PubMed

Wang, Liuxin; Kong, Weixiang; Liu, Bing; Zhang, Xueqing

2018-08-01

Proliferating cell nuclear antigen (PCNA) functions as a bridging molecule, which targets proteins that have distinct roles in cell growth. The expression of PCNA is dysregulated in some tumors and takes part in the progression of oncogenesis. However, the roles of PCNA in the progression of non-small cell lung cancer (NSCLC) remain unknown. The present study aimed to investigate the function of PCNA in the occurrence and development of NSCLC and its underlying molecular mechanisms. Western blotting, RT-PCR, and immunohistochemistry assays were used to detect the expression pattern of PCNA in NSCLC tissues and cells. A log rank test was performed to compare the overall survival (OS) of patients with high/low expression of PCNA. Besides, the relationship between PCNA and signal transducer and activator of transcription-3 (STAT3) proteins were evaluated. Then, MTT, flow cytometry, clonal formation, and in vivo xenograft assays were conducted to investigate the effects of PCNA/STAT3 on cell growth, clonal formation, apoptosis, and tumorigenesis. Results showed that PCNA expression was elevated in NSCLC tissues and cells and it could combine with STAT3 and increased its expression and phosphorylation. Moreover, the expression of PCNA showed a positive correlation with the TNM grade and occurrence rate of the lymphatic metastasis and poor prognosis of NSCLC patients. Overexpression of PCNA promoted cell proliferation, clonal formation, and tumorigenesis in lung cancer cells and inhibited cell apoptosis. In contrast, these effects were inhibited when knockdown of STAT3. In conclusion, this study demonstrates that PCNA functions as an oncogene in the progression of NSCLC through up-regulation of STAT3. These findings point to a potentially new therapeutic strategy for NSCLC. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

PubMed Central

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-01-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

PubMed

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-08-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

Regulation and spatial organization of PCNA in Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaufmann, Doris; Gassen, Alwine; Maiser, Andreas

2012-03-23

Highlights: Black-Right-Pointing-Pointer Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). Black-Right-Pointing-Pointer TbPCNA is a suitable marker to detect replication in T. brucei. Black-Right-Pointing-Pointer TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. bruceimore » (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.« less

Cell proliferation and plant development under novel altered gravity environments.

PubMed

Herranz, R; Medina, F J

2014-01-01

Gravity is a key factor for life on Earth. It is the only environmental factor that has remained constant throughout evolution, and plants use it to modulate important physiological activities; gravity removal or alteration produces substantial changes in essential functions. For root gravitropism, gravity is sensed in specialised cells, which are capable of detecting magnitudes of the g vector lower than 10(-3) . Then, the mechanosignal is transduced to upper zones of the root, resulting in changes in the lateral distribution of auxin and in the rate of auxin polar transport. Gravity alteration has consequences for cell growth and proliferation rates in root meristems, which are the basis of the developmental programme of a plant, in which regulation via auxin is involved. The effect is disruption of meristematic competence, i.e. the strict coordination between cell proliferation and growth, which characterises meristematic cells. This effect can be related to changes in the transport and distribution of auxin throughout the root. However, similar effects of gravity alteration have been found in plant cell cultures in vitro, in which neither specialised structures for gravity sensing and signal transduction, nor apparent gravitropism have been described. We postulate that gravity resistance, a general mechanism of cellular origin for developing rigid structures in plants capable of resisting the gravity force, could also be responsible for the changes in cell growth and proliferation parameters detected in non-specialised cells. The mechanisms of gravitropism and graviresistance are complementary, the first being mostly sensitive to the direction of the gravity vector, and the second to its magnitude. At a global molecular level, the consequence of gravity alteration is that the genome should be finely tuned to counteract a type of stress that plants have never encountered before throughout evolution. Multigene families and redundant genes present an advantage in that they can experience changes without the risk of being deleterious and, for this reason, they should play a key role in the response to gravitational stress. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Seasonal variation in telencephalon cell proliferation in adult female tsinling dwarf skinks (Scincella tsinlingensis).

PubMed

Yang, Chun; Wang, Limin; Xing, Xiangyang; Gao, Yanyan; Guo, Li

2017-05-01

In adult mammals, neurogenesis is limited to specific niches in the brain, but considerable adult neurogenesis occurs in many brain regions in non-mammalian vertebrates. Non-mammalian vertebrates provide invaluable comparative material for understanding the core mechanisms of adult neural stem cell maintenance and fate, but phylogenetic differences in adult neurogenesis remain poorly understood. Here we examine cell proliferation seasonality in the telencephalon of adult female tsinling dwarf skinks (Scincella tsinlingensis) by injecting wild animals caught in summer, autumn and spring, and animals caught in autumn and raised under winter conditions, with 5-Bromo-2'-deoxyuridine (BrdU). Then, 24h, 7d and 28d after BrdU administration we examined brain tissue and quantified BrdU-labeled cells as a marker of neuronal proliferation. The highest number of labeled cells in the telencephalon was found in the 7d group. BrdU-positive cells were widely distributed in the anterior olfactory nucleus (AON), medial cortex (MC), dorsal cortex (DC), lateral cortex (LC), dorsal ventricular ridge (DVR), septum (SP), striatum (STR) and nucleus sphericus (NS). No BrdU-positive cells were detected in olfactory bulbs or elsewhere in the telencephalon. The highest proliferative levels were found in the AON in autumn. The NS exhibited relatively high levels of cell proliferation. The proliferative rate in the AON fluctuated seasonally as autumn>summer>spring>winter. Glial fibrillary acidic protein-positive cells were widely distributed in the telencephalon and their fibrous processes extended into brain parenchyma and anchored in the meninges. Doublecortin-positive newborn neurons of the subventricular zone appeared to migrate into the cerebral cortex via the radial migratory stream. Cell proliferation in the telencephalon of adult female S. tsinlingensis fluctuates seasonally, especially in regions related to olfactory memory. This is the first demonstration of proliferative activity in the telencephalon of a skink. Copyright © 2017 Elsevier B.V. All rights reserved.

Concurrent Fowlpox and Candidiasis Diseases in Backyard Chickens with Unusual Pox Lesions in the Bursa of Fabricius.

PubMed

Ogasawara, Fusae; Yamamoto, Yu; Sato, Yasuo; Fukunari, Kazuhiro; Murata, Ken-Ichi; Yaegashi, Gakuji; Goto, Makiko; Murakami, Ryukoh

2016-09-01

Concurrent fowlpox and candidiasis diseases occurred in a backyard chicken flock. Four deceased chickens (one Nagoya breed and three white silkie chickens) were examined for diagnosis. At necropsy, white curd-like plaques were observed in the crop. Fungal elements that stained positive for Candida albicans with immunohistochemistry were distributed throughout the tongue, choanal mucosa, esophagus, and crop. Typical fowlpox lesions, composed of proliferating epithelial cells with ballooning degeneration and viral intracytoplasmic inclusions, were observed in the conjunctiva, nasal mucosa, and skin around the cloaca. Interestingly, hyperplastic interfollicular epithelium with rare virus inclusions was observed in the bursa of Fabricius (BF). Some bursal follicles were replaced by proliferating epithelial cells. These proliferating cells immunohistochemically stained positive for cytokeratin. PCR and subsequent genetic sequencing detected the C. albicans gene in the crop, and fowlpox virus genes in the BF. These results indicate that this outbreak was a rare presentation of fowlpox in spontaneously infected chickens, with unusual pox lesions in the BF.

RACK1 promotes radiation resistance in esophageal cancer via regulating AKT pathway and Bcl-2 expression.

PubMed

Liu, Bowen; Wang, Cong; Chen, Pengxiang; Wang, Lu; Cheng, Yufeng

2017-09-23

RACK1 is a seven Trp-Asp 40 repeat protein, which interacts with a wide range of kinases and proteins. RACK1 plays an important role in the proliferation and progression of various cancers. The aim of this study is to detect the role of RACK1 in the radioresistance in esophageal cancer. The results indicated that downregulation of RACK1 reduced the colony formation ability, proliferation ability and resistance of cells to radiation effection through regulating the radiation-related proteins including pAKT, Bcl-2 and Bim; whereas upregulation of RACK1 promoted the ability and radioresistance of ESCC cells. Our findings suggest that RACK1 promotes proliferation and radioresistance in ESCC cells by activating the AKT pathway, upregulating Bcl-2 expression and downregulating protein levels of Bim. Our study fills in gaps in the field of RACK1 and radiation resistance and may provide new possibilities for improving strategies of radiotherapy in esophageal cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Transitional change in rat fetal cell proliferation in response to ghrelin and des-acyl ghrelin during the last stage of pregnancy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, Yoshiyuki; Nakahara, Keiko; Kangawa, Kenji

Expression of mRNA for the ghrelin receptor, GHS-R1a, was detected in various peripheral and central tissues of fetal rats, including skin, bone, heart, liver, gut, brain and spinal cord, on embryonic day (ED)15 and ED17. However, its expression in skin, bone, heart and liver, but not in gut, brain and spinal cord, became relatively weak on ED19 and disappeared after birth (ND2). Ghrelin and des-acyl ghrelin facilitated the proliferation of cultured fetal (ED17, 19), but not neonatal (ND2), skin cells. On the other hand, with regard to cells from the spinal cord and hypothalamus, the proliferative effect of ghrelin continuedmore » after birth, whereas the effect of des-acyl ghrelin on neurogenesis in these tissues was lost at the ED19 fetal and ND2 neonatal stages. Immunohistochemistry revealed that the cells in the hypothalamus induced to proliferate by ghrelin at the ND2 stage were positive for nestin and glial fibrillary acidic protein. These results suggest that in the period immediately prior to, and after birth, rat fetal cells showing proliferation in response to ghrelin and des-acyl ghrelin are at a transitional stage characterized by alteration of the expression of GHS-R1a and an undefined des-acyl ghrelin receptor, their responsiveness varying among different tissues.« less

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

PubMed

Krajňáková, Jana; Sutela, Suvi; Aronen, Tuija; Gömöry, Dušan; Vianello, Angelo; Häggman, Hely

2011-08-01

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities. Copyright © 2011 Elsevier Inc. All rights reserved.

Semiquantitative immunohistochemical marker staining and localization in canine thyroid carcinoma and normal thyroid gland.

PubMed

Pessina, P; Castillo, V; Sartore, I; Borrego, J; Meikle, A

2016-09-01

Immunoreactive proteins in follicular cells, fibroblasts and endothelial cells were assessed in canine thyroid carcinomas and healthy thyroid glands. No differences were detected in thyrotropin receptor and thyroglobulin staining between cancer and normal tissues, but expression was higher in follicular cells than in fibroblasts. Fibroblast growth factor-2 staining was more intense in healthy follicular cells than in those of carcinomas. Follicular cells in carcinomas presented two- to three-fold greater staining intensity of thyroid transcription factor-1 and proliferating cell nuclear antigen, respectively, than healthy cells, and a similar trend was found for the latter antigen in fibroblasts. Vascular endothelial growth factor staining was more intense in the endothelial cells of tumours than in those of normal tissues. In conclusion, greater expression of factors related to proliferation and angiogenesis was demonstrated in several cell types within thyroid carcinomas compared to healthy tissues, which may represent mechanisms of tumour progression in this disease. © 2014 John Wiley & Sons Ltd.

Reconstructing the in vivo dynamics of hematopoietic stem cells from telomere length distributions

PubMed Central

Werner, Benjamin; Beier, Fabian; Hummel, Sebastian; Balabanov, Stefan; Lassay, Lisa; Orlikowsky, Thorsten; Dingli, David; Brümmendorf, Tim H; Traulsen, Arne

2015-01-01

We investigate the in vivo patterns of stem cell divisions in the human hematopoietic system throughout life. In particular, we analyze the shape of telomere length distributions underlying stem cell behavior within individuals. Our mathematical model shows that these distributions contain a fingerprint of the progressive telomere loss and the fraction of symmetric cell proliferations. Our predictions are tested against measured telomere length distributions in humans across all ages, collected from lymphocyte and granulocyte sorted telomere length data of 356 healthy individuals, including 47 cord blood and 28 bone marrow samples. We find an increasing stem cell pool during childhood and adolescence and an approximately maintained stem cell population in adults. Furthermore, our method is able to detect individual differences from a single tissue sample, i.e. a single snapshot. Prospectively, this allows us to compare cell proliferation between individuals and identify abnormal stem cell dynamics, which affects the risk of stem cell related diseases. DOI: http://dx.doi.org/10.7554/eLife.08687.001 PMID:26468615

Inhibition of green tea polyphenol EGCG((-)-epigallocatechin-3-gallate) on the proliferation of gastric cancer cells by suppressing canonical wnt/β-catenin signalling pathway.

PubMed

Yang, Chenggang; Du, Wenfeng; Yang, Daogui

2016-11-01

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, could affect carcinogenesis and development of many cancers. However, the effects and underlying mechanisms of EGCG on gastric cancer remain unclear. We found that EGCG significantly inhibited proliferation and increased apoptosis of SGC-7901 cells in vitro. The decreased expressions of p-β-catenin(Ser552), p-GSK3β(S9) and β-catenin target genes were detected in SGC-7901 cells after treated by EGCG. XAV939 and β-catenin plasmid were further used to demonstrate the inhibition of EGCG on canonical Wnt/β-catenin signalling. Moreover, EGCG significantly inhibited gastric tumour growth in vivo by inhibiting Wnt/β-catenin signalling. Taken together, our findings establish that EGCG suppressed gastric cancer cell proliferation and demonstrate that this inhibitory effect is related to canonical Wnt/β-catenin signalling. This study raises a new insight into gastric cancer prevention and therapy, and provides evidence that green tea could be used as a nutraceutical beverage.

[The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells].

PubMed

Luan, Yan; Deqin, Yang

2017-08-01

Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

Osteopontin Promotes Invasion, Migration and Epithelial-Mesenchymal Transition of Human Endometrial Carcinoma Cell HEC-1A Through AKT and ERK1/2 Signaling.

PubMed

Li, Yinghua; Xie, Yunpeng; Cui, Dan; Ma, Yanni; Sui, Linlin; Zhu, Chenyang; Kong, Hui; Kong, Ying

2015-01-01

Osteopontin (OPN) is an Extracellular Matrix (ECM) molecule and is involved in many physiologic and pathologic processes, including cell adhesion, angiogenesis and tumor metastasis. OPN is a well-known multifunctional factor involved in various aspects of cancer progression, including endometrial cancer. In this study, we examined the significance of OPN in endometrial cancer. The proliferation, migration and invasion ability of HEC-1A cells were detected by Cell Counting Kit-8 (CCK-8), Wound scratch assay and transwell. Western blots were employed to detect the expression of Matrix metalloproteinase-2 (MMP-2) and epithelial-mesenchymal transition (EMT)-related factors in HEC-1A cells treated with rhOPN. rhOPN promotes cell proliferation, migration and invasion in HEC-1A cells. rhOPN influenced EMT-related factors and MMP-2 expression in HEC-1A cells. rhOPN promoted HEC-1A cells migration, invasion and EMT through protein kinase B (PKB/AKT) and Extracellular regulated protein kinases (ERK1/2) signaling pathway. These results may open up a novel therapeutic strategy for endometrial cancer: namely, rhOPN have important roles in controlling growth of endometrial of cancer cells and suggest a novel target pathway for treatment of this cancer. © 2015 The Author(s) Published by S. Karger AG, Basel.

Effects of Electromagnetic Radiation from Smartphones on Learning Ability and Hippocampal Progenitor Cell Proliferation in Mice

PubMed Central

Choi, Yu-Jin; Choi, Yun-Sik

2015-01-01

Objectives Nonionizing radiation is emitted from electronic devices, such as smartphones. In this study, we intended to elucidate the effect of electromagnetic radiation from smartphones on spatial working memory and progenitor cell proliferation in the hippocampus. Methods Both male and female mice were randomly separated into two groups (radiated and control) and the radiated group was exposed to electromagnetic radiation for 9 weeks and 11 weeks for male and female mice, respectively. Spatial working memory was examined with a Y maze, and proliferation of hippocampal progenitor cells were examined by 5-bromo-2′-deoxyuridine administration and immunohistochemical detection. Results When spatial working memory on a Y maze was examined in the 9th week, there was no significant difference in the spontaneous alternation score on the Y maze between the two groups. In addition, there was no significant difference in hippocampal progenitor cell proliferation. However, immunoreactivity to glial fibrillary acidic protein was increased in exposed animals. Next, to test the effect of recovery following chronic radiation exposure, the remaining female mice were further exposed to electromagnetic radiation for 2 more weeks (total 11 weeks), and spontaneous alternation was tested 4 weeks later. In this experiment, although there was no significant difference in the spontaneous alternation scores, the number of arm entry was significantly increased. Conclusion These data indicate that although chronic electromagnetic radiation does not affect spatial working memory and hippocampal progenitor cell proliferation it can mediate astrocyte activation in the hippocampus and delayed hyperactivity-like behavior. PMID:26981337

[Shikimic acid inhibits the degranulation and histamine release in RBL-2H3 cells].

PubMed

Chen, Xianyong; Zheng, Qianqian; Liu, Wei; Yu, Lingling; Wang, Jinling; Li, Shigang

2017-05-01

Objective To study the effects of shikimic acid on the proliferation of rat RBL-2H3 cells and the degranulation of the cells induced by C48/80 and its mechanism. Methods MTT assay was performed to measure the proliferation of RBL-2H3 cells treated with 3, 10, 30 μg/mL shikimic acid. Toluidine blue staining was used to observe the degranulation of RBL-2H3 cells. The release of β-hexosaminidase from RBL-2H3 cells treated with 0, 12.5, 25, 50, 80, 100 μg/mL C48/80 was determined by substrate assay. ELISA was used to detect the histamine content in the supernatant of each treated group. Results Shikimic acid at 3, 10, 300 μg/mL had no obvious inhibitory effect on the proliferation of RBL-2H3 cells. There was a dose-effect relationship between the degranulation of RBL-2H3 cells and C48/80 concentration. Shikimic acid inhibited the degranulation of RBL-2H3 cells compared with the positive control group, the β-hexosaminidase release rate and histamine release were significantly reduced in RBL-2H3 cells treated with shikimic acid and C48/80. Conclusion Shikimic acid can inhibit the degranulation of RBL-2H3 cells and reduce histamine release.

Utilization of nuclear structural proteins for targeted therapy and detection of proliferative and differentiation disorders

DOEpatents

Lelievre, Sophie; Bissell, Mina

2001-01-01

The localization of nuclear apparatus proteins (NUMA) is used to identify tumor cells and different stages in the tumor progression and differentiation processes. There is a characteristic organization of NuMA in tumor cells and in phenotypically normal cells. NuMA distribution patterns are significantly less diffuse in proliferating non-malignant cells compared to malignant cells. The technique encompasses cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

Progesterone inhibits proliferation and modulates expression of proliferation-Related genes in classical progesterone receptor-negative human BxPC3 pancreatic adenocarcinoma cells.

PubMed

Goncharov, Alexey I; Maslakova, Aitsana A; Polikarpova, Anna V; Bulanova, Elena A; Guseva, Alexandra A; Morozov, Ivan A; Rubtsov, Petr M; Smirnova, Olga V; Shchelkunova, Tatiana A

2017-01-01

Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 μM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 μM and 20 μM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Ping, E-mail: fanpinggoodluck@163.com; He, Lan; Pu, Dan

Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertolimore » cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-{gamma}-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-{alpha} induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.« less

Wnt5b-associated exosomes promote cancer cell migration and proliferation.

PubMed

Harada, Takeshi; Yamamoto, Hideki; Kishida, Shosei; Kishida, Michiko; Awada, Chihiro; Takao, Toshifumi; Kikuchi, Akira

2017-01-01

Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post-translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured-cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC-1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild-type PANC-1 cells, but not those from Wnt5b-knockout PANC-1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b-associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco-2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco-2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b-associated exosomes from Caco-2/Wnt5b cells and inhibited Wnt5b-dependent cell proliferation. Exosomes secreted from Caco-2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

CTGF Mediates Smad-Dependent Transforming Growth Factor β Signaling To Regulate Mesenchymal Cell Proliferation during Palate Development

PubMed Central

Parada, Carolina; Li, Jingyuan; Iwata, Junichi; Suzuki, Akiko

2013-01-01

Transforming growth factor β (TGF-β) signaling plays crucial functions in the regulation of craniofacial development, including palatogenesis. Here, we have identified connective tissue growth factor (Ctgf) as a downstream target of the TGF-β signaling pathway in palatogenesis. The pattern of Ctgf expression in wild-type embryos suggests that it may be involved in key processes during palate development. We found that Ctgf expression is downregulated in both Wnt1-Cre; Tgfbr2fl/fl and Osr2-Cre; Smad4fl/fl palates. In Tgfbr2 mutant embryos, downregulation of Ctgf expression is associated with p38 mitogen-activated protein kinase (MAPK) overactivation, whereas loss of function of Smad4 itself leads to downregulation of Ctgf expression. We also found that CTGF regulates its own expression via TGF-β signaling. Osr2-Cre; Smad4fl/fl mice exhibit a defect in cell proliferation similar to that of Tgfbr2 mutant mice, as well as cleft palate. We detected no alteration in bone morphogenetic protein (BMP) downstream targets in Smad4 mutant palates, suggesting that the reduction in cell proliferation is due to defective transduction of TGF-β signaling via decreased Ctgf expression. Significantly, an exogenous source of CTGF was able to rescue the cell proliferation defect in both Tgfbr2 and Smad4 mutant palates. Collectively, our data suggest that CTGF regulates proliferation as a mediator of the canonical pathway of TGF-β signaling during palatogenesis. PMID:23816882

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

PubMed Central

Rastegar, Farbod; Gao, Jian-Li; Shenaq, Deana; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Jiang, Wei; Wagner, Eric R.; Huang, Enyi; Gao, Yanhong; Shen, Jikun; Yang, Ke; He, Bai-Cheng; Chen, Liang; Zuo, Guo-Wei; Luo, Jinyong; Luo, Xiaoji; Bi, Yang; Liu, Xing; Li, Mi; Hu, Ning; Wang, Linyuan; Luther, Gaurav; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan

2010-01-01

Background Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. Methodology/Principal Findings Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. Conclusions/Significance Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors. PMID:21152068

The p-ERK–p-c-Jun–cyclinD1 pathway is involved in proliferation of smooth muscle cells after exposure to cigarette smoke extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Tianjia; Song, Ting; Ni, Leng

Highlights: • Smooth muscle cells proliferated after exposure to cigarette smoke extract. • The p-ERK, p-c-Jun, and cyclinD1 expressions increased in the process. • The p-ERK inhibitor, U0126, can reverse these effects. • The p-ERK → p-c-Jun → cyclinD1 pathway is involved in the process. - Abstract: An epidemiological survey has shown that smoking is closely related to atherosclerosis, in which excessive proliferation of vascular smooth muscle cells (SMCs) plays a key role. To investigate the mechanism underlying this unusual smoking-induced proliferation, cigarette smoke extract (CSE), prepared as smoke-bubbled phosphate-buffered saline (PBS), was used to induce effects mimicking those exertedmore » by smoking on SMCs. As assessed by Cell Counting Kit-8 detection (an improved MTT assay), SMC viability increased significantly after exposure to CSE. Western blot analysis demonstrated that p-ERK, p-c-Jun, and cyclinD1 expression increased. When p-ERK was inhibited using U0126 (inhibitor of p-ERK), cell viability decreased and the expression of p-c-Jun and cyclinD1 was reduced accordingly, suggesting that p-ERK functions upstream of p-c-Jun and cyclinD1. When a c-Jun over-expression plasmid was transfected into SMCs, the level of cyclinD1 in these cells increased. Moreover, when c-Jun was knocked down by siRNA, cyclinD1 levels decreased. In conclusion, our findings indicate that the p-ERK–p-c-Jun–cyclinD1 pathway is involved in the excessive proliferation of SMCs exposed to CSE.« less

Cysteine-Rich Intestinal Protein 1 Silencing Inhibits Migration and Invasion in Human Colorectal Cancer.

PubMed

He, Guoyang; Zou, Liyuan; Zhou, Lin; Gao, Peiqiong; Qian, Xinlai; Cui, Jing

2017-01-01

Cysteine-rich intestinal protein 1 (CRIP1), a member of the LIM/double zinc finger protein family, is abnormally expressed in several tumour types. However, few data are available on the role of CRIP1 in cancer. In the present study, we aimed to investigate the expression profile and functions of CRIP1 in colorectal cancer. To examine the protein expression level of CRIP1, immunohistochemistry (IHC) was performed on 56 pairs of colon cancer tissue samples. Western blotting was performed to investigate CRIP1 protein expression in four colon cancer cell lines. The endogenous expression of CRIP1 was suppressed using short interfering RNAs (siRNAs). Cell proliferation assays were used to determine whether CRIP1 silencing affected cell proliferation. Flow cytometry analysis was used to detect cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion was detected using the transwell and wound-healing assays. IHC analysis showed that protein level of CRIP1 was significantly higher in tumour tissue samples than in paired non-tumour tissue samples and that the CRIP1 level was higher in metastatic tissue samples than in non-metastatic tissue samples. In addition, protein levels of CRIP1 were higher in highly metastatic colon cancer cell lines than in colon cancer cell lines with low metastasis. Further, CRIP1 silencing had no effect on cell proliferation or apoptosis in SW620 and HT29 cells. CRIP1 silencing suppressed cell migration and invasion obviously in SW620 and HT29 cells. The present study provides new evidence that abnormal expression of CRIP1 might be related to the degree of metastasis in colorectal cancer and that CRIP1 silencing could effectively inhibit migration and invasion during colorectal cancer development. These findings might aid the development of a biomarker for colon cancer prognosis and metastasis, and thus help to treat this common type of cancer. © 2017 The Author(s). Published by S. Karger AG, Basel.

Rapid Assessment of Genotoxicity by Flow Cytometric Detection of Cell Cycle Alterations.

PubMed

Bihari, Nevenka

2017-01-01

Flow cytometry is a convenient method for the determination of genotoxic effects of environmental pollution and can reveal genotoxic compounds in unknown environmental mixtures. It is especially suitable for the analyses of large numbers of samples during monitoring programs. The speed of detection is one of the advantages of this technique which permits the acquisition of 10 4 -10 5 cells per sample in 5 min. This method can rapidly detect cell cycle alterations resulting from DNA damage. The outcome of such an analysis is a diagram of DNA content across the cell cycle which indicates cell proliferation, G 2 arrests, G 1 delays, apoptosis, and ploidy.Here, we present the flow cytometric procedure for rapid assessment of genotoxicity via detection of cell cycle alterations. The described protocol simplifies the analysis of genotoxic effects in marine environments and is suitable for monitoring purposes. It uses marine mussel cells in the analysis and can be adapted to investigations on a broad range of marine invertebrates.

Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duan, Lincan; Shen, Hongmei; Zhao, Guangqiang

2014-04-18

Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition ofmore » cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.« less

Hippo pathway regulates somatic development and cell proliferation of silkworm.

PubMed

Li, Niannian; Tong, Xiaoling; Zeng, Jie; Meng, Gang; Sun, Fuze; Hu, Hai; Song, Jiangbo; Lu, Cheng; Dai, Fangyin

2018-03-01

Hippo signaling pathway (signaling pathway Hippo, hereinafter referred to as the Hippo pathway) was the earliest found in Drosophila (Schneck [1]), which can regulate the development of tissues and organs, even some components of the pathway were identified as tumor suppressor [2]. The pathway was more concerned in fruit flies and mice (Schneck [1]), but little attention has been given in silkworm, an important economic and lepidopteran model insect. In this manuscript, we identified major Hippo pathway related genes (Hippo, Salvador, Warts, Mats, Yorkie) in silkworm and named BmHpo, BmSav, BmWts, BmMats, BmYki. The domain organization of these genes was highly conserved in silkworm and other organisms suggesting that they could use similar protein-protein interactions to construct the Hippo kinase cascades. The expression profiles of these genes in silkworm during embryonic, larval, wandering, pupal and adult stages were analyzed, 14 tissues/organs of the day 3, 5th instar larvae (L5D3) as well. Experimental results showed that the expression of Hippo pathway had some influence on the development of silkworm. In order to find out the mechanism of Hippo pathway affecting silkworm development, BmHpo and BmYki were up-regulated and de-regulated in the cell line of Bombyx mori-BmN-SWU1(NS), and the changes of cell proliferation activity and cell cycle were detected. The distribution of BmYki was detected by immunofluorescence technique. This study provides insights into the genes of Hippo pathway which have a certain effect on somatic development and cell proliferation in silkworm. Copyright © 2018 Elsevier Inc. All rights reserved.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Overexpression of HepaCAM inhibits cell viability and motility through suppressing nucleus translocation of androgen receptor and ERK signaling in prostate cancer.

PubMed

Song, Xuedong; Wang, Yin; Du, Hongfei; Fan, Yanru; Yang, Xue; Wang, Xiaorong; Wu, Xiaohou; Luo, Chunli

2014-07-01

HepaCAM is suppressed in a variety of human cancers, and involved in cell adhesion, growth, migration, invasion, and survival. However, the expression and function of HepaCAM in prostate cancer are still unknown. HepaCAM expression has been detected by RT-PCR, Western blotting and immunohistochemistry staining in prostate cell lines RWPE-1, LNCap, DU145, PC3, and in 75 human prostate tissue specimens, respectively. Meanwhile, the cell proliferation ability was detected by WST-8 assay. The role of HepaCAM in prostate cancer cell migration and invasion was examined by wound healing and transwell assay. And flow cytometry was used to observe the apoptosis of prostate cancer cells. Then we detected changes of Androgen Receptor translocation and ERK signaling using immunofluorescence staining and western blot after overexpression of HepaCAM. The HepaCAM expression was significantly down-regulated in prostate cancer tissues and undetected in prostate cancer cells. However, the low HepaCAM expression was not statistically associated with clinicopathological characteristics of prostate cancer. Overexpression of HepaCAM in prostate cancer cells decreased the cell proliferation, migration and invasion, and induced the cell apoptosis. Meanwhile, HepaCAM prevented the androgen receptor translocation from the cytoplasm to the nucleus and down-regulated the MAPK/ERK signaling. Our results suggested that HepaCAM acted as a tumor suppressor in prostate cancer. HepaCAM inhibited cell viability and motility which might be through suppressing the nuclear translocation of Androgen Receptor and down-regulating the ERK signaling. Therefore, it was indicated that HepaCAM may be a potential therapeutic target for prostate cancer. © 2014 Wiley Periodicals, Inc.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells.

PubMed

Chen, Jian; Mu, Qitian; Li, Xia; Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-06-20

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells

PubMed Central

Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-01-01

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT. PMID:28454099

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Li-An; Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an; Yuan, Guohua

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show thatmore » transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.« less

Curcumin Suppresses the Colon Cancer Proliferation by Inhibiting Wnt/β-Catenin Pathways via miR-130a.

PubMed

Dou, Huiqiang; Shen, Renhui; Tao, Jianxin; Huang, Longchang; Shi, Haoze; Chen, Hang; Wang, Yixin; Wang, Tong

2017-01-01

Curcumin exhibits anti-tumor effects in several cancers, including colorectal carcinoma (CRC), but the detailed mechanisms are still unclear. Here we studied the mechanisms underlying the anti-tumor effect of curcumin in colon cancer cells. SW480 cells were injected into mice to establish the xenograft tumor model, followed by evaluation of survival rate with the treatment of curcumin. The expression levels of β-catenin, Axin and TCF4 were measured in the SW480 cells in the absence or presence of curcumin. Moreover, miRNAs related to the curcumin treatment were also detected in vitro . Curcumin could suppress the growth of colon cancer cells in the mouse model. This anti-tumor activity of curcumin was exerted by inhibiting cell proliferation rather than promoting cell apoptosis. Further study suggested that curcumin inhibited cell proliferation by suppressing the Wnt/β-catenin pathway. MiR-130a was down-regulated by curcumin treatment, and overexpressing miR-130a could abolish the anti-tumor activity of curcumin. Our study confirms that curcumin is able to inhibit colon cancer by suppressing the Wnt/β-catenin pathways via miR-130a. MiR-130a may serve as a new target of curcumin for CRC treatment.

Persistence of T-cell immune response induced by two acellular pertussis vaccines in children five years after primary vaccination.

PubMed

Palazzo, Raffaella; Carollo, Maria; Bianco, Manuela; Fedele, Giorgio; Schiavoni, Ilaria; Pandolfi, Elisabetta; Villani, Alberto; Tozzi, Alberto E; Mascart, Françoise; Ausiello, Clara M

2016-01-01

The resurgence of pertussis suggests the need for greater efforts to understand the long-lasting protective responses induced by vaccination. In this paper we dissect the persistence of T memory responses induced by primary vaccination with two different acellular pertussis (aP) vaccines, hexavalent Hexavac® vaccine (Hexavac) (Sanofi Pasteur MSD) and Infanrix hexa® (Infanrix) (Glaxo-SmithKline Biologicals). We evaluated magnitude and duration of T-cell responses to pertussis toxin (PT) by measuring T-cell proliferation, cytokines (IL-2 and IFNγ) production and memory subsets in two groups of children 5 years after primary vaccination. Some of the enrolled children received only primary vaccination, while others had the pre-school boost dose. Positive T-cell responses to PT were detected in 36% of children. Percentage of responsive children, T-cell proliferation and CD4IL-2+ cells were significantly higher in the children primed with Hexavac than in those who received Infanrix vaccine. No major effects of the boost on PT-specific proliferation were observed. Overall, our data documented a persistence of T-cell memory against PT in a minor fraction of children 5 years after primary vaccination. The different responses induced by Hexavac and Infanrix vaccine could rely on differences in PT inactivation process or excipients/adjuvants formulations.