Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

Glioma-Associated Oncogene Homolog Inhibitors Have the Potential of Suppressing Cancer Stem Cells of Breast Cancer.

PubMed

Jeng, Kuo-Shyang; Jeng, Chi-Juei; Sheen, I-Shyan; Wu, Szu-Hua; Lu, Ssu-Jung; Wang, Chih-Hsuan; Chang, Chiung-Fang

2018-05-05

Overexpression of Sonic Hedgehog signaling (Shh) pathway molecules is associated with invasiveness and recurrence in breast carcinoma. Therefore, inhibition of the Shh pathway downstream molecule Glioma-associated Oncogene Homolog (Gli) was investigated for its ability to reduce progression and invasiveness of patient-derived breast cancer cells and cell lines. Human primary breast cancer T2 cells with high expression of Shh signaling pathway molecules were compared with breast cancer line MDA-MB-231 cells. The therapeutic effects of Gli inhibitors were examined in terms of the cell proliferation, apoptosis, cancer stem cells, cell migration and gene expression. Blockade of the Shh signaling pathway could reduce cell proliferation and migration only in MDA-MB-231 cells. Hh pathway inhibitor-1 (HPI-1) increased the percentages of late apoptotic cells in MDA-MB-231 cells and early apoptotic cells in T2 cells. It reduced Bcl2 expression for cell proliferation and increased Bim expression for apoptosis. In addition, Gli inhibitor HPI-1 decreased significantly the percentages of cancer stem cells in T2 cells. HPI-1 worked more effectively than GANT-58 against breast carcinoma cells. In conclusion, HPI-1 could inhibit cell proliferation, reduce cell invasion and decrease cancer stem cell population in breast cancer cells. To target Gli-1 could be a potential strategy to suppress breast cancer stem cells.

Reduced expression of HSP27 following HAD-B treatment is associated with Her2 downregulation in NIH:OVCAR-3 human ovarian cancer cells.

PubMed

Li, Kuo Chu; Heo, Kyun; Ambade, Nitin; Kim, Min Kyung; Kim, Kyung-Hee; Yoo, Byong Chul; Yoo, Hwa-Seung

2015-09-01

The Korean traditional medicine, HangAmDan (HAD), was developed in 1996 for use as an antitumor agent, and has since been modified to HAD‑B (an altered form of HAD), in order to potentiate its therapeutic effects. In the present study, the effect of HAD‑B on the proliferation and invasion of NIH:OVCAR‑3 and SKOV‑3 human ovarian cancer cell lines was investigated. In addition, the expression of major signal transduction molecules and changes in the proteome in these cells were measured. HAD‑B treatment effectively induced a reduction in the levels of cell proliferation in serum‑free conditioned media. However, unaltered levels of PARP and caspase‑3 indicated that HAD‑B does not reduce proliferation by inducing apoptotic cell death. Fluorescence‑activated cell sorting analysis revealed no significant change in apoptosis following HAD-B treatment. Invasion assay results indicated a reduced rate of invasion following HAD‑B treatment. HAD‑B also influenced the expression of major signal transduction molecules; the phosphorylation of mTOR and AKT was reduced, while that of ERK was increased. Alterations in the proteomes of the two cell lines were investigated following HAD‑B treatment. Among the 9 proteins with differential expression, heat‑shock protein β‑1 (HSP27) was downregulated in NIH:OVCAR‑3 cells treated with HAD‑B. The reduced expression of HSP27 was associated with human epidermal growth factor receptor 2 (Her2) downregulation in these cells. In conclusion, the results of the current proteome assessment suggest that HAD‑B has the potential to suppress the proliferation and invasion of human ovarian cancer cells. HAD‑B treatment of NIH:OVCAR‑3 cells suppressed HSP27 expression and was also associated with Her2 downregulation.

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PubMed Central

2017-01-01

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity.

PubMed

Wang, Yanyan; Xu, Han; Zheng, Xiaodong; Wei, Haiming; Sun, Rui; Tian, Zhigang

2007-10-01

Human umbilical cord blood (CB) has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). It was reported that the activity of CB NK cells was lower than that of adult peripheral blood (PB) NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-gamma, TNF-alpha and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.

Effect of dexamethasone on expression of glucocorticoid receptor in human monocyte cell line THP-1.

PubMed

Li, Bo; Bai, Xiangjun; Wanh, Haiping

2006-01-01

The effect of dexamethasone with different concentrations and different stimulating periods on the expression of glucocorticoid receptors (GRalpha, GRbeta) protein was investigated in human monocyte cell line THP-1. The cultured human monocyte line THP-1 cells were stimulated by dexamethasone with different concentrations and different periods. The expression of GRalpha and GRbeta protein was detected by Western blotting. The results showed that the expression of GRalpha and GRbeta was detected in the THP-1 cells. The quantity of GRalpha expression was reduced by dexamethasone under the same concentration with the prolongation of the stimulating periods. The quantity of GRbeta expression was increased by dexamethasone treatment in a time- and dose-dependent manner. It was concluded that dexamethasone stimulation time-dependently reduced the GRalpha expression in THP-1 cells. Dexamethasone stimulation time- and dose-dependently increased the GRbeta expression in THP-1 cells. The expression of GRalpha and GRbeta was regulated by glucocorticoid.

Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

PubMed

Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P

2013-03-01

Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo

PubMed Central

Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo

2016-01-01

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development. PMID:26771605

Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

PubMed

Xie, Ying; Koch, Mia Lee; Zhang, Xin; Hamblen, Melanie J; Godinho, Frank J; Fujiwara, Yuko; Xie, Huafeng; Klusmann, Jan-Henning; Orkin, Stuart H; Li, Zhe

2017-07-01

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785. © 2017 AlphaMed Press.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

PubMed

Sun, Jingping; Law, Gabriela P; McKallip, Robert J

2012-03-01

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Heat shock protein 90{beta}: A novel mediator of vitamin D action

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

Angelo, Giana; Mineral Bioavailability Laboratory, 711 Washington Street, Boston, MA 02111; Lamon-Fava, Stefania

2008-03-14

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90{beta} expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90{beta} by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%,more » respectively, in Hsp90{beta}-deficient cells. Nuclear protein for VDR and RXR{alpha}, its heterodimer partner, were not reduced in Hsp90{beta}-deficient cells. These findings indicate that Hsp90{beta} is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90{beta} in VDR signaling.« less

microRNA-26a suppresses recruitment of macrophages by down-regulating macrophage colony-stimulating factor expression through the PI3K/Akt pathway in hepatocellular carcinoma.

PubMed

Chai, Zong-Tao; Zhu, Xiao-Dong; Ao, Jian-Yang; Wang, Wen-Quan; Gao, Dong-Mei; Kong, Jian; Zhang, Ning; Zhang, Yuan-Yuan; Ye, Bo-Gen; Ma, De-Ning; Cai, Hao; Sun, Hui-Chuan

2015-05-29

microRNAs (miRNAs) have been reported to modulate macrophage colony-stimulating factor (M-CSF) and macrophages. The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages. Hepatocellular carcinoma (HCC) cell lines with decreased or increased expression of miR-26a were established in a previous study. M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay, and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro. Real-time PCR measured a panel of mRNAs expressed by macrophages. Xenograft models were used to observe tumor growth. Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC. Ectopic expression of miR-26a reduced expression of M-CSF. The conditioned medium (CM) from HepG2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate (PMA) increased expression of interleukin (IL)-12b or IL-23 mRNA and decreased expression of chemokine (C-C motif) ligand (CCL)22, CCL17, and IL-10 mRNA, in comparison to the medium from the parental HepG2 cells. These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002. Ectopic expression of miR-26a in HCC cells suppressed tumor growth, M-CSF expression, and infiltration of macrophages in tumors. Similar results were also found when using HCCLM3 cells. Furthermore, the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC. miR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

FOXP3 over-expression inhibits melanoma tumorigenesis via effects on proliferation and apoptosis.

PubMed

Tan, BeeShin; Anaka, Matthew; Deb, Siddhartha; Freyer, Claudia; Ebert, Lisa M; Chueh, Anderly C; Al-Obaidi, Sheren; Behren, Andreas; Jayachandran, Aparna; Cebon, Jonathan; Chen, Weisan; Mariadason, John M

2014-01-15

The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conflicting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers

NASA Astrophysics Data System (ADS)

Song, Kwang Hoon; Lee, Jaehyun; Park, Hyoungjun; Kim, Hye Mi; Park, Jeehun; Kwon, Keon Woo; Doh, Junsang

2016-03-01

Stiff nuclei in cell-dense microenvironments may serve as distinct biomechanical cues for cell migration, but such a possibility has not been tested experimentally. As a first step addressing this question, we altered nuclear stiffness of endothelial cells (ECs) by reducing the expression of A-type lamins using siRNA, and investigated the migration of T cells on and under EC layers. While most T cells crawling on control EC layers avoided crossing over EC nuclei, a significantly higher fraction of T cells on EC layers with reduced expression of A-type lamins crossed over EC nuclei. This result suggests that stiff EC nuclei underlying T cells may serve as “duro-repulsive” cues to direct T cell migration toward less stiff EC cytoplasm. During subendothelial migration under EC layers with reduced expression of A-type lamins, T cells made prolonged contact and substantially deformed EC nuclei, resulting in reduced speed and directional persistence. This result suggests that EC nuclear stiffness promotes fast and directionally persistent subendothelial migration of T cells by allowing minimum interaction between T cells and EC nuclei.

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection.

PubMed

Venkatasubramanian, Sambasivan; Tripathi, Deepak; Tucker, Torry; Paidipally, Padmaja; Cheekatla, Satyanarayana; Welch, Elwyn; Raghunath, Anjana; Jeffers, Ann; Tvinnereim, Amy R; Schechter, Melissa E; Andrade, Bruno B; Mackman, Nizel; Idell, Steven; Vankayalapati, Ramakrishna

2016-02-01

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2-like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HDM2 promotes WIP1-mediated medulloblastoma growth

PubMed Central

Buss, Meghan C.; Read, Tracy-Ann; Schniederjan, Matthew J.; Gandhi, Khanjan; Castellino, Robert C.

2012-01-01

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma. PMID:22379189

ERAP1 reduces accumulation of aberrant and disulfide-linked forms of HLA-B27 on the cell surface.

PubMed

Tran, Tri M; Hong, Sohee; Edwan, Jehad H; Colbert, Robert A

2016-06-01

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. Published by Elsevier Ltd.

ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

PubMed Central

Tran, Tri; Hong, Sohee; Edwan, Jehad; Colbert, Robert A.

2016-01-01

Objective Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. Methods ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. Results ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Conclusions Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. PMID:27107845

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells.

PubMed

Okamoto, A; Lovett, M; Payan, D G; Bunnett, N W

1994-05-01

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

PubMed

Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

2017-08-01

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells.

PubMed

Chui, Amy; Gunatillake, Tilini; Brennecke, Shaun P; Ignjatovic, Vera; Monagle, Paul T; Whitelock, John M; van Zanten, Dagmar E; Eijsink, Jasper; Wang, Yao; Deane, James; Borg, Anthony J; Stevenson, Janet; Erwich, Jan Jaap; Said, Joanne M; Murthi, Padma

2017-06-01

Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. APPROACH AND RESULTS: BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks' gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 ( ANGPT4 ), platelet-derived growth factor receptor α ( PDGFRA ), tumor necrosis factor superfamily member 15 ( TNFSF15 ), angiogenin ( ANG ), serpin family C member 1 ( SERPIN1 ), angiopoietin 2 ( ANGPT2 ), and CXC motif chemokine 12 ( CXCL12 ) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies. © 2017 American Heart Association, Inc.

Modulation of Mcl-1 expression reduces age-related cochlear degeneration

PubMed Central

Yang, Wei Ping; Xu, Yang; Guo, Wei Wei; Liu, Hui Zhan; Hu, Bo Hua

2013-01-01

Mcl-1 is an anti-apoptotic member of the Bcl-2 family that modulates apoptosis-related signaling pathways and promotes cell survival. We have previously demonstrated a reduction of Mcl-1 expression in aging cochleae. To investigate whether restoring Mcl-1 expression would reduce aging-related cochlear degeneration, we developed a rat model of Mcl-1 overexpression. A plasmid encoding human Mcl-1/enhanced green fluorescent protein was applied to the round window of the cochlea. This in vivo treatment transfected both the sensory and supporting cells of the cochlear sensory epithelium and enhanced Mcl-1 expression at both the mRNA and the protein level. The upregulation of Mcl-1 expression reduced the progression of age-related cochlear dysfunction and sensory cell death. Furthermore, the transfection of Mcl-1 exerted its protective effect by suppressing cochlear apoptosis at the mitochondrial level. This study demonstrates that the genetic modulation of Mcl-1 expression reduces the progression of age-related cochlear degeneration. PMID:23790646

[The role of BDNF pathway in lambda-cyhalothrin disrupting the promotion of 17β-Estradiol on Post-synaptic Density 95 protein expression in HT22 cell].

PubMed

Li, N; Wang, Q N; Wu, D J; Yang, C W; Luo, B B

2016-07-20

Objective: To explore the effect of BDNF pathway on lambda-cyhalothrin interfering estrogen promoting the expression of PSD95 in hippocampus neurons. Methods: HT22 cell line were used to, treating with lambda-cyhalothrin (LCT, 50 μmol/L) , 17β-Estradiol (E2, 10 nmol/L) , LCT (50 μmol/L) +TrkB FC (20 μg/ml) , E2 (10 nmol/L) +TrkB FC (20 μg/ml) , LCT (50 μmol/L) +ICI182 780 (1 μmol/L) , E2 (10 nmol/L) + ICI182 780 (1 μmol/L) , LCT (50 μmol/L) +E2 (10 nmol/L) for 24 h. MTT kit was used to detect cell viability. Post-synaptic Density 95 protein expression was measured by western blot. ELISA assay was used to detect the level of brain derived neurotrophic factor (BDNF) of culture supernatant and cell. Results: Campared to Sham, LCT or E2 could promote the expression of PSD95 LCT+ICI could reduce the expresion of BDNF ( P <0.05) , campared to LCT, LCT+TrkB FC could reduce the expression of PSD95 and LCT+ICI cound reduce the expresion of BDNF ( P <0.05) , campared to E2, E2+TrkB FC could reduce the expression of PSD95 and E 2 +ICI could reduce the expression of BDNF ( P <0.05) , campared to E2, LCT+ E2 could reduce the expression of PSD95 and BDNF ( P <0.05) . Conclusion: BDNF pathway plays a key role in E2 promoting the expression of PSD95 in neural cells. Although LCT alone has a similar effect on E2. LCT could disrupt the promotion of E2 on PSD95 expression via BDNF pathway.

Loss of p21{sup Sdi1} expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21{sup Sdi1} gene promoter

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

Choi, Ok Ran; Lim, In Kyoung, E-mail: iklim@ajou.ac.kr

2011-04-08

Highlights: {yields} Reduced p21 expression in senescent cells treated with DNA damaging agents. {yields} Increase of [{sup 3}H]thymidine and BrdU incorporations in DNA damaged-senescent cells. {yields} Upregulation of miR-93 expression in senescent cells in response to DSB. {yields} Failure of p53 binding to p21 promoter in senescent cells in response to DSB. {yields} Molecular mechanism of increased cancer development in aged than young individuals. -- Abstract: To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin ormore » X-ray irradiation. Response to the damage was different between young and old cells; loss of p21{sup sdi1} expression in spite of p53{sup S15} activation in old cells along with [{sup 3}H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21{sup sdi1} expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.« less

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

PubMed

Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

2017-01-01

In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

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

Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion

2005-12-16

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly,more » PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.« less

FOXP3 over-expression inhibits melanoma tumorigenesis via effects on proliferation and apoptosis.

PubMed Central

Tan, BeeShin; Anaka, Matthew; Deb, Siddhartha; Freyer, Claudia; Ebert, Lisa M.; Chueh, Anderly C.; Al-Obaidi, Sheren; Behren, Andreas; Jayachandran, Aparna; Cebon, Jonathan; Chen, Weisan; Mariadason, John M.

2014-01-01

The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conficting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma. PMID:24406338

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Epithelial to mesenchymal transition in human endocrine islet cells

PubMed Central

Moreno-Amador, José Luis; Téllez, Noèlia; Marin, Sandra; Aloy-Reverté, Caterina; Semino, Carlos; Nacher, Montserrat

2018-01-01

Background β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. Objective To investigate whether EMT takes place in the endocrine non-β cells of human islets. Methodology Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. Results Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. Conclusions In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype. PMID:29360826

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

PubMed

Anastasia, Luigi; Holguera, Javier; Bianchi, Anna; D'Avila, Francesca; Papini, Nadia; Tringali, Cristina; Monti, Eugenio; Villar, Enrique; Venerando, Bruno; Muñoz-Barroso, Isabel; Tettamanti, Guido

2008-03-01

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

EMP2 is a novel therapeutic target for endometrial cancer stem cells

PubMed Central

Kiyohara, Meagan H.; Dillard, Christen; Tsui, Jessica; Kim, Sara Ruth; Lu, Jianyi; Sachdev, Divya; Goodglick, Lee; Tong, Maomeng; Torous, Vanda Farahmand; Aryasomayajula, Chinmayi; Wang, Wei; Najafzadeh, Parisa; Gordon, Lynn K.; Braun, Jonathan; McDermott, Sean; Wicha, Max S.; Wadehra, Madhuri

2017-01-01

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against CSCs in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells. PMID:28604744

Rapid modulation of the silent information regulator 1 by melatonin after hypoxia-ischemia in the neonatal rat brain.

PubMed

Carloni, Silvia; Riparini, Giulia; Buonocore, Giuseppe; Balduini, Walter

2017-10-01

Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia-ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin-treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI-induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin-induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Myocilin Regulates Cell Proliferation and Survival*

PubMed Central

Joe, Myung Kuk; Kwon, Heung Sun; Cojocaru, Radu; Tomarev, Stanislav I.

2014-01-01

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway. PMID:24563482

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

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

Kuan, Man I; O’Dowd, John M.; Fortunato, Elizabeth

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introductionmore » of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.« less

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

NASA Astrophysics Data System (ADS)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

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

Kover, Karen, E-mail: kkover@cmh.edu; University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108; Yan, Yun

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up tomore » 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP expression in beta cells.« less

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the PARP1 inhibitor niraparib to kill ovarian cancer cells.

PubMed

Booth, Laurence; Roberts, Jane L; Samuel, Peter; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Poklepovic, Andrew; Dent, Paul

2018-06-03

The irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.

The Bmi-1 helix–turn and ring finger domains are required for Bmi-1 antagonism of (–) epigallocatechin-3-gallate suppression of skin cancer cell survival

PubMed Central

Balasubramanian, Sivaprakasam; Scharadin, Tiffany M.; Han, Bingshe; Xu, Wen; Eckert, Richard L.

2016-01-01

The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (–) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21Cip1 and p27Kip1 expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix–turn–helix–turn–helix–turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents. PMID:25843776

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

Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activitymore » by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction.« less

ERK1 is important for Th2 differentiation and development of experimental asthma

PubMed Central

Goplen, Nicholas; Karim, Zunayet; Guo, Lei; Zhuang, Yonghua; Huang, Hua; Gorska, Magdalena M.; Gelfand, Erwin; Pagés, Gilles; Pouysségur, Jacques; Alam, Rafeul

2012-01-01

The ERK1/2 signaling pathway regulates a variety of T-cell functions. We observed dynamic changes in the expression of ERK1/2 during T-helper cell differentiation. Specifically, the expression of ERK1/2 was decreased and increased by IL-12 and IL-4, respectively. To address this subject further, we examined the specific role of ERK1 in Th2 differentiation and development of experimental asthma using ERK1−/− mice. ERK1−/− mice were unable to mount airway inflammation and hyperreactivity in two different models of asthma, acute and chronic. ERK1−/− mice had reduced expression of Th2 cytokines IL-4 and IL-5 but not IL-17A or IFN-γ. They had reduced levels of allergen-specific IgE and blood eosinophils. T cells from immunized ERK1−/− mice manifested reduced proliferation in response to the sensitizing allergen. ERK1−/− T cells had reduced and short-lived expression of JunB following TCR stimulation, which likely contributed to their impaired Th2 differentiation. Immunized ERK1−/− mice showed reduced numbers of CD44high CD4 T cells in the spleen. In vitro studies demonstrated that Th2 but not Th1 cells from ERK1−/− mice had reduced numbers of CD44high cells. Finally, CD4 T cells form ERK1−/− mice expressed higher levels of BIM under growth factor-deprived conditions and reduced Mcl-1 on stimulation. As a result, the survival of CD4 T cells, especially CD44high Th2 cells, was much reduced in ERK1−/− mice. We conclude that ERK1 plays a nonredundant role in Th2 differentiation and development of experimental asthma. ERK1 controls Th2 differentiation and survival through its effect on JunB and BIM, respectively.—Goplen, N., Karim, Z., Guo, L., Zhuang, Y., Huang, H., Gorska, M. M., Gelfand, E., Pagés, G., Pouysségur, J., Alam, R. ERK1 is important for Th2 differentiation and development of experimental asthma. PMID:22262639

Reduced Ang2 expression in aging endothelial cells.

PubMed

Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells.

PubMed Central

Okamoto, A; Lovett, M; Payan, D G; Bunnett, N W

1994-01-01

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR. Images Figure 1 Figure 2 PMID:7514869

Long noncoding RNA BC200 regulates cell growth and invasion in colon cancer.

PubMed

Wu, Kaiming; Xu, Kaiwu; Liu, Kuanzhi; Huang, Jiehong; Chen, Jianhui; Zhang, Jian; Zhang, Ning

2018-06-01

Colon cancer is the third most commonly diagnosed and deadly cancer worldwide. Efforts have been made to characterize its pathological mechanisms and to explore new therapeutic targets of this disease. Aberrant expression of long noncoding RNAs (lncRNAs) has been associated with the pathogenesis of colon cancer. In the current study, we aimed to define the biological mechanism of the lncRNA BC200 in colon cancer. Here, we found that expression of BC200 was up-regulated in colon cancer tissues as compared with adjacent non-cancerous tissues. The BC200 level was positively correlated with advanced TNM stage. The Kaplan-Meier method indicated that the cumulative survival rate was significantly lower in patients with high BC200 expression than in those with low BC200 expression. Interestingly, we found that knockdown of BC200 inhibited proliferation of HCT-116 and HT29 colon cancer cell lines and reduce the expression of cell proliferation markers, such as Ki-67 and PCNA. In addition, silencing of BC200 could induce obvious G0/G1 arrest and cause apoptosis in HCT-116 and HT29 cells and reduced the expression of cyclin D1, cyclin E, and c-Myc through inhibiting the expression of β-catenin. Importantly, we found that knockdown of BC200 reduced invasion of HCT-116 and HT29 cells and epithelial-mesenchymal transition (EMT) by reducing the expression of MMP-2 and MMP-9. Mechanistically, silencing of BC200 significantly reduced the phosphorylation of STAT3. Overall, the findings presented here suggest that lncRNA BC200 may serve as a novel oncogene and a new therapeutic target for colon cancer. Copyright © 2018. Published by Elsevier Ltd.

Hormonal Regulation and Distinct Functions of Semaphorin-3B and Semaphorin-3F in Ovarian Cancer

PubMed Central

Joseph, Doina; Ho, Shuk-Mei; Syed, Viqar

2009-01-01

Semaphorins comprise a family of molecules that influence neuronal growth and guidance. Class-3 semaphorins, semaphorin-3B (SEMA3B) and semaphorin-3F (SEMA3F) illustrate their effects by forming a complex with neuropilins (NP-1 or NP-2) and plexins. We examined the status and regulation of semaphorins and their receptors in human ovarian cancer cells. A significantly reduced expression of SEMA3B (83 kD), SEMA3F (90 kD), and plexin-A3 was observed in ovarian cancer (OVCA) cell lines when compared to normal human ovarian surface epithelial (HOSE) cells. The expression of NP-1, NP-2 and plexin-A1 was not altered in HOSE and OVCA cells. The decreased expression of SEMA3B, SEMA3F, and plexin-A3 was confirmed in stage 3 ovarian tumors. Treatment of OVCA cells with luteinizing hormone, follicle-stimulating hormone, and estrogen induced a significant upregulation of SEMA3B, whereas SEMA3F was upregulated only by estrogen. Co-treatment of cell lines with a hormone and its specific antagonist blocked the effect of the hormone. Ectopic expression of SEMA3B or SEMA3F reduced soft-agar colony formation, adhesion, and cell invasion of OVCA cell cultures. Forced expression of SEMA3B, but not SEMA3F, inhibited viability of OVCA cells. Overexpression of SEMA3B and SEMA3F reduced focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase (MMP)-2 and -9 expression in OVCA cells. Forced expression of SEMA3F, but not SEMA3B in OVCA cells, significantly inhibited endothelial cell tube formation. Collectively, our results suggest loss of SEMA3 expression could be a hallmark of cancer progression. Furthermore, gonadotropin- and/or estrogen-mediated maintenance of SEMA3 expression could control ovarian cancer angiogenesis and metastasis. PMID:20124444

Transcription factor Mohawk and the pathogenesis of human anterior cruciate ligament degradation

PubMed Central

Nakahara, Hiroyuki; Hasegawa, Akihiko; Otabe, Koji; Ayabe, Fumiaki; Matsukawa, Tetsuya; Onizuka, Naoko; Ito, Yoshiaki; Ozaki, Toshifumi; Lotz, Martin K.; Asahara, Hiroshi

2013-01-01

Objective To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissues and ligament cells from normal and osteoarthritis-affected knees. Methods Knee joints were obtained at autopsy within 24-48 hours postmortem from 13 normal donors (age 36.9±11.0 years), 16 OA donors (age 79.7±11.4 years) and 8 old donors without OA (age 76.9±12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative PCR. ACL-derived cells were used to study regulation of MKX expression by IL-1β. MKX was knocked down by siRNA to analyze function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells. Results The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry showed that MKX positive cells were significantly reduced in ACL tissues from OA donors in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX gene expression and reduced ligament ECM genes, COL1A1 and TNXB. On the other hand, SOX9, chondrocyte master transcription factor, was up regulated by IL-1β treatment. Importantly, knock down of MKX expression by siRNA upregulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB were decreased. Conclusion Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints and this may be in part mediated by IL-1β. MKX appears necessary to maintain the tissue specific cellular differentiation status and ECM production in adult human tendons and ligaments. PMID:23686683

Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

PubMed

Mebratu, Yohannes A; Schwalm, Kurt; Smith, Kevin R; Schuyler, Mark; Tesfaigzi, Yohannes

2011-06-01

Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. We screened for dysregulated expression of the Bcl-2 family members. We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

Cigarette Smoke Suppresses Bik To Cause Epithelial Cell Hyperplasia and Mucous Cell Metaplasia

PubMed Central

Mebratu, Yohannes A.; Schwalm, Kurt; Smith, Kevin R.; Schuyler, Mark; Tesfaigzi, Yohannes

2011-01-01

Rationale: Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. Objectives: To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. Methods: We screened for dysregulated expression of the Bcl-2 family members. Measurements and Main Results: We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. Conclusions: These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis. PMID:21317312

The role of human chorionic gonadotropin in regulation of naïve and memory T cells activity in vitro.

PubMed

Zamorina, S A; Litvinova, L S; Yurova, K A; Khaziakhmatova, O G; Timganova, V P; Bochkova, M S; Khramtsov, P V; Rayev, M B

2018-01-01

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA+) and CD25 expression on the memory T cells (CD45R0+). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0+cells. In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression

PubMed Central

Rao, Jasti S.

2013-01-01

Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2. PMID:23222817

Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

PubMed Central

Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

2016-01-01

Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

PubMed

Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation. These events are manifest in inflammatory diseases, such as atherosclerosis. Therefore, our results suggest that DBMSCs could be usefully employed as a therapeutic strategy for atherosclerosis.

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression.

PubMed

Martínez, Juan A; Tavárez, José J; Oliveira, Caroline M; Banerjee, Dipak K

2006-05-01

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

Effect of microculture on cell metabolism and biochemistry: do cells get stressed in microchannels?

PubMed

Su, Xiaojing; Theberge, Ashleigh B; January, Craig T; Beebe, David J

2013-02-05

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

PubMed

Richter, D U; Jeschke, U; Bergemann, C; Makovitzky, J; Lüthen, F; Karsten, U; Briese, V

2005-01-01

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

Iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/ t‑BID/cytochrome c/caspase-3 signaling pathway.

PubMed

Wang, Yansheng; Liu, Changqing; Wang, Jianchun; Zhang, Yang; Chen, Linlin

2017-09-01

The aim of this study was to elucidate the effects of iodine-131 on the induction of apoptosis in human cardiac muscle cells and the underlying molecular mechanisms. We found that iodine-131 reduced cell proliferation, induced apoptosis, induced p53, PIDD, t-BID (mitochondria) protein expression, suppressed cytochrome c (mitochondria) protein expression, and increased Bax protein expression, and promoted caspase-2, -3 and -9 expression levels in human cardiac muscle cells. Meanwhile, si-p53 inhibited the effects of iodine-131 on the reduction in cell proliferation and induction of apoptosis in human cardiac muscle cells through regulation of Bax/cytochrome c/caspase-3 and PIDD/caspase‑2/t-BID/cytochrome c/caspase-3 signaling pathway. After si-Bax reduced the effects of iodine-131, it reduced cell proliferation and induced apoptosis in human cardiac muscle cells through the cytochrome c/caspase-3 signaling pathway. However, si-caspase-2 also reduced the effects of iodine-131 on the reduction of cell proliferation and induction of apoptosis in human cardiac muscle cells through the t-BID/cytochrome c/caspase-3 signaling pathway. These findings demonstrated that iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/t-BID/cytochrome c/caspase-3 signaling pathway.

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

PubMed

Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

2015-11-18

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

miR-137 regulates the constitutive androstane receptor and modulates doxorubicin sensitivity in parental and doxorubicin-resistant neuroblastoma cells

PubMed Central

Takwi, Apana A; Wang, Yue-Ming; Wu, Jing; Michaelis, Martin; Cinatl, Jindrich; Chen, Taosheng

2013-01-01

Chemotherapy is the most common treatment for cancer. However, multidrug resistance (MDR) remains a major obstacle to effective chemotherapy, limiting the efficacy of both conventional chemotherapeutic and novel biologic agents. The constitutive androstane receptor (CAR), a xenosensor, is a key regulator of MDR. It functions in xenobiotic detoxification by regulating the expression of phase I drug metabolizing enzymes and ATP-binding cassette (ABC) transporters, whose overexpression in cancers and whose role in drug resistance make them potential therapeutic targets for reducing MDR. MicroRNAs (miRNAs) are endogenous negative regulators of gene expression and have been implicated in most cellular processes, including drug resistance. Here we report the inversely related expression of miR-137 and CAR in parental and doxorubicin-resistant neuroblastoma cells, wherein miR-137 is down-regulated in resistant cells. miR-137 over-expression resulted in down-regulation of CAR protein and mRNA (via mRNA degradation); it sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and increased G2-phase cell cycle arrest) and reduced the in vivo growth rate of neuroblastoma xenografts. We observed similar results in cellular models of hepatocellular and colon cancers, indicating that the doxorubicin-sensitizing effect of miR-137 is not tumor type-specific. Finally, we show for the first time a negative feedback loop whereby miR-137 down-regulates CAR expression and CAR down-regulates miR-137 expression. Hypermethylation of the miR-137 promoter and negative regulation of miR-137 by CAR contribute in part to reduced miR-137 expression and increased CAR and MDR1 expression in doxorubicin-resistant neuroblastoma cells. These findings demonstrate that miR-137 is a crucial regulator of cancer response to doxorubicin treatment, and they identify miR-137 as a highly promising target to reduce CAR-driven doxorubicin resistance. PMID:23934188

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

PubMed

Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

2018-06-14

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

Serotonin- and Dopamine-Related Gene Expression in db/db Mice Islets and in MIN6 β-Cells Treated with Palmitate and Oleate.

PubMed

Cataldo, L R; Mizgier, M L; Busso, D; Olmos, P; Galgani, J E; Valenzuela, R; Mezzano, D; Aranda, E; Cortés, V A; Santos, J L

2016-01-01

High circulating nonesterified fatty acids (NEFAs) concentration, often reported in diabetes, leads to impaired glucose-stimulated insulin secretion (GSIS) through not yet well-defined mechanisms. Serotonin and dopamine might contribute to NEFA-dependent β-cell dysfunction, since extracellular signal of these monoamines decreases GSIS. Moreover, palmitate-treated β-cells may enhance the expression of the serotonin receptor Htr2c, affecting insulin secretion. Additionally, the expression of monoamine-oxidase type B (Maob) seems to be lower in islets from humans and mice with diabetes compared to nondiabetic islets, which may lead to increased monoamine concentrations. We assessed the expression of serotonin- and dopamine-related genes in islets from db/db and wild-type (WT) mice. In addition, the effect of palmitate and oleate on the expression of such genes, 5HT content, and GSIS in MIN6 β-cell was determined. Lower Maob expression was found in islets from db/db versus WT mice and in MIN6 β-cells in response to palmitate and oleate treatment compared to vehicle. Reduced 5HT content and impaired GSIS in response to palmitate (-25%; p < 0.0001) and oleate (-43%; p < 0.0001) were detected in MIN6 β-cells. In conclusion, known defects of GSIS in islets from db/db mice and MIN6 β-cells treated with NEFAs are accompanied by reduced Maob expression and reduced 5HT content.

Transdifferentiation between Luminal- and Basal-Type Cancer Cells

DTIC Science & Technology

2013-12-01

aggressiveness of MCF7/shPKD1 cells. More importantly, the MCF7/shPKD1 cells show reduced expression of estrogen receptor (ERα/ ESR1 , Fig 3B-D). Clinically, the...express Her2, the lack of expression of ESR1 and PgR (Fig 3D and Table 1) literally make the MCF7/shPKD1 cell a triple-negative cancer cell. Fig 3

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

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

Tomioka, Yukiko, E-mail: ytomi@muses.tottori-u.ac.jp; Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553; Morimatsu, Masami, E-mail: mmorimat@vetmed.hokudai.ac.jp

Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9more » (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.« less

The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis.

PubMed

Shao, Weijuan; Xiong, Xiaoquan; Ip, Wilfred; Xu, Fenghao; Song, Zhuolun; Zeng, Kejing; Hernandez, Marcela; Liang, Tao; Weng, Jianping; Gaisano, Herbert; Nostro, M Cristina; Jin, Tianru

2015-04-01

Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding. TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass. Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.

Effect of heat stress and recovery on viability, oxidative damage, and heat shock protein expression in hepatic cells of grass carp (Ctenopharyngodon idellus).

PubMed

Cui, Yanting; Liu, Bo; Xie, Jun; Xu, Pao; Habte-Tsion, H-Michael; Zhang, Yuanyuan

2014-06-01

In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34 °C for 0.5 h and then immediately incubated at 27 °C in 5 % CO2 for 6, 12, 24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32 °C, the expression of HSP60 after recovery for 1, 2, and 4 h (P < 0.05), the expression of HSP70 after recovery for 0.5 and 1 h (P < 0.01), and the expression of HSP90 throughout recovery were significantly higher (P < 0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium

PubMed Central

Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B.; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Purpose Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch’s membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Methods Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Results Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Conclusions Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases. PMID:27293373

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium.

PubMed

Dardik, Rima; Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch's membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes

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

Gonzalez-Rubio, Sandra; Linares, Clara I.; Bello, Rosario I.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca{sup 2+} on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca{sup 2+} pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca{sup 2+} entrance was induced by A23187 in HepG2. Cell death, Ca{sup 2+} mobilization, NOS-1, -2 and -3 expression, AP-1 activation,more » and NO production were evaluated. GCDCA reduced intracellular Ca{sup 2+} concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and L-NAME enhanced, GCDCA-induced cell death. The reduction of Ca{sup 2+} entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca{sup 2+} entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca{sup 2+} concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca{sup 2+}-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.« less

Thymoquinone suppresses migration of LoVo human colon cancer cells by reducing prostaglandin E2 induced COX-2 activation.

PubMed

Hsu, Hsi-Hsien; Chen, Ming-Cheng; Day, Cecilia Hsuan; Lin, Yueh-Min; Li, Shin-Yi; Tu, Chuan-Chou; Padma, Viswanadha Vijaya; Shih, Hui-Nung; Kuo, Wei-Wen; Huang, Chih-Yang

2017-02-21

To identify potential anti-cancer constituents in natural extracts that inhibit cancer cell growth and migration. Our experiments used high dose thymoquinone (TQ) as an inhibitor to arrest LoVo (a human colon adenocarcinoma cell line) cancer cell growth, which was detected by cell proliferation assay and immunoblotting assay. Low dose TQ did not significantly reduce LoVo cancer cell growth. Cyclooxygenase 2 (COX-2) is an enzyme that is involved in the conversion of arachidonic acid into prostaglandin E2 (PGE2) in humans. PGE2 can promote COX-2 protein expression and tumor cell proliferation and was used as a control. Our results showed that 20 μmol/L TQ significantly reduced human LoVo colon cancer cell proliferation. TQ treatment reduced the levels of p-PI3K, p-Akt, p-GSK3β, and β-catenin and thereby inhibited the downstream COX-2 expression. Results also showed that the reduction in COX-2 expression resulted in a reduction in PGE2 levels and the suppression of EP2 and EP4 activation. Further analysis showed that TG treatment inhibited the nuclear translocation of β-catenin in LoVo cancer cells. The levels of the cofactors LEF-1 and TCF-4 were also decreased in the nucleus following TQ treatment in a dose-dependent manner. Treatment with low dose TQ inhibited the COX-2 expression at the transcriptional level and the regulation of COX-2 expression efficiently reduced LoVo cell migration. The results were further verified in vivo by confirming the effects of TQ and/or PGE2 using tumor xenografts in nude mice. TQ inhibits LoVo cancer cell growth and migration, and this result highlights the therapeutic advantage of using TQ in combination therapy against colorectal cancer.

BAG3 increases the invasiveness of uterine corpus carcinoma cells by suppressing miR-29b and enhancing MMP2 expression.

PubMed

Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

2015-05-01

Approximately 30% of uterine corpus carcinomas are diagnosed at an advanced stage and have a poor prognosis. Our previous study indicated that BCL2-associated athanogene 3 (BAG3) enhances matrix metalloproteinase-2 (MMP2) expression and binds to MMP2 to positively regulate the process of cell invasion in ovarian cancer cells. Recently, altered miRNA expression patterns were observed in several groups of patients with endometrial cancers. One of the altered miRNAs, miR-29b, reportedly reduces tumor invasiveness by suppressing MMP2 expression. Our aim in the present study was to examine the relationships among BAG3, miR-29b and MMP2 in endometrioid adenocarcinoma cells. We found that BAG3 suppresses miR-29b expression and enhances MMP2 expression, which in turn increases cell motility and invasiveness. Moreover, restoration of miR-29b through BAG3 knockdown reduced MMP2 expression, as well as cell motility and invasiveness. Collectively, our findings indicate that BAG3 enhances MMP2 expression by suppressing miR-29b, thereby increasing the metastatic potential of endometrioid adenocarcinomas.

BRCA1/p220 loss triggers BRCA1-IRIS overexpression via mRNA stabilization in breast cancer cells

PubMed Central

Shimizu, Yoshiko; Mullins, Nicole; Blanchard, Zannel; ElShamy, Wael M.

2012-01-01

BRCA1/p220-assocaited and triple negative/basal-like (TN/BL) tumors are aggressive and incurable breast cancer diseases that share among other features the no/low BRCA1/p220 expression. Here we show that BRCA1/p220 silencing in normal human mammary epithelial (HME) cells reduces expression of two RNA-destabilizing proteins, namely AUF1 and pCBP2, both proteins bind and destabilize BRCA1-IRIS mRNA. BRCA1-IRIS overexpression in HME cells triggers expression of several TN/BL markers, e.g., cytokeratins 5 and 17, p-cadherin, EGFR and cyclin E as well as expression and activation of the pro-survival proteins; AKT and survivin. BRCA1-IRIS silencing in the TN/BL cell line, SUM149 or restoration of BRCA1/p220 expression in the mutant cell line, HCC1937 reduced expression of TN/BL markers, AKT, survivin, and induced cell death. Collectively, we propose that BRCA1/p220 loss of expression or function triggers BRCA1-IRIS overexpression through a post-transcriptional mechanism, which in turn promotes formation of aggressive and invasive breast tumors by inducing expression of TN/BL and survival proteins. PMID:22431556

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts.

PubMed

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-09-15

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation.

Lipopolysaccharide inhibits the self-renewal of spermatogonial stem cells in vitro via downregulation of GDNF expression in Sertoli cells.

PubMed

Zhang, Xiaoli; Shi, Kun; Li, Yi; Zhang, Haiyu; Hao, Jing

2014-06-01

Lipopolysaccharide (LPS) can reduce sperm count and sperm quality. The molecular mechanisms underlying this process are not fully understood. In this report, we investigated the effects of LPS-treated Sertoli cells on self-renewal and differentiation of spermatogoinial stem cells (SSCs). Sertoli cell cultures were established and incubated with LPS (10μg/ml) for 1, 2 or 3 days, respectively. The culture media were collected and used as conditioned media (CM) to culture SSCs. The expression of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF) and bone morphogenetic protein 4 (BMP4) in Sertoli cells treated with LPS was analyzed by RT-PCR and Western blotting. The results showed that the expression of SSC differentiation markers, c-kit and Sohlh2, was increased, while the expression of SSC self-renewal markers, plzf, oct4, and PCNA, was repressed when cultured in CM from LPS-treated Sertoli cells. GDNF levels in Sertoli cells and CM reduced dramatically after LPS treatments, while SCF and BMP4 levels did not show any significant changes. Moreover, correlated with the GDNF levels in CM, GDNF target genes, Bcl6b and Etv5, were reduced markedly in SSCs. Our results suggest that LPS inhibits the expression of GDNF in Sertoli cells, and might prevent the SSC self-renewal via down-regulation of GDNF target genes. Copyright © 2014 Elsevier Inc. All rights reserved.

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

PubMed Central

Peiris, Heshan; Duffield, Michael D.; Fadista, Joao; Kashmir, Vinder; Genders, Amanda J.; McGee, Sean L.; Martin, Alyce M.; Saiedi, Madiha; Morton, Nicholas; Carter, Roderick; Cousin, Michael A.; Oskolkov, Nikolay; Volkov, Petr; Hough, Tertius A.; Fisher, Elizabeth M. C.; Tybulewicz, Victor L. J.; Busciglio, Jorge; Coskun, Pinar E.; Becker, Ann; Belichenko, Pavel V.; Mobley, William C.; Ryan, Michael T.; Chan, Jeng Yie; Laybutt, D. Ross; Coates, P. Toby; Yang, Sijun; Ling, Charlotte; Groop, Leif; Pritchard, Melanie A.; Keating, Damien J.

2016-01-01

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D. PMID:27195491

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

PubMed Central

Klamer, Sofieke E; Kuijk, Carlijn GM; Hordijk, Peter L; van der Schoot, C Ellen; von Lindern, Marieke; van Hennik, Paula B; Voermans, Carlijn

2013-01-01

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis. PMID:24152593

MiR-24 alleviates cardiomyocyte apoptosis after myocardial infarction via targeting BIM.

PubMed

Pan, L-J; Wang, X; Ling, Y; Gong, H

2017-07-01

Ischemia hypoxia induces cardiomyocyte (CM) apoptosis in the process of acute myocardial infarction (AMI). It was showed that pro-apoptosis factor BIM participates in regulating tumor cell apoptosis under ischemia or hypoxia condition, while its role in CM apoptosis after AMI is still unclear. It was revealed that miR-24 expression was significantly reduced in myocardial tissue after AMI. Bioinformatics analysis exhibits that miR-24 is targeted to the 3'-UTR of BIM. This study aims to investigate the role of miR-24 in mediating BIM expression and CM apoptosis. Dual-luciferase assay was used to confirm the targeted regulation between miR-24 and BIM. Cells were cultured under ischemia hypoxia for 12 h after transfection for 48 h. Cell apoptosis was tested by using flow cytometry. The caspase activity was detected by using spectrophotometry. Wistar rats were divided into four groups, including Sham, AMI, AMI + agomir-control, and AMI + agomir-24 groups. Cardiac function was evaluated by using echocardiography. CM apoptosis was determined by using TUNEL. Infarction area was measured by using evans blue staining. MiR-24 targeted suppressed BIM expression. MiR-24 mimic and/or si-BIM transfection significantly declined the BIM expression, inhibited caspase-9 and caspase-3 activities, and reduced cell apoptosis in H9C2 cells. MiR-24 expression was decreased, while BIM levels were up-regulated in myocardium after AMI. Agomir-24 injection down-regulated the BIM expression in myocardium, reduced CM apoptosis, narrowed infarction area, and improved cardiac function in rats. MiR-24 was reduced, whereas BIM was enhanced in the CM after AMI. MiR-24 up-regulation plays a critical role in decreasing BIM expression, reducing CM apoptosis, and improving cardiac function after AMI.

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma.

PubMed

Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

2014-07-30

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

PubMed Central

Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

2014-01-01

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

Low gene expression levels of activating receptors of natural killer cells (NKG2E and CD94) in patients with fulminant type 1 diabetes.

PubMed

Nakata, Shinsuke; Imagawa, Akihisa; Miyata, Yugo; Yoshikawa, Atsushi; Kozawa, Junji; Okita, Kohei; Funahashi, Tohru; Nakamura, Seiji; Matsubara, Kenichi; Iwahashi, Hiromi; Shimomura, Iichiro

2013-01-01

Fulminant type 1 diabetes is an independent subtype of type 1 diabetes characterized by extremely rapid onset and absence of islet-related autoantibodies. However, detailed pathophysiology of this subtype is poorly understood. In this study, a comprehensive approach was applied to understand the pathogenesis of fulminant type 1 diabetes. We determined the genes that were differentially expressed in fulminant type 1 diabetes compared with type 1A diabetes and healthy control, using gene expression microarray in peripheral blood cells. Using volcano plot analysis, we found reduced expression of killer cell lectin-like receptor subfamily C, member 3 (KLRC3) which encodes NKG2E, a natural killer (NK) cell activating receptor, in fulminant type 1 diabetes, compared with healthy controls. This difference was confirmed by real-time RT-PCR among NK-enriched cells. The expression of KLRD1 (CD94), which forms heterodimer with NKG2E (KLRC3), was also reduced in NK-enriched cells in fulminant type 1 diabetes. Furthermore, flow cytometry showed significantly lower proportion of NK cells among peripheral blood mononuclear cells (PBMCs) in fulminant type 1 diabetes than in healthy controls. In patients with fulminant type 1 diabetes, the relative proportion of NK cells correlated significantly with the time period between onset of fever to the appearance of hyperglycemic-related symptoms. We conclude the presence of reduced NK activating receptor gene expression and low proportion of NK cells in fulminant type 1 diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription.

PubMed

Fernández, Jaime G; Rodríguez, Diego A; Valenzuela, Manuel; Calderon, Claudia; Urzúa, Ulises; Munroe, David; Rosas, Carlos; Lemus, David; Díaz, Natalia; Wright, Mathew C; Leyton, Lisette; Tapia, Julio C; Quest, Andrew Fg

2014-09-09

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

PubMed

Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

2018-04-01

Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

[Curcumine inhibits migration and invasion of hepatic stellate cells by reducing MMP-2 expression and activity].

PubMed

Huang, Jian-xian; Zhu, Bao-he; He, De; Huang, Lin; Hu, Ke; Huang, Bo

2009-11-01

To investigate the molecular mechanism of the inhibitory effect of curcumine on the migration and invasion of hepatic stellate cells (HSC). Rat hepatic stellate cells were cultured and activated with ConA. Matrix metalloproteinase-2 (MMP-2) expression and activity was determined by Western blot and gelatin zymography. Migration and invasion of HSC was assessed by wound healing assay and modified Boyden chamber assay. Curcumine reduced the level and activity of MMP-2 expression in activated HSC in a dose-dependent manner. When treated with 25, 50 or 100 micromol/L curcumine, the expression of MMP-2 was reduced by 21.8%+/-5.1%, 65.5%+/-9.2% or 87.9%+/-11.5% (P < 0.05), and the activity of MMP-2 was also significantly reduced by curcumine. Migration and invasion of activated HSC was also inhibited by curcumine in a dose-dependent way. When treated with 25, 50 or 100 micromol/L curcumine, the migration of activated HSC was reduced by 27.5%+/-5.8%, 54.4%+/-7.6% or 67.1%+/-9.3% (P < 0.05), and the invasion of activated HSC was also significantly reduced by curcumine. Curcumine inhibits migration and invasion of activated HSC by reducing MMP-2 expression and activity.

Bitter Melon Reduces Head and Neck Squamous Cell Carcinoma Growth by Targeting c-Met Signaling

PubMed Central

Nerurkar, Pratibha; Gonzalez, Juan G.; Crawford, Susan; Varvares, Mark; Ray, Ratna B.

2013-01-01

Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat, and despite of advances in treatment, the overall survival rate has only modestly improved over the past several years. Thus, there is an urgent need for additional therapeutic modalities. We hypothesized that treatment of HNSCC cells with a dietary product such as bitter melon extract (BME) modulates multiple signaling pathways and regresses HNSCC tumor growth in a preclinical model. We observed a reduced cell proliferation in HNSCC cell lines. The mechanistic studies reveal that treatment of BME in HNSCC cells inhibited c-Met signaling pathway. We also observed that BME treatment in HNSCC reduced phosphoStat3, c-myc and Mcl-1 expression, downstream signaling molecules of c-Met. Furthermore, BME treatment in HNSCC cells modulated the expression of key cell cycle progression molecules leading to halted cell growth. Finally, BME feeding in mice bearing HNSCC xenograft tumor resulted in an inhibition of tumor growth and c-Met expression. Together, our results suggested that BME treatment in HNSCC cells modulates multiple signaling pathways and may have therapeutic potential for treating HNSCC. PMID:24147107

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model.

PubMed

Zhu, Wan; Chen, Wanqiu; Zou, Dingquan; Wang, Liang; Bao, Chen; Zhan, Lei; Saw, Daniel; Wang, Sen; Winkler, Ethan; Li, Zhengxi; Zhang, Meng; Shen, Fanxia; Shaligram, Sonali; Lawton, Michael; Su, Hua

2018-05-01

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. bAVMs were induced through induction of brain focal activin-like kinase 1 ( Alk1 , an AVM causative gene) gene deletion and angiogenesis in adult Alk1 -floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68 + cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression. © 2018 American Heart Association, Inc.

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

PubMed

Shi, Yanhong; Chichung Lie, D; Taupin, Philippe; Nakashima, Kinichi; Ray, Jasodhara; Yu, Ruth T; Gage, Fred H; Evans, Ronald M

2004-01-01

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

Suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression and TNFα-mediated NFκB activation in piceatannol-treated human leukemia U937 cells.

PubMed

Liu, Wen-Hsin; Chang, Long-Sen

2012-09-01

To address the mechanism of piceatannol in inhibiting TNFα-mediated pathway, studies on piceatannol-treated human leukemia U937 cells were conducted. Piceatannol treatment reduced TNFα shedding and NFκB activation and decreased the release of soluble TNFα into the culture medium of U937 cells. Moreover, ADAM17 expression was down-regulated in piceatannol-treated cells. Over-expression of ADAM17 abrogated the ability of piceatannol to suppress TNFα-mediated NFκB activation. Piceatannol-evoked β-TrCP up-regulation promoted Sp1 degradation, thus reducing transcriptional level of ADAM17 gene in U937 cells. Piceatannol treatment induced p38 MAPK phosphorylation but inactivation of Akt and ERK. In contrast to p38 MAPK inhibitor or restoration of ERK activation, transfection of constitutive active Akt abolished the effect of piceatannol on β-TrCP, Sp1 and ADAM17 expression. Piceatannol-elicited down-regulation of miR-183 expression was found to cause β-TrCP up-regulation. Inactivation of Akt resulted in Foxp3 down-regulation and reduced miR-183 expression in piceatannol-treated cells. Knock-down of Foxp3 and chromatin immunoprecipitating revealed that Foxp3 genetically regulated transcription of miR-183 gene. Taken together, our data indicate that suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression in piceatannol-treated U937 cells. Consequently, piceatannol suppresses TNFα shedding, leading to inhibition of TNFα/NFκB pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

Downregulation of CXCR4 Expression and Functionality After Zoledronate Exposure in Canine Osteosarcoma.

PubMed

Byrum, M L; Pondenis, H C; Fredrickson, R L; Wycislo, K L; Fan, T M

2016-07-01

The establishment and progression of metastases remains the life-limiting factor for dogs diagnosed with osteosarcoma (OS). The pattern of metastases is likely regulated through interactions between chemokine receptors and chemokines, and perturbations in these signaling cascades responsible for cytoskeletal organization and directional migration have the potential to alter metastatic cell trafficking behaviors. Zoledronate will impair directional migration of OS cells through downregulation of chemokine (C-X-C motif) receptor 4 (CXCR4) expression and functionality. Nineteen archived tumor specimens and plasma from 20 dogs with OS. Prospectively, the expressions of CXCR4 were studied in OS cell lines and spontaneous tumor samples. The effect of zoledronate on CXCR4 expression and functionality was investigated by characterizing responses in 3 OS cell lines. In 19 OS specimens and 20 dogs with OS, changes in CXCR4 expression and circulating CXCR4 concentrations were characterized in response to zoledronate therapy respectively. All canine OS cells express CXCR4, and zoledronate reduces CXCR4 expression and functionality by 27.7% (P < .0001), through augmented proteasome degradation and reduced prenylation of heterotrimeric G-proteins in 33% of tumor cell lines evaluated. In OS-bearing dogs, zoledronate reduces CXCR4 expressions by 40% within the primary tumor compared to untreated controls (P = .03) and also decreases the circulating concentrations of CXCR4 in 18 of 20 dogs with OS. Zoledronate can alter CXCR4 expression and functionality in OS cells, and consequent perturbations in CXCR4 intracellular signaling cascades might influence patterns of metastases. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

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

Malinowsky, Katharina; Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg; Luksza, Julia

2008-06-20

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tailmore » required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of virus-cell fusion confirming the correlation between post-translational modification of tubulin and virus-cell fusion. These results thus identify tubulin and its post-translational modification as a new cellular target for interference with HIV-cell fusion.« less

The role of MMP-1 in breast cancer growth and metastasis to the brain in a xenograft model.

PubMed

Liu, Hui; Kato, Yukinari; Erzinger, Stephanie A; Kiriakova, Galina M; Qian, Yongzhen; Palmieri, Diane; Steeg, Patricia S; Price, Janet E

2012-12-07

Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis. Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability. Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells. Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

Role of Bruton’s tyrosine kinase in myeloma cell migration and induction of bone disease

PubMed Central

Bam, Rakesh; Ling, Wen; Khan, Sharmin; Pennisi, Angela; Venkateshaiah, Sathisha Upparahalli; Li, Xin; van Rhee, Frits; Usmani, Saad; Barlogie, Bart; Shaughnessy, John; Epstein, Joshua; Yaccoby, Shmuel

2014-01-01

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton’s tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL) –6– or stroma–dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. PMID:23456977

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

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

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

PubMed

Valero, María Ll; Mello de Queiroz, Fernanda; Stühmer, Walter; Viana, Félix; Pardo, Luis A

2012-01-01

Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.

Prebiotic oligosaccharides reduce proinflammatory cytokines in intestinal Caco-2 cells via activation of PPARγ and peptidoglycan recognition protein 3.

PubMed

Zenhom, Marwa; Hyder, Ayman; de Vrese, Michael; Heller, Knut J; Roeder, Thomas; Schrezenmeir, Jürgen

2011-05-01

Prebiotic oligosaccharides modulate the intestinal microbiota and beneficially affect the human body by reducing intestinal inflammation. This immunomodulatory effect was assumed to be bacterial in origin. However, some observations suggest that oligosaccharides may exert an antiinflammatory effect per se. We hypothesized that oligosaccharides affect the intestinal immunity via activation of peptidoglycan recognition protein 3 (PGlyRP3), which reduces the expression of proinflammatory cytokines. Caco-2 cells were treated with the oligosaccharides, α3-sialyllactose, or fructooligosaccharides (Raftilose p95), and the effects of these treatments on PGlyRP3 and PPARγ expression, the release and expression of some proinflammatory cytokines, and NF-κB translocation were tested. Both oligosaccharides had antiinflammatory activity; they significantly reduced IL-12 secretion in Caco-2 cells and gene expression of IL-12p35, IL-8, and TNFα. They also reduced the gene expression and nuclear translocation of NF-κB. Both oligosaccharides dose and time dependently induced the production of PGlyRP3, the silencing of which by transfection of Caco-2 cells with specific small interfering RNA targeting PGlyRP3 abolished the antiinflammatory role of both oligosaccharides. Incubation of Caco-2 cells with both oligosaccharides induced PPARγ. Antagonizing PPARγ by culturing the cells with GW9662 for 24 h inhibited the oligosaccharide-induced PGlyRP3 production and the antiinflammatory effect of the oligosaccharides. We conclude that oligosaccharides may exert an antiinflammatory effect by inducing the nuclear receptor PPARγ, which regulates the antiinflammatory PGlyRP3.

Decursin and decursinol from Angelica gigas inhibit the lung metastasis of murine colon carcinoma.

PubMed

Son, Seung Hwa; Park, Kwang-Kyun; Park, Sun Kyu; Kim, Young Choong; Kim, Yeong Shik; Lee, Sang-Kook; Chung, Won-Yoon

2011-07-01

The principal objective of the present study was to evaluate the antimetastatic activity of decursin and decursinol isolated from Angelica gigas. Decursin and decursinol inhibited the proliferation and invasion of CT-26 colon carcinoma cells. The expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in cells and the activities in the culture medium were also reduced by decursin and decursinol treatment. In CT-26 cells, the extracellular signal-regulated kinase (ERK) inhibitor inhibited cell proliferation, invasion and MMP-9 expression, and the c-Jun N-terminal kinase (JNK) inhibitor suppressed the expression of both MMPs, as well as cell proliferation and cell invasion. The phosphatidylinositol-3 kinase (PI3K) inhibitor reduced only the expression of MMP-2. In addition, the invasion of CT-26 cells was inhibited by the treatment with anti-MMP-9 antibody, rather than anti-MMP-2 antibody. These results indicate that MMP-9 expression via ERK and JNK plays a critical role for the invasion of CT26 cells. Decursin and decursinol downregulated ERK and JNK phosphorylation. Moreover, oral administration of decursin and decursinol reduced the formation of tumor nodules in the lungs and the increase in lung weight caused by CT-26 metastases. Therefore, both decursin and decursinol may be beneficial antimetastatic agents, targeting MMPs and their upstream signaling molecules. Copyright © 2011 John Wiley & Sons, Ltd.

Epigenetic silencing of Na,K-ATPase β 1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma.

PubMed

Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

2014-04-01

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β 1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.

Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function.

PubMed

Lortz, S; Lenzen, S; Mehmeti, I

2015-08-01

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing. © 2015 Society for Endocrinology.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants[OPEN

PubMed Central

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Havaux, Michel; Schmülling, Thomas

2016-01-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. PMID:27354555

Effect of polysaccharide of dendrobium candidum on proliferation and apoptosis of human corneal epithelial cells in high glucose

PubMed Central

Li, Qiangxiang; Chen, Jing; Li, Yajia; Chen, Ting; Zou, Jing; Wang, Hua

2017-01-01

Abstract Background: The aim of the study was to observe the effect of polysaccharide of dendrobium candidum (PDC) and high glucose on proliferation, apoptosis of human corneal epithelial cells (HCEC). Methods: The MTT method was used to screen and take the optimal high-glucose concentration, treatment time, and PDC concentration using HCEC and divide it into 4 groups: control group (C), high glucose group (HG), PDC group, and HG + PDC group. We observed and compared the effect of the 4 groups on HCEC proliferation by MTT, apoptosis by Annexin V-FITC/PI double fluorescent staining and flow cytometry (FCM), and expression of bax mRNA and bcl-2 mRNA by RT-qPCR. Results: Compared with the control group, proliferative activity of HCEC cells was reduced; the cells apoptosis ratio was increased; the expression of bax mRNA was increased, and the expression of bcl-2 mRNA was reduced in the HG group. Proliferative activity of HCEC cells in the PDC group was increased, and the expression of bcl-2 mRNA was increased but that of bax mRNA was decreased. Proliferative activity of HCEC cells in the HG + PDC group was increased, but it could not restore to the normal level; the expression of bax mRNA was significantly decreased but the expression of bcl-2 mRNA was significantly increased. Conclusions: Our results demonstrate that high glucose can inhibit proliferative activity and induce apoptosis of HCEC. PDC can improve the proliferative activity of HCEC cells under the high glucose environment and reduce the apoptosis of cells by regulating the expression of bax and bcl-2. PDC play a very important role on protecting and repairing of corneal epithelial cells damage in high glucose. PMID:28796073

Rare TREM2 variants associated with Alzheimer's disease display reduced cell surface expression.

PubMed

Sirkis, Daniel W; Bonham, Luke W; Aparicio, Renan E; Geier, Ethan G; Ramos, Eliana Marisa; Wang, Qing; Karydas, Anna; Miller, Zachary A; Miller, Bruce L; Coppola, Giovanni; Yokoyama, Jennifer S

2016-09-02

Rare variation in TREM2 has been associated with greater risk for Alzheimer's disease (AD). TREM2 encodes a cell surface receptor expressed on microglia and related cells, and the R47H variant associated with AD appears to affect the ability of TREM2 to bind extracellular ligands. In addition, other rare TREM2 mutations causing early-onset neurodegeneration are thought to impair cell surface expression. Using a sequence kernel association (SKAT) analysis in two independent AD cohorts, we found significant enrichment of rare TREM2 variants not previously characterized at the protein level. Heterologous expression of the identified variants showed that novel variants S31F and R47C displayed significantly reduced cell surface expression. In addition, we identified rare variant R136Q in a patient with language-predominant AD that also showed impaired surface expression. The results suggest rare TREM2 variants enriched in AD may be associated with altered TREM2 function and that AD risk may be conferred, in part, from altered TREM2 surface expression.

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

PubMed Central

Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

2015-01-01

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

PubMed Central

Ju, Xiao-Ning; Mu, Wei-Na; Liu, Yuan-Tao; Wang, Mei-Hong; Kong, Feng; Sun, Chao; Zhou, Qing-Bo

2015-01-01

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. PMID:26823714

Inhibition of cell surface expression of endothelial adhesion molecules by ursolic acid prevents intimal hyperplasia of venous bypass grafts in rats

PubMed Central

Zeller, Iris; Wiedemann, Dominik; Schwaiger, Stefan; Stelzmüller, Marlies; Kreutmayer, Simone; Leberfing, Oliver; Stuppner, Hermann; Bernhard, David

2012-01-01

OBJECTIVES Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts. METHODS The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry. RESULTS The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 μm, UA group: 18 μM–8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NFκB and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis. CONCLUSIONS Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts. PMID:22551965

The apoptotic effect of simvastatin via the upregulation of BIM in nonsmall cell lung cancer cells.

PubMed

Lee, Hwa Young; Kim, In Kyoung; Lee, Hye In; Mo, Jin Young; Yeo, Chang Dong; Kang, Hyeon Hui; Moon, Hwa Sik; Lee, Sang Haak

2016-01-01

Statins are known to have pleiotropic effects that induce cell death in certain cancer cells. BIM is a member of the bcl-2 gene family, which promotes apoptotic cell death. This study investigated the hypothesis that simvastatin has pro-apoptotic effects in epidermal growth factor receptor (EGFR)-mutated lung cancer cell lines via the upregulation of the expression of the BIM protein. The cytotoxic effects of simvastatin on gefitinib-sensitive (HCC827, E716-A750del) and -resistant (H1975, T790M + L858R) nonsmall cell lung cancer (NSCLC) cells were compared. Cell proliferation and expression of apoptosis-related and EGFR downstream signaling proteins were evaluated. Expression of BIM was compared in H1975 cells after treatment with simvastatin or gefitinib. SiRNA-mediated BIM depletion was performed to confirm whether the cytotoxicity of simvastatin was mediated by the expression of BIM. H1975 cells showed significantly reduced viability compared with HCC827 cells after treatment with simvastatin (2 μM) for 48 hours. In simvastatin-treated H1975 cells, expression of pro-apoptotic proteins was increased and the phosphorylation of ERK 1/2 (p-ERK 1/2) was reduced. Expression of BIM was suppressed by gefitinib (1 μM) treatment in H1975 cells, but it was significantly increased by treatment with simvastatin. BIM depletion by siRNA transfection enhanced the viability of H1975 cells that received simvastatin treatment and increased their expression of anti-apoptotic proteins. Simvastatin restored the expression of BIM to induce apoptotic cell death in NSCLC cells harboring an EGFR-resistant mutation. Our study suggests the potential utility of simvastatin as a BIM-targeted treatment for NSCLC.

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

PubMed Central

Bohlken, A; Cheung, B B; Bell, J L; Koach, J; Smith, S; Sekyere, E; Thomas, W; Norris, M; Haber, M; Lovejoy, D B; Richardson, D R; Marshall, G M

2009-01-01

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism. PMID:19127267

VISA is Required for B Cell Expression of TLR7

PubMed Central

Xu, Liang-Guo; Jin, Lei; Zhang, Bi-Cheng; Akerlund, Janie L.; Shu, Hong-Bing; Cambier, John C.

2011-01-01

B cells play a critical role in the initialization and development of the Systemic Lupus Erythematosus (SLE) that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the Type I IFN secreted by Plasmacytoid Dendritic Cells (PDC). Here we report that VISA, also known as MAVS, IPS-1 and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from VISA−/− mouse express reduced TLR7, but normal basal levels of Type I IFN. We also show that while IFNβ and TLR7 agonists synergize to promote TLR7 expression in VISA−/− B cells, they do not fully complement the defect seen in VISA−/− cells. Cell transfer experiments revealed that the observed effects of VISA−/− are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced up-regulation of activation markers CD69 and CD86, cell proliferation, production of IFNα, TNF, IL-12 and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA−/− mice, since VISA−/− B cells differ in CD23 and TLR7 expression when on C57BL/6 vs 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity. PMID:22105994

VISA is required for B cell expression of TLR7.

PubMed

Xu, Liang-Guo; Jin, Lei; Zhang, Bi-Cheng; Akerlund, Linda J; Shu, Hong-Bing; Cambier, John C

2012-01-01

B cells play a critical role in the initialization and development of the systemic lupus erythematosus that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the type I IFN secreted by plasmacytoid dendritic cells. In this article, we report that VISA, also known as MAVS, IPS-1, and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from a VISA(-/-) mouse express reduced TLR7 but normal basal levels of type I IFN. We also show that although IFN-β and TLR7 agonists synergize to promote TLR7 expression in VISA(-/-) B cells, they do not fully complement the defect seen in VISA(-/-) cells. Cell transfer experiments revealed that the observed effects of VISA(-/-) are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced upregulation of activation markers CD69 and CD86, cell proliferation, production of IFN-α, TNF, and IL-12, and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA(-/-) mice, because VISA(-/-) B cells differ in CD23 and TLR7 expression when on C57BL/6 versus 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity.

Magnolol reduced TNF-α-induced vascular cell adhesion molecule-1 expression in endothelial cells via JNK/p38 and NF-κB signaling pathways.

PubMed

Liang, Chan-Jung; Lee, Chiang-Wen; Sung, Hsin-Ching; Chen, Yung-Hsiang; Wang, Shu-Huei; Wu, Pei-Jhen; Chiang, Yao-Chang; Tsai, Jaw-Shiun; Wu, Chau-Chung; Li, Chi-Yuan; Chen, Yuh-Lien

2014-01-01

Expression of cell adhesion molecules by the endothelium and the attachment of leukocytes to these cells play major roles in inflammation and cardiovascular disorders. Magnolol, a major active component of Magnolia officinalis, has antioxidative and anti-inflammatory properties. In the present study, the effects of magnolol on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAECs) and the related mechanisms were investigated. TNF-α induced VCAM-1 protein expression and mRNA stability were significantly decreased in HAECs pre-treated with magnolol. Magnolol significantly reduced the phosphorylation of ERK, JNK, and p38 in TNF-α-treated HAECs. The decrease in VCAM-1 expression in response to TNF-α treatment was affected by JNK and p38 inhibitors, not by an ERK inhibitor. Magnolol also attenuates NF-κB activation and the translocation of HuR (an RNA binding protein) in TNF-α-stimulated HAECs. The VCAM-1 expression was weaker in the aortas of TNF-α-treated apo-E deficient mice with magnolol treatment. These data demonstrate that magnolol inhibits TNF-α-induced JNK/p38 phosphorylation, HuR translocation, NF-κB activation, and thereby suppresses VCAM-1 expression resulting in reduced leukocyte adhesion. Taken together, these results suggest that magnolol has an anti-inflammatory property and may play an important role in the prevention of atherosclerosis and inflammatory responses.

hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis

PubMed Central

Park, Sun-Ji; Kim, Tae-Shin; Park, Choon-Keun; Lee, Sang-Hee; Kim, Jin-Man; Lee, Kyu-Sun; Lee, In-kyu; Park, Jeen-Woo; Lawson, Mark A; Lee, Dong-Seok

2014-01-01

Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. PMID:23256993

Osteoprotegerin expression in triple-negative breast cancer cells promotes metastasis.

PubMed

Weichhaus, Michael; Segaran, Prabu; Renaud, Ashleigh; Geerts, Dirk; Connelly, Linda

2014-10-01

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells.

PubMed

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio; Párraga, Mario; Villena, Joan

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions . These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells

PubMed Central

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells. PMID:29552079

Eomesodermin(lo) CTLA4(hi) Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4 Immunoglobulin-Treated Nonhuman Primates.

PubMed

Ezzelarab, Mohamed B; Lu, Lien; Guo, Hao; Zahorchak, Alan F; Shufesky, William F; Cooper, David K C; Morelli, Adrian E; Thomson, Angus W

2016-01-01

Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in nonhuman primates. We evaluated Eomes and coinhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin, both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+ T cells. By contrast, CD8+ T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes(lo)CTLA4(hi) cells displayed higher levels of CD25 and Foxp3 than Eomes(hi)CTLA4(lo) CD8+ T cells. After allostimulation, distinct proliferating Eomes(lo)CTLA4(hi) and Eomes(hi)CTLA4(lo) CD8+ T cell populations were identified, with a high proportion of Tcm being Eomes(lo)CTLA4(hi). CB with CTLA4Ig during allostimulation of CD8+ T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes(lo)CTLA4(hi) cells. After transplantation with CB and rapamycin, donor-reactive Eomes(lo)CTLA4(hi) CD8+ T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes(lo)CTLA4(hi) Tcm.

Eomesoderminlo CTLA4hi Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4Ig-Treated Non-Human Primates

PubMed Central

Ezzelarab, Mohamed B.; Lu, Lien; Guo, Hao; Zahorchak, Alan F.; Shufesky, William F.; Cooper, David K.C.; Morelli, Adrian E.; Thomson, Angus W.

2015-01-01

Background Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in non-human primates. Methods We evaluated Eomes and co-inhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin (Ig), both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. Results In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+T cells. By contrast, CD8+T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, EomesloCTLA4hi cells displayed higher levels of CD25 and Foxp3 than EomeshiCTLA4lo CD8+ T cells. Following allostimulation, distinct proliferating EomesloCTLA4hi and EomeshiCTLA4lo CD8+ T cell populations were identified, with a high proportion of Tcm being EomesloCTLA4hi. CB with CTLA4Ig during allostimulation of CD8+T cells reduced CTLA4 but not Eomes expression, significantly reducing EomesloCTLA4hi cells. After transplantation with CB and rapamycin, donor-reactive EomesloCTLA4hi CD8+T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Conclusions Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive EomesloCTLA4hi Tcm. PMID:26680373

Wnt signaling regulates pancreatic β cell proliferation

PubMed Central

Rulifson, Ingrid C.; Karnik, Satyajit K.; Heiser, Patrick W.; ten Berge, Derk; Chen, Hainan; Gu, Xueying; Taketo, Makoto M.; Nusse, Roel; Hebrok, Matthias; Kim, Seung K.

2007-01-01

There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet β cell proliferation. The addition of purified Wnt3a protein to cultured β cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of β cell cycle progression, and led to increased β cell proliferation in vitro. Conditional pancreatic β cell expression of activated β-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to β cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional β cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by β cells, resulting in reduced neonatal β cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet β cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function. PMID:17404238

Cyclooxygenase 2 Promotes Parathyroid Hyperplasia in ESRD

PubMed Central

Zhang, Qian; Qiu, Junsi; Li, Haiming; Lu, Yanwen; Wang, Xiaoyun; Yang, Junwei; Wang, Shaoqing; Zhang, Liyin; Gu, Yong; Hao, Chuan-Ming

2011-01-01

Hyperplasia of the PTG underlies the secondary hyperparathyroidism (SHPT) observed in CKD, but the mechanism underlying this hyperplasia is incompletely understood. Because aberrant cyclooxygenase 2 (COX2) expression promotes epithelial cell proliferation, we examined the effects of COX2 on the parathyroid gland in uremia. In patients with ESRD who underwent parathyroidectomy, clusters of cells within the parathyroid glands had increased COX2 expression. Some COX2-positive cells exhibited two nuclei, consistent with proliferation. Furthermore, nearly 78% of COX2-positive cells expressed proliferating cell nuclear antigen (PCNA). In the 5/6-nephrectomy rat model, rats fed a high-phosphate diet had significantly higher serum PTH levels and larger parathyroid glands than sham-operated rats. Compared with controls, the parathyroid glands of uremic rats exhibited more PCNA-positive cells and greater COX2 expression in the chief cells. Treatment with COX2 inhibitor celecoxib significantly reduced PCNA expression, attenuated serum PTH levels, and reduced the size of the glands. In conclusion, COX2 promotes the pathogenesis of hyperparathyroidism in ESRD, suggesting that inhibiting the COX2 pathway could be a potential therapeutic target. PMID:21335517

Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

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

Hong, Gia-Ming; Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637; Bain, Lisa J., E-mail: lbain@clemson.edu

2012-05-01

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site ofmore » myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2 and Dnmt3a localization to the myogenin promoter is induced by arsenic.« less

Inhibition of Tumorigenesis by the Thyroid Hormone Receptor β in Xenograft Models

PubMed Central

Kim, Won Gu; Zhao, Li; Kim, Dong Wook; Willingham, Mark C.

2014-01-01

Background: Previous studies showed a close association between several types of human cancers and somatic mutations of thyroid hormone receptor β (TRβ) and reduced expression of TRβ due to epigenetic inactivation and/or deletion of the THRB gene. These observations suggest that TRβ could act as a tumor suppressor in carcinogenesis. However, the mechanisms by which TRβ could function to inhibit tumorigenesis are less well understood. Methods: We used the human follicular thyroid cancer cell lines (FTC-133 and FTC-236 cells) to elucidate how functional expression of the THRB gene could affect tumorigenesis. We stably expressed the THRB gene in FTC cells and evaluated the effects of the expressed TRβ on cancer cell proliferation, migration, and tumor growth in cell-based studies and xenograft models. Results: Expression of TRβ in FTC-133 cells, as compared with control FTC cells without TRβ, reduced cancer cell proliferation and impeded migration of tumor cells through inhibition of the AKT-mTOR-p70 S6K pathway. TRβ expression in FTC-133 and FTC-236 led to less tumor growth in xenograft models. Importantly, new vessel formation was significantly suppressed in tumors induced by FTC cells expressing TRβ compared with control FTC cells without TRβ. The decrease in vessel formation was mediated by the downregulation of vascular endothelial growth factor in FTC cells expressing TRβ. Conclusions: These findings indicate that TRβ acts as a tumor suppressor through downregulation of the AKT-mTOR-p70 S6K pathway and decreased vascular endothelial growth factor expression in FTC cells. The present results raise the possibility that TRβ could be considered as a potential therapeutic target for thyroid cancer. PMID:23731250

Reduced B Lymphoid Kinase (Blk) Expression Enhances Proinflammatory Cytokine Production and Induces Nephrosis in C57BL/6-lpr/lpr Mice

PubMed Central

Papillion, Amber M.; Tatum, Arthur H.; Princiotta, Michael F.; Hayes, Sandra M.

2014-01-01

BLK, which encodes B lymphoid kinase, was recently identified in genome wide association studies as a susceptibility gene for systemic lupus erythematosus (SLE), and risk alleles mapping to the BLK locus result in reduced gene expression. To determine whether BLK is indeed a bona fide susceptibility gene, we developed an experimental mouse model, namely the Blk+/−.lpr/lpr (Blk+/−.lpr) mouse, in which Blk expression levels are reduced to levels comparable to those in individuals carrying a risk allele. Here, we report that Blk is expressed not only in B cells, but also in IL-17-producing γδ and DN αβ T cells and in plasmacytoid dendritic cells (pDCs). Moreover, we found that solely reducing Blk expression in C57BL/6-lpr/lpr mice enhanced proinflammatory cytokine production and accelerated the onset of lymphoproliferation, proteinuria, and kidney disease. Together, these findings suggest that BLK risk alleles confer susceptibility to SLE through the dysregulation of a proinflammatory cytokine network. PMID:24637841

γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation.

PubMed

Sakai, Satoshi; Murata, Takahisa; Tsubosaka, Yoshiki; Ushio, Hideki; Hori, Masatoshi; Ozaki, Hiroshi

2012-04-04

γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

Suppressor of cytokine signaling 1 modulates invasion and metastatic potential of colorectal cancer cells.

PubMed

David, Muriel; Naudin, Cécile; Letourneur, Martine; Polrot, Mélanie; Renoir, Jack-Michel; Lazar, Vladimir; Dessen, Philippe; Roche, Serge; Bertoglio, Jacques; Pierre, Josiane

2014-07-01

Suppressor of cytokine signaling (SOCS) 1 is an inducible negative regulator of cytokine signaling but its role in human cancer is not completely established. Here we report that, while SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas, its level decreases during progression of colon adenocarcinomas, the lowest level being found in the most aggressive stage and least differentiated carcinomas. Forced expression of SOCS1 in metastatic colorectal SW620 cells reverses many characteristics of Epithelial-Mesenchymal Transition (EMT), as highlighted by the disappearance of the transcription factor ZEB1 and the mesenchymal form of p120ctn and the re-expression of E-cadherin. Furthermore, miRNA profiling indicated that SOCS1 also up-regulates the expression of the mir-200 family of miRNAs, which can promote the mesenchymal-epithelial transition and reduce tumor cell migration. Accordingly, overexpression of SOCS1 induced cell morphology changes and dramatically reduced tumor cell invasion in vitro. When injected in nude mice, SOCS1-expressing SW620 cells induced metastases in a smaller number of animals than parental SW620 cells, and did not generate any adrenal gland or bone metastasis. Overall, our results suggest that SOCS1 controls metastatic progression of colorectal tumors by preventing the mesenchymal-epithelial transition (MET), including E-cadherin expression. This pathway may be associated with survival to colorectal cancer by reducing the capacity of generating metastases. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Bmi-1 helix-turn and ring finger domains are required for Bmi-1 antagonism of (-) epigallocatechin-3-gallate suppression of skin cancer cell survival.

PubMed

Balasubramanian, Sivaprakasam; Scharadin, Tiffany M; Han, Bingshe; Xu, Wen; Eckert, Richard L

2015-07-01

The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (-) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21(Cip1) and p27(Kip1) expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix-turn-helix-turn-helix-turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents. Copyright © 2015 Elsevier Inc. All rights reserved.

[Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration.

PubMed

Booth, Laurence; Roberts, Jane L; Rais, Rumeesa; Kirkwood, John; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Poklepovic, Andrew; Dent, Paul

2018-01-19

The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo , prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.

[Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration

PubMed Central

Booth, Laurence; Roberts, Jane L.; Rais, Rumeesa; Kirkwood, John; Avogadri-Connors, Francesca; Cutler, Richard E.; Lalani, Alshad S.; Poklepovic, Andrew; Dent, Paul

2018-01-01

The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo, prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies. PMID:29464055

Ruxolitinib synergizes with DMF to kill via BIM+BAD-induced mitochondrial dysfunction and via reduced SOD2/TRX expression and ROS.

PubMed

Tavallai, Mehrad; Booth, Laurence; Roberts, Jane L; McGuire, William P; Poklepovic, Andrew; Dent, Paul

2016-04-05

We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition*

PubMed Central

Rico-Leo, Eva M.; Alvarez-Barrientos, Alberto; Fernandez-Salguero, Pedro M.

2013-01-01

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR+/+ and AhR−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells. PMID:23382382

Elimination of Kalrn Expression in POMC Cells Reduces Anxiety-Like Behavior and Contextual Fear Learning

PubMed Central

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A.; Mains, Richard E.

2014-01-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. PMID:25014196

SOX12: a novel potential target for acute myeloid leukaemia.

PubMed

Wan, Haixia; Cai, Jiayi; Chen, Fangyuan; Zhu, Jianyi; Zhong, Jihua; Zhong, Hua

2017-02-01

The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34 + cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-12 weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML. © 2016 John Wiley & Sons Ltd.

TRPV2 channel negatively controls glioma cell proliferation and resistance to Fas-induced apoptosis in ERK-dependent manner.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Farfariello, Valerio; Ricci-Vitiani, Lucia; Caprodossi, Sara; Arcella, Antonella; Santoni, Matteo; Giangaspero, Felice; De Maria, Ruggero; Santoni, Giorgio

2010-05-01

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 2 (TRPV2) in human glioma cells. By Real-Time-PCR and western blot analysis, we found that TRPV2 messenger RNA (mRNA) and protein were expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased (n = 49 cases), and in U87MG cells and in MZC, FCL and FSL primary glioma cells. To investigate the function of TRPV2 in glioma, small RNA interfering was used to silence TRPV2 expression in U87MG cells. As evaluated by RT-Profiler PCR array, siTRPV2-U87MG transfected cells displayed a marked downregulation of Fas and procaspase-8 mRNA expression, associated with upregulation of cyclin E1, cyclin-dependent kinase 2, E2F1 transcriptor factor 1, V-raf-1 murine leukemia viral oncogene homolog 1 and Bcl-2-associated X protein (Bcl-X(L)) mRNA expression. TRPV2 silencing increased U87MG cell proliferation as shown by the increased percentage of cells incorporating 5-bromo-2-deoxyuridine expressing beta(III)-tubulin and rescued glioma cells to Fas-induced apoptosis. These events were dependent on extracellular signal-regulated kinase (ERK) activation: indeed inhibition of ERK activation in siTRPV2-U87MG transfected cells by treatment with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, reduced Bcl-X(L) protein levels, promoted Fas expression, and restored Akt/protein kinase B pathway activation leading to reduced U87MG cell survival and proliferation, and increased sensitivity to Fas-induced apoptosis. In addition, transfection of TRPV2 in MZC glioma cells, by inducing Fas overexpression, resulted in a reduced viability and an increased spontaneous and Fas-induced apoptosis. Overall, our findings indicate that TRPV2 negatively controls glioma cell survival and proliferation, as well as resistance to Fas-induced apoptotic cell death in an ERK-dependent manner.

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

PubMed

Juárez, Mariana; González-De la Rosa, Claudia H; Memún, Elisa; Sigala, Juan-Carlos; Lara, Alvaro R

2017-03-01

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hormonal Regulation of Dendritic Cell Differentiation in the Thymus.

PubMed

Shirshev, S V; Orlova, E G; Loginova, O A; Nekrasova, I V; Gorbunova, O L; Maslennikova, I L

2018-06-19

We studied the effect of hormones estriol, ghrelin, kisspeptin, and chorionic gonadotropin in concentrations corresponding to their content in the peripheral blood in each trimester of pregnancy on the expression of membrane molecules on myeloid and plasmacytoid dendritic cells of the thymus. It was found that thymic myeloid dendritic cells are sensitive to the action of estriol and kisspeptin. Estriol in a concentration of the first trimester of pregnancy reduces the number of myeloid dendritic cells expressing receptor for thymic stromal lymphopoietin (CD11c+TSLP-R + ) and inhibitory molecule B7-H3 (CD11c + CD276 + ). In contrast to estriol, kisspeptin regulates the processes of differentiation of thymic myeloid dendritic cells in concentrations typical of the second-third trimesters and reduced their total number (CD11c + ) and the number of cells expressing TSLP-R (CD11c + TSLP-R + ). Estriol and kisspeptin do not affect the total number of plasmacytoid dendritic cells (CD303 + ) and expression of TSLP-R and CD276 by these cells. Ghrelin and chorionic gonadotropin in the studied concentrations had no significant effect on the total number of thymic myeloid and plasmacytoid dendritic cells and on the expression of membrane molecules of TSLP-R and CD276.

[Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension].

PubMed

Rylova, Iu V; Buravkova, L B

2013-01-01

We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.

Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis

PubMed Central

Chen, Liye; Ridley, Anna; Hammitzsch, Ariane; Al-Mossawi, Mohammad Hussein; Bunting, Helen; Georgiadis, Dimitris; Chan, Antoni; Kollnberger, Simon; Bowness, Paul

2016-01-01

Objective Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. Results The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. Conclusions ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment. PMID:26130142

α-Solanine inhibits invasion of human prostate cancer cell by suppressing epithelial-mesenchymal transition and MMPs expression.

PubMed

Shen, Kun-Hung; Liao, Alex Chien-Hwa; Hung, Jui-Hsiang; Lee, Wei-Jiunn; Hu, Kai-Chieh; Lin, Pin-Tsen; Liao, Ruei-Fang; Chen, Pin-Shern

2014-08-11

α-Solanine, a naturally occurring steroidal glycoalkaloid found in nightshade (Solanum nigrum Linn.), was found to inhibit proliferation and induce apoptosis of tumor cells. However, the mechanism involved in suppression of cancer cell metastasis by α-solanine remains unclear. This study investigates the suppression mechanism of α-solanine on motility of the human prostate cancer cell PC-3. Results show that α-solanine reduces the viability of PC-3 cells. When treated with non-toxic doses of α-solanine, cell invasion is markedly suppressed by α-solanine. α-Solanine also significantly elevates epithelial marker E-cadherin expression, while it concomitantly decreases mesenchymal marker vimentin expression, suggesting it suppresses epithelial-mesenchymal transition (EMT). α-Solanine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9 and extracellular inducer of matrix metalloproteinase (EMMPRIN), but increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK), and tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Immunoblotting assays indicate α-solanine is effective in suppressing the phosphorylation of phosphatidylinositide-3 kinase (PI3K), Akt and ERK. Moreover, α-solanine downregulates oncogenic microRNA-21 (miR-21) and upregulates tumor suppressor miR-138 expression. Taken together, the results suggest that inhibition of PC-3 cell invasion by α-solanine may be, at least in part, through blocking EMT and MMPs expression. α-Solanine also reduces ERK and PI3K/Akt signaling pathways and regulates expression of miR-21 and miR-138. These findings suggest an attractive therapeutic potential of α-solanine for suppressing invasion of prostate cancer cell.

Spatial Control of Cell Transfection Using Soluble or Solid-Phase Redox Agents and a Redox-Active Ferrocenyl Lipid

PubMed Central

Aytar, Burcu S.; Muller, John P. E.; Kondo, Yukishige; Abbott, Nicholas L.; Lynn, David M.

2013-01-01

We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes. PMID:23965341

Spatial control of cell transfection using soluble or solid-phase redox agents and a redox-active ferrocenyl lipid.

PubMed

Aytar, Burcu S; Muller, John P E; Kondo, Yukishige; Abbott, Nicholas L; Lynn, David M

2013-09-11

We report principles for active, user-defined control over the locations and timing with which DNA is expressed in cells. Our approach exploits unique properties of a ferrocenyl cationic lipid that is inactive when oxidized, but active when chemically reduced. We show that methods that exert spatial control over the administration of reducing agents can lead to local activation of lipoplexes and spatial control over gene expression. The versatility of this approach is demonstrated using both soluble and solid-phase reducing agents. These methods provide control over cell transfection, including methods for remote activation and the patterning of expression using solid-phase redox agents, that are difficult to achieve using conventional lipoplexes.

Hypoxia reduces the E-cadherin expression and increases OSCC cell migration regardless of the E-cadherin methylation profile.

PubMed

Domingos, Patrícia Luciana Batista; Souza, Marcela Gonçalves; Guimarães, Talita Antunes; Santos, Eliane Sobrinho; Farias, Lucyana Conceição; de Carvalho Fraga, Carlos Alberto; Jones, Kimberly Marie; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena

2017-05-01

The purpose of the current study is to investigate the association between E-cadherin methylation status, hypoxia and OSCC. HaCat and SCC9 cell lines were submitted to hypoxic treatment, followed by methylation profile analysis (MS-PCR) and analysis of the expression of mRNA gene E-cadherin (RT-PCR). Study group samples comprise individuals affected by potentially malignant lesions Potential Malignant Oral Lesion (PMOL, n=18) and oral squamous cell carcinoma (OSCC, n=28). The control group oral mucosa (OM, n=15) of patients with an oral mucocele. Cell migration ability was evaluated a scratch wound assay in SCC9 and HaCat cell lines RESULTS: E-cadherin mRNA expression in the cell lines SCC9 and HaCat was significantly reduced under hypoxia, regardless of the methylation profile, when compared to the control group. No differences in methylation profile of the E-cadherin were observed among the groups OM, PMOL and OSCC. HaCat and SCC9 presented increases in cell migration rates under hypoxia. The current study demonstrates that hypoxia reduces E-cadherin expression and increase cell migration, regardless of the methylation profile. Additionally, no differences in E-cadherin methylation patterns were observed among OM, PMOL and OSCC. Copyright © 2017 Elsevier GmbH. All rights reserved.

Targeting filamin A reduces K-RAS–induced lung adenocarcinomas and endothelial response to tumor growth in mice

PubMed Central

2012-01-01

Background Many human cancer cells express filamin A (FLNA), an actin-binding structural protein that interacts with a diverse set of cell signaling proteins, but little is known about the biological importance of FLNA in tumor development. FLNA is also expressed in endothelial cells, which may be important for tumor angiogenesis. In this study, we defined the impact of targeting Flna in cancer and endothelial cells on the development of tumors in vivo and on the proliferation of fibroblasts in vitro. Methods First, we used a Cre-adenovirus to simultaneously activate the expression of oncogenic K-RAS and inactivate the expression of Flna in the lung and in fibroblasts. Second, we subcutaneously injected mouse fibrosarcoma cells into mice lacking Flna in endothelial cells. Results Knockout of Flna significantly reduced K-RAS–induced lung tumor formation and the proliferation of oncogenic K-RAS–expressing fibroblasts, and attenuated the activation of the downstream signaling molecules ERK and AKT. Genetic deletion of endothelial FLNA in mice did not impact cardiovascular development; however, knockout of Flna in endothelial cells reduced subcutaneous fibrosarcoma growth and vascularity within tumors. Conclusions We conclude that FLNA is important for lung tumor growth and that endothelial Flna impacts local tumor growth. The data shed new light on the biological importance of FLNA and suggest that targeting this protein might be useful in cancer therapeutics. PMID:22857000

Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

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

Sun, Lihua; Wang, Wensheng; Xiao, Weidong

2012-08-10

Highlights: Black-Right-Pointing-Pointer Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. Black-Right-Pointing-Pointer The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. Black-Right-Pointing-Pointer GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. Black-Right-Pointing-Pointer GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. Inmore » the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.« less

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition.

PubMed

Afgar, Ali; Fard-Esfahani, Pezhman; Mehrtash, Amirhosein; Azadmanesh, Kayhan; Khodarahmi, Farnaz; Ghadir, Mahdis; Teimoori-Toolabi, Ladan

2016-11-01

It is observed that upregulation of DNMT3B enzyme in some cancers, including colon cancer, could lead to silencing of tumor suppressor genes. MiR-339 and miR-766 have been predicted to target 3'UTR of DNMT3B gene. Luciferase reporter assay validated that individual and co-transfection of miR-766 and miR-339 into the HEK293T cell reduced luciferase activity to 26% ± 0.41%, 43% ± 0.42 and 64% ± 0.52%, respectively, compared to the control (P < 0.05). Furthermore, transduction of miR-339 and miR-766 expressing viruses into colon cancer cell lines (SW480 and HCT116) decreased DNMT3B expression (1.5, 3-fold) and (3, 4-fold), respectively. In addition, DNA methylation of some tumor suppressor genes decreased. Expression of these genes such as SFRP1 (2 and 1.6-fold), SFRP2 (0.07 and 4-fold), WIF1 (0.05 and 4-fold), and DKK2 (2 and 4-fold) increased in SW-339 and SW-766 cell lines; besides, expression increments for these genes in HCT-339 and HCT-766 cell lines were (2.8, 4-fold), (0.005, 1.5-fold), (1.7 and 3-fold) and (0.04, 1.7-fold), respectively. Also, while in SW-766, cell proliferation reduced to 2.8% and 21.7% after 24 and 48 hours, respectively, SW-339 showed no reduced proliferation. Meanwhile, HCT-766 and HCT-339 showed (3.5%, 12.8%) and (18.8%, 33.9%) reduced proliferation after 24 and 48 hours, respectively. Finally, targeting DNMT3B by these miRs, decreased methylation of tumor suppressor genes such as SFRP1, SFRP2, WIF1 and DKK2 in the mentioned cell lines, and returned the expression of these tumor suppressor genes which can contribute to lethal effect on colon cancer cells and reducing tumorigenicity of these cells.

Upregulation of GRAIL is associated with impaired CD4 T cell proliferation in sepsis.

PubMed

Aziz, Monowar; Yang, Weng-Lang; Matsuo, Shingo; Sharma, Archna; Zhou, Mian; Wang, Ping

2014-03-01

The loss of numbers and functionality of CD4 T cells is observed in sepsis; however, the mechanism remains elusive. Gene related to anergy in lymphocytes (GRAIL) is critical for the impairment of CD4 T cell proliferation. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during sepsis. Sepsis was induced in 10-wk-old male C57BL/6 mice by cecal ligation and puncture. Splenocytes were isolated and subjected to flow cytometry to determine CD4 T cell contents. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenocytes was measured by immunohistochemistry, real-time PCR, and flow cytometry. The expressions of IL-2 and early growth response-2 were determined by real-time PCR. As compared with shams, the numbers of CD4 T cells were significantly reduced in spleens. Septic CD4 T cells were less efficient in proliferation than shams. The IL-2 expression was significantly reduced, whereas the GRAIL expression was significantly increased in septic mice splenocytes as compared with shams. The small interfering RNA-mediated knockdown of GRAIL expression re-established the CD4 T cell proliferation ability ex vivo. Similarly, the treatment with recombinant murine IL-2 to the septic CD4 T cells restored their proliferation ability by downregulating GRAIL expression. Our findings reveal a novel association of the increased GRAIL expression with impaired CD4 T cell proliferation, implicating an emerging therapeutic tool in sepsis.

Synergistic antitumor effect of combining metronomic chemotherapy with adoptive cell immunotherapy in nude mice.

PubMed

Shi, Shujing; Tao, Leilei; Song, Haizhu; Chen, Longbang; Huang, Guichun

2014-05-01

Adoptive cell immunotherapy with cytokine-induced killer cell (CIK cell) represents a promising non-toxic anticancer therapy. However, the clinical efficacy of CIK cells is limited because of abnormal tumor vasculature. Metronomic chemotherapy shows promising anticancer activity by its potential antiangiogenic effect and reduced toxicity. We hypothesized that metronomic chemotherapy with paclitaxel could improve the antitumor effect of adoptive CIK cell immunotherapy. Mice health status was analyzed by measuring mice weight and observing mice behavior. Immunohistochemistry was used to investigate the recruitment of CIK cells, the expression of endothelial cell molecules, as well as the hypoxic tumor area. Metronomic paclitaxel synergized with adoptive CIK cell immunotherapy to inhibit the growth of non-small cell lung cancer (NSCLC). Metronomic paclitaxel reduced hypoxic tumor area and increased CIK cell infiltration. Hypoxia impeded the adhesion of CIK cells and reduced the expression of endothelial cell adhesion molecules. In vivo studies demonstrated that more CIK cells were found in endothelial cell adhesion molecules high expressed area. Our study provides a new rationale for combining metronomic chemotherapy with adoptive cell immunotherapy in the treatment of xenograft NSCLC tumors in immunodeficient mice. Further clinical trials integrating translational research are necessary to better evaluate the clinical benefit of this promising approach. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

PubMed

Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-03-01

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. © 2016 Federation of European Biochemical Societies.

The inhibitor of kappa B kinase-epsilon regulates MMP-3 expression levels and can promote lung metastasis.

PubMed

Seccareccia, E; Pinard, M; Wang, N; Li, S; Burnier, J; Dankort, D; Brodt, P

2014-08-18

The factors that determine the ability of metastatic tumor cells to expand and grow in specific secondary site(s) are not yet fully understood. Matrix metalloproteinases (MMP) were identified as potential regulators of the site-specificity of metastasis. We found that lung carcinoma cells ectopically expressing high levels of the receptor for the type I insulin like growth factor receptor (M27(R) cells) had a significant reduction in MMP-3 expression levels and this coincided with reduced metastasis to the lung. We used these cells to further investigate signaling pathways regulating MMP-3 expression and the role that MMP-3 plays in lung metastasis. We show that ectopic IκB kinase ɛ (IKKɛ) expression in these cells partly restored MMP-3 expression levels and also sensitized MMP-3 transcription to induction by phorbol 12-myristate 13-acetate (PMA). This increase in MMP-3 production was due to increased activation of several signal transduction mediators, including protein kinase C alpha, ERK2, Akt and the transcription factor p65. Furthermore, reconstitution of MMP-3 expression in M27(R) cells restored their ability to colonize the lung whereas silencing of MMP-3 in M27 cells reduced metastases. Collectively, our results implicate IKKɛ as a central regulator of PMA-induced cell signaling and MMP-3 expression and identify MMP-3 as an enabler of tumor cell expansion in the lung.Oncogenesis (2014) 3, e116; doi:10.1038/oncsis.2014.28; published online 18 August 2014.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

PubMed

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

2018-01-01

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

PubMed Central

Kurek, Kyle; Del Mare, Sara; Salah, Zaidoun; Abdeen, Suhaib; Sadiq, Hussain; Lee, Sukhee; Gaudio, Eugenio; Zanesi, Nicola; Jones, Kevin B.; DeYoung, Barry; Amir, Gail; Gebhardt, Mark; Warman, Matthew; Stein, Gary S.; Stein, Janet L.; Lian, Jane B.; Aqeilan, Rami I.

2011-01-01

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P< 0.0001). Compared to the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels, relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorgenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas Runx2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease. PMID:20530675

EMMPRIN regulates tumor growth and metastasis by recruiting bone marrow-derived cells through paracrine signaling of SDF-1 and VEGF.

PubMed

Chen, Yanke; Gou, Xingchun; Kong, Derek Kai; Wang, Xiaofei; Wang, Jianhui; Chen, Zeming; Huang, Chen; Zhou, Jiangbing

2015-10-20

EMMPRIN, a cell adhesion molecule highly expressed in a variety of tumors, is associated with poor prognosis in cancer patients. Mechanistically, EMMPRIN has been characterized to contribute to tumor development and progression by controlling the expression of MMPs and VEGF. In the present study, by using fluorescently labeled bone marrow-derived cells (BMDCs), we found that the down-regulation of EMMPRIN expression in cancer cells reduces tumor growth and metastasis, and is associated with the reduced recruitment of BMDCs. Further protein profiling studies suggest that EMMPRIN controls BMDC recruitment through regulating the secretion of soluble factors, notably, VEGF and SDF-1. We demonstrate that the expression and secretion of SDF-1 in tumor cells are regulated by EMMPRIN. This study reveals a novel mechanism by which EMMPRIN promotes tumor growth and metastasis by recruitment of BMDCs through controlling secretion and paracrine signaling of SDF-1 and VEGF.

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

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

Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APPmore » has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.« less

Human bone marrow-derived clonal mesenchymal stem cells inhibit inflammation and reduce acute pancreatitis in rats.

PubMed

Jung, Kyung Hee; Song, Sun U; Yi, Tacghee; Jeon, Myung-Shin; Hong, Sang-Won; Zheng, Hong-Mei; Lee, Hee-Seung; Choi, Myung-Joo; Lee, Don-Haeng; Hong, Soon-Sun

2011-03-01

Acute pancreatitis (AP) has a high mortality rate; repetitive AP induces chronic AP and pancreatic adenocarcinoma. Mesenchymal stem cells (MSCs) have immunoregulatory effects and reduce inflammation. We developed a protocol to isolate human bone marrow-derived clonal MSCs (hcMSCs) from bone marrow aspirate and investigated the effects of these cells in rat models of mild and severe AP. Mild AP was induced in Sprague-Dawley rats by 3 intraperitoneal injections of cerulein (100 μg/kg), given at 2-hour intervals; severe AP was induced by intraparenchymal injection of 3% sodium taurocholate solution. hcMSCs were labeled with CM-1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchloride and administered to rats through the tail vein. hcMSCs underwent self-renewal and had multipotent differentiation capacities and immunoregulatory functions. Greater numbers of infused hcMSCs were detected in pancreas of rats with mild and severe AP than of control rats. Infused hcMSCs reduced acinar-cell degeneration, pancreatic edema, and inflammatory cell infiltration in each model of pancreatitis. The hcMSCs reduced expression of inflammation mediators and cytokines in rats with mild and severe AP. hcMSCs suppressed the mixed lymphocyte reaction and increased expression of Foxp3(+) (a marker of regulatory T cells) in cultured rat lymph node cells. Rats with mild or severe AP that were given infusions of hcMSCs had reduced numbers of CD3(+) T cells and increased expression of Foxp3(+) in pancreas tissues. hcMSCs reduced inflammation and damage to pancreatic tissue in a rat model of AP; they reduced levels of cytokines and induced numbers of Foxp3(+) regulatory T cells. hcMSCs might be developed as a cell therapy for pancreatitis. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

PubMed

Arabzadeh, Azadeh; McGregor, Kevin; Breton, Valérie; Van Der Kraak, Lauren; Akavia, Uri David; Greenwood, Celia M T; Beauchemin, Nicole

2017-11-28

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6 , we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1's role should be considered in the presence of other CEACAM family members.

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

Doi, Keiko; Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka; Central Research Institute of Life Sciences for the Next Generation of Women Scientists, Fukuoka University, Fukuoka

Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in themore » immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.« less

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

PubMed Central

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

Unripe Rubus coreanus Miquel suppresses migration and invasion of human prostate cancer cells by reducing matrix metalloproteinase expression.

PubMed

Kim, Yesl; Lee, Seung Min; Kim, Jung-Hyun

2014-01-01

Rubus coreanus Miquel (RCM) is used to promote prostate health and has been shown to have anti-oxidant and anti-carcinogenic activities. However, the effects and mechanisms of RCM on prostate cancer metastasis remain unclear. PC-3 and DU 145 cells were treated with ethanol or water extract of unripe or ripe RCM and examined for cell invasion, migration, and matrix metalloproteinases (MMPs) activity and expression. Phosphoinositide 3-kinase (PI3K) and Akt activities were examined. Unripe RCM extracts exerted significant inhibitory effects on cell migration, invasion, and MMPs activities. A significant reduction in MMPs activities by unripe RCM ethanol extract treatment (UE) was associated with reduction of MMPs expression and induction of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, PI3K/Akt activity was diminished by UE treatment. In this study, we demonstrated that UE decreased metastatic potential of prostate cancer cells by reducing MMPs expression through the suppression of PI3K/Akt phosphorylation, thereby decreasing MMP activity and enhancing TIMPs expression.

Alpha-linolenic acid regulates Cox2/VEGF/MAP kinase pathway and decreases the expression of HPV oncoproteins E6/E7 through restoration of p53 and Rb expression in human cervical cancer cell lines.

PubMed

Deshpande, Rashmi; Mansara, Prakash; Kaul-Ghanekar, Ruchika

2016-03-01

Cervical cancer represents the largest cause of mortality in women worldwide. In our previous report, we have shown how alpha-linolenic acid (ALA), an omega-3 fatty acid, regulated the growth of cervical cancer cells. The present study aimed to explore mechanistic details for the anticancer activity of ALA in cervical cancer cell lines, SiHa and HeLa. ALA significantly modulated the growth kinetics of the cells and reduced cell migration with concomitant decrease in the expression of VEGF, MMP-2, and MMP-9 proteins. Besides this, ALA significantly decreased the expression of phosphorylated p38, pERK1/2, c-JUN, NFκB, and COX2, proteins. Most importantly, ALA reduced the expression of HPV onco-proteins E6 and E7, resulting into restoration of expression of tumor suppressor proteins, p53 and Rb. These results suggested that ALA could be explored for its therapeutic potential in cervical cancer.

Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway

PubMed Central

Riganti, Chiara; Salaroglio, Iris Chiara; Caldera, Valentina; Campia, Ivana; Kopecka, Joanna; Mellai, Marta; Annovazzi, Laura; Bosia, Amalia; Ghigo, Dario; Schiffer, Davide

2013-01-01

Background Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. Methods A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. Results Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine–phosphate–guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. Conclusions Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling. PMID:23897632

Interactions between IGF-I, estrogen receptor-α (ERα), and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells.

PubMed

Mendoza, Rhone A; Enriquez, Marlene I; Mejia, Sylvia M; Moody, Emily E; Thordarson, Gudmundur

2011-01-01

Understanding of the interactions between estradiol (E₂) and IGF-I is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating noninterfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions, and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human GH plus epidermal growth factor, but E₂ did not cause an increase in the number of the IGF-IR.low cells compared to controls. The proliferation rate of IGF-IR.low cells was only reduced in response to E₂ compared to controls, whereas their basal and hormone-stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E₂, without affecting control cells. Furthermore, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. In conclusion, suppressing IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK, which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma.

PubMed

Zhou, Yong; Zha, Jie; Lin, Zhijuan; Fang, Zhihong; Zeng, Hanyan; Zhao, Jintao; Luo, Yiming; Li, Zhifeng; Xu, Bing

2018-01-15

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4 + T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19 + cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19 + cells than patients who did not show recurrence. Examining cytotoxic CD4 + T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4 + T cells. Also, frequency of cytotoxic CD4 + T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4 + T cells against autologous CD19 + cells was investigated. We found that the cytotoxic potential of CD4 + T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19 + cells presented a significant reduction after longer incubation with cytotoxic CD4 + T cells, suggesting that cytotoxic CD4 + T cells preferentially eliminated MHC II-expressing CD19 + cells. Blocking MHC II on CD19 + cells significantly reduced the cytolytic capacity of CD4 + T cells. Despite these discoveries, the frequency of cytotoxic CD4 + T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4 + T cells presented an MHC II-dependent cytotoxic potential against autologous CD19 + cells and could potentially represent a future treatment option for DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.

Jolkinolide B inhibits glycolysis by downregulating hexokinase 2 expression through inactivating the Akt/mTOR pathway in non-small cell lung cancer cells.

PubMed

Gao, Xiang; Han, Han

2018-06-01

Jolkinolide B (JB), a bioactive compound isolated from herbal medicine, has been found to inhibit tumor growth by altering glycolysis. However, whether glycolysis is influenced by JB in non-small cell lung cancer (NSCLC) cells and the mechanism remain unknown. The aim of the present study was to evaluate the effect of JB on the glycolysis in NSCLC cells and the underlying molecular mechanism. The results showed that JB treatment inhibited cell viability of A549 and H1299 cells in a concentration-dependent manner. JB reduced the glucose consumption, lactate production, and HK2 expression. The expressions of p-Akt and p-mTOR were also decreased by JB treatment. Knockdown of HK2 reduced glucose consumption and lactate production. Inhibition of the Akt/mTOR pathway decreased HK2 expression and inhibited glycolysis. In conclusion, the results indicated that JB inhibits glycolysis by down-regulating HK2 expression through inactivating the Akt/mTOR pathway in NSCLC cells, suggesting that JB might be a potential therapeutic agent for the treatment of NSCLC. © 2018 Wiley Periodicals, Inc.

Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis

PubMed Central

Wormley, Floyd L.; Chaiban, Joseph; Fidel, Paul L.

2001-01-01

Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection. PMID:11447188

CD151-mediated adhesion is crucial to osteosarcoma pulmonary metastasis

PubMed Central

Sun, Mengxiong; Zhou, Chenghao; Chen, Jian; Yin, Fei; Wang, Hongsheng; Lin, Binhui; Zuo, Dongqing; Li, Suoyuan; Feng, Lijin; Duan, Zhenfeng; Cai, Zhengdong; Hua, Yingqi

2016-01-01

CD151, a tetraspanin family protein involved in cell-cell and cell-extracellular matrix interaction, is differentially expressed in osteosarcoma cell membranes. Thus, this study aimed to investigate the role of CD151 in osteosarcoma metastasis. We analyzed CD151 expression in patient tissue samples using immunohistochemistry. CD151 expression was also silenced with shRNA in osteosarcoma cells of high metastatic potential, and cell adhesion, migration and invasion were evaluated in vitro and pulmonary metastasis was investigated in vivo. Mediators of cell signaling pathways were also examined following suppression of CD151 expression. Overall survival for patients with low versus high CD151 expression level was 94 vs. 41 months (p=0.0451). CD151 expression in osteosarcoma cells with high metastatic potential was significantly higher than in those with low metastatic potential (p<0.001). shRNA-mediated silencing of CD151 did not influence cell viability or proliferation; however, cell adhesion, migration and invasion were all inhibited (all p<0.001). In mice inoculated with shRNA-transduced osteosarcoma cells, the number and size of lung metastatic lesions were reduced compared to the mice inoculated with control-shRNA transduced cells (p<0.001). In addition, CD151 knockdown significantly reduced Akt, p38, and p65 phosphorylation as well as focal adhesion kinase, integrin β1, p70s6, and p-mTOR levels. Taken together, CD151 induced osteosarcoma metastasis likely by regulating cell function through adhesion signaling. Further studies are necessary to fully explore the diagnostic and prognostic value of determining CD151 expression in osteosarcoma patients. PMID:27556355

GATA3 Inhibits Lysyl Oxidase Mediated Metastases of Human Basal Triple-Negative Breast Cancer Cells

PubMed Central

Chu, Isabel M.; Michalowski, Aleksandra M.; Hoenerhoff, Mark; Szauter, Kornelia M.; Luger, Dror; Sato, Misako; Flanders, Kathy; Oshima, Akira; Csiszar, Katalin; Green, Jeffrey E.

2011-01-01

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative type breast cancers (BTNBC) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that the expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of Lysyl oxidase (LOX), a metastasis-promoting matrix remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by siRNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between high LOX and low GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBC to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBC. PMID:21892208

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

PubMed Central

Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

2014-01-01

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na+ and uptake of K+ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression. PMID:24452105

Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency

PubMed Central

Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.

2012-01-01

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097

ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

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

Ungerer, Christopher; Doberstein, Kai; Buerger, Claudia

2010-10-22

Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far.more » In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.« less

The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells.

PubMed

Fortunato, Angelo

2017-08-01

The transition of cells from the epithelial to the mesenchymal state (EMT) plays an important role in tumor progression. EMT allows cells to acquire mobility, stem-like behavior and resistance to apoptosis and drug treatment. These features turn EMT into a central process in tumor biology. Ion channels are attractive targets for the treatment of cancer since they play critical roles in controlling a wide range of physiological processes that are frequently deregulated in cancer. Here, we investigated the role of ether-a-go-go-related 1 (hERG1) ion channels in the EMT of colorectal cancer cells. We studied the epithelial-mesenchymal profile of different colorectal cancer-derived cell lines and the expression of hERG1 potassium channels in these cell lines using real-time PCR. Next, we knocked down hERG1 expression in HCT116 cells using lentivirus mediated RNA interference and characterized the hERG1 silenced cells in vitro and in vivo. Finally, we investigated the capacity of riluzole, an ion channel-modulating drug used in humans to treat amyotrophic lateral sclerosis, to reduce the resistance of the respective colorectal cancer cells to the chemotherapeutic drug cisplatin. We found that of the colorectal cancer-derived cell lines tested, HCT116 showed the highest mesenchymal profile and a high hERG1 expression. Subsequent hERG1 expression knockdown induced a change in cell morphology, which was accompanied by a reduction in the proliferative and tumorigenic capacities of the cells. Notably, we found that hERG1expression knockdown elicited a reversion of the EMT profile in HCT116 cells with a reacquisition of the epithelial-like profile. We also found that riluzole increased the sensitivity of HCT116 cisplatin-resistant cells to cisplatin. Our data indicate that hERG1 plays a role in the EMT of colorectal cancer cells and that its knockdown reduces the proliferative and tumorigenic capacities of these cells. In addition, we conclude that riluzole may be used in combination with cisplatin to reduce chemo-resistance in colorectal cancer cells.

Reducing FLI1 Levels in the MRL/lpr Lupus Mouse Model Impacts T Cell Function by Modulating Glycosphingolipid Metabolism

PubMed Central

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A.; Basher, Fahmin; Roddy, Patrick O.; Siskind, Leah J.; Nietert, Paul J.; Nowling, Tamara K.

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1 +/+ or Fli1 +/- T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1 +/- lupus T cells compared to animals receiving Fli1 +/+ lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1 +/- T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1 +/+ T cells. Moreover, the Fli1 +/- T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1 +/+ T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus. PMID:24040398

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

PubMed

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A; Basher, Fahmin; Roddy, Patrick O; Siskind, Leah J; Nietert, Paul J; Nowling, Tamara K

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.

PubMed

Nitschke, Silvia; Cortleven, Anne; Iven, Tim; Feussner, Ivo; Havaux, Michel; Riefler, Michael; Schmülling, Thomas

2016-07-01

The circadian clock helps plants measure daylength and adapt to changes in the day-night rhythm. We found that changes in the light-dark regime triggered stress responses, eventually leading to cell death, in leaves of Arabidopsis thaliana plants with reduced cytokinin levels or defective cytokinin signaling. Prolonged light treatment followed by a dark period induced stress and cell death marker genes while reducing photosynthetic efficiency. This response, called circadian stress, is also characterized by altered expression of clock and clock output genes. In particular, this treatment strongly reduced the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Intriguingly, similar changes in gene expression and cell death were observed in clock mutants lacking proper CCA1 and LHY function. Circadian stress caused strong changes in reactive oxygen species- and jasmonic acid (JA)-related gene expression. The activation of the JA pathway, involving the accumulation of JA metabolites, was crucial for the induction of cell death, since the cell death phenotype was strongly reduced in the jasmonate resistant1 mutant background. We propose that adaptation to circadian stress regimes requires a normal cytokinin status which, acting primarily through the AHK3 receptor, supports circadian clock function to guard against the detrimental effects of circadian stress. © 2016 American Society of Plant Biologists. All rights reserved.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

PubMed

Rodriguez-Perez, N; Schiavi, E; Frei, R; Ferstl, R; Wawrzyniak, P; Smolinska, S; Sokolowska, M; Sievi, N A; Kohler, M; Schmid-Grendelmeier, P; Michalovich, D; Simpson, K D; Hessel, E M; Jutel, M; Martin-Fontecha, M; Palomares, O; Akdis, C A; O'Mahony, L

2017-11-01

Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Role of Extracellular miR-122 in Breast Cancer Metastasis

DTIC Science & Technology

2016-02-01

expression by miR-122 reduced the level of the GLUT1 causing reduced glucose uptake; and 4) anti-miR-122 therapy suppressed metastasis in a xenograft mouse...metastatic niche selection by circulating tumor cells. 100% completed.  Major Task 3: Orthotopic xenograft tumors expressing high miR-122 and...aforementioned cell lines and used to treat NSG mice i.v (Fig. 5). Xenograft tumors were established in NSG mice for over-expression of miR-122 in

6-Mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1.

PubMed

Marinković, Goran; Hamers, Anouk A J; de Vries, Carlie J M; de Waard, Vivian

2014-09-01

Inflammatory bowel disease is characterized by chronic intestinal inflammation. Azathioprine and its metabolite 6-mercaptopurine (6-MP) are effective immunosuppressive drugs that are widely used in patients with inflammatory bowel disease. However, established understanding of their immunosuppressive mechanism is limited. Azathioprine and 6-MP have been shown to affect small GTPase Rac1 in T cells and endothelial cells, whereas the effect on macrophages and gut epithelial cells is unknown. Macrophages (RAW cells) and gut epithelial cells (Caco-2 cells) were activated by cytokines and the effect on Rac1 signaling was assessed in the presence or absence of 6-MP. Rac1 is activated in macrophages and epithelial cells, and treatment with 6-MP resulted in Rac1 inhibition. In macrophages, interferon-γ induced downstream signaling through c-Jun-N-terminal Kinase (JNK) resulting in inducible nitric oxide synthase (iNOS) expression. iNOS expression was reduced by 6-MP in a Rac1-dependent manner. In epithelial cells, 6-MP efficiently inhibited tumor necrosis factor-α-induced expression of the chemokines CCL2 and interleukin-8, although only interleukin-8 expression was inhibited in a Rac1-dependent manner. In addition, activation of the transcription factor STAT3 was suppressed in a Rac1-dependent fashion by 6-MP, resulting in reduced proliferation of the epithelial cells due to diminished cyclin D1 expression. These data demonstrate that 6-MP affects macrophages and gut epithelial cells beneficially, in addition to T cells and endothelial cells. Furthermore, mechanistic insight is provided to support development of Rac1-specific inhibitors for clinical use in inflammatory bowel disease.

Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning.

PubMed

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A; Mains, Richard E

2014-07-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. Copyright © 2014 Elsevier Inc. All rights reserved.

Silencing NKD2 by Promoter Region Hypermethylation Promotes Esophageal Cancer Progression by Activating Wnt Signaling.

PubMed

Cao, Baoping; Yang, Weili; Jin, Yongshuai; Zhang, Meiying; He, Tao; Zhan, Qimin; Herman, James G; Zhong, Guanglin; Guo, Mingzhou

2016-11-01

Naked cuticle homolog 2 (NKD2) was found to be frequently methylated in human breast and gastric cancers. However, the epigenetic changes and mechanisms of NKD2 in human esophageal cancer remain unclear. Nine esophageal cancer cell lines and 154 cases of primary esophageal cancer samples were analyzed using methylation-specific polymerase chain reaction, immunohistochemical analysis, Western blot, and xenograft mouse models. Loss of NKD2 expression and complete methylation were found in KYSE150 and TE1 cells. Reduced NKD2 expression and partial methylation of the promoter region were observed in KYSE30, KYSE70, KYSE410, KYSE140, and COLO680 cells. High levels of NKD2 expression and unmethylation were detected in KYSE450 and TE8 cells. Reexpression of NKD2 was induced by 5-aza-2'-deoxycytidine in cells in which NKD2 was not expressed or cells in which NKD2 expression was reduced. NKD2 was methylated in 53.2% of human primary esophageal cancer samples (82 of 154), and promoter region hypermethylation was significantly associated with reduced expression of NKD2 (p < 0.01). NKD2 methylation was associated with tumor, node, and metastasis stage and lymph node metastasis (p < 0.01). Our results suggest that NKD2 is regulated by promoter region methylation and that methylation of NKD2 may serve as a prognostic marker in esophageal cancer. Our further studies demonstrate that NKD2 suppresses cell proliferation, colony formation, cell invasion, and migration and also induces G1/S checkpoint arrest in esophageal cancer cells. NKD2 suppressed xenograft tumor growth and inhibited Wnt signaling in human esophageal cancer cells. NKD2 is frequently methylated in human esophageal cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses esophageal cancer progression by inhibiting Wnt signaling both in vitro and in vivo. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Macrophage-Specific Expression of IL-37 in Hyperlipidemic Mice Attenuates Atherosclerosis.

PubMed

McCurdy, Sara; Baumer, Yvonne; Toulmin, Emma; Lee, Bog-Hieu; Boisvert, William A

2017-11-15

Atherosclerosis, the progressive buildup of plaque within arterial blood vessels, can lead to fatal downstream events, such as heart attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of monocytes and its associated inflammation, as well as the formation of lipid-laden macrophage foam cells within the vessel wall. IL-37 is recognized as an important anti-inflammatory cytokine expressed especially by immune cells. This study was undertaken to elucidate the role of macrophage-expressed IL-37 in reducing the production and effects of proinflammatory cytokines, preventing foam cell formation, and reducing the development of atherosclerosis. Expression of human IL-37 was achieved with a macrophage-specific overexpression system, using the CD68 promoter in mouse primary bone marrow-derived macrophages via retroviral transduction. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted protein production (e.g., IL-6, M-CSF, and ICAM-1) of key inflammatory mediators. IL-37 expression also inhibited macrophage proliferation, apoptosis, and transmigration, as well as reduced lipid uptake, compared with controls in vitro. The in vivo effects of macrophage-expressed IL-37 were investigated through bone marrow transplantation of transduced hematopoietic stem cells into irradiated atherosclerosis-prone Ldlr -/- mice. After 10 wk on a high-fat/high-cholesterol diet, mice with IL-37-expressing macrophages showed reduced disease pathogenesis, which was demonstrated by significantly less arterial plaque development and systemic inflammation compared with control mice. The athero-protective effect of macrophage-expressed IL-37 has implications for development of future therapies to treat atherosclerosis, as well as other chronic inflammatory diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

Frequent downregulation of miR-34 family in human ovarian cancers.

PubMed

Corney, David C; Hwang, Chang-Il; Matoso, Andres; Vogt, Markus; Flesken-Nikitin, Andrea; Godwin, Andrew K; Kamat, Aparna A; Sood, Anil K; Ellenson, Lora H; Hermeking, Heiko; Nikitin, Alexander Yu

2010-02-15

The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC.

Budesonide increases TLR4 and TLR2 expression in Treg lymphocytes of allergic asthmatics.

PubMed

Pace, Elisabetta; Di Sano, Caterina; Ferraro, Maria; Bruno, Andreina; Caputo, Valentina; Gallina, Salvatore; Gjomarkaj, Mark

2015-06-01

Reduced innate immunity responses as well as reduced T regulatory activities characterise bronchial asthma. In this study the effect of budesonide on the expression of TLR4 and TLR2 in T regulatory lymphocyte sub-population was assessed. TLR4 and TLR2 expression in total peripheral blood mononuclear cells (PBMC), in CD4+/CD25+ and in CD4+/CD25- was evaluated, by flow cytometric analysis, in mild intermittent asthmatics (n = 14) and in controls (n = 11). The in vitro effects of budesonide in modulating: TLR4 and TLR2 expression in controls and in asthmatics; IL-10 expression and cytokine release (IL-6 and TNF-α selected by a multiplex assay) in asthmatics were also explored. TLR4 and TLR2 were reduced in total PBMC from asthmatics in comparison to PBMC from controls. CD4+CD25+ cells expressed at higher extent TLR2 and TLR4 in comparison to CD4+CD25- cells. Budesonide was able to increase the expression of TLR4, TLR2 and IL-10 in CD4+/CD25 highly+ cells from asthmatics. TLR4 ligand, LPS induced Foxp3 expression. Budesonide was also able to reduce the release of IL-6 and TNF-α by PBMC of asthmatics. Budesonide potentiates the activity of Treg by increasing TLR4, TLR2 and IL-10 expression. This event is associated to the decreased release of IL-6 and TNF-α in PBMC treated with budesonide. These findings shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells

PubMed Central

Zhang, Bin; Li, Ling; Ho, Yinwei; Li, Min; Marcucci, Guido

2016-01-01

Chronic myelogenous leukemia (CML) results from transformation of a long-term hematopoietic stem cell (LTHSC) by expression of the BCR-ABL fusion gene. However, BCR-ABL–expressing LTHSCs are heterogeneous in their capacity as leukemic stem cells (LSCs). Although discrepancies in proliferative, self-renewal, and differentiation properties of normal LTHSCs are being increasingly recognized, the mechanisms underlying heterogeneity of leukemic LTHSCs are poorly understood. Using a CML mouse model, we identified gene expression differences between leukemic and nonleukemic LTHSCs. Expression of the thrombopoietin (THPO) receptor MPL was elevated in leukemic LTHSC populations. Compared with LTHSCs with low MPL expression, LTHSCs with high MPL expression showed enhanced JAK/STAT signaling and proliferation in response to THPO in vitro and increased leukemogenic capacity in vivo. Although both G0 and S phase subpopulations were increased in LTHSCs with high MPL expression, LSC capacity was restricted to quiescent cells. Inhibition of MPL expression in CML LTHSCs reduced THPO-induced JAK/STAT signaling and leukemogenic potential. These same phenotypes were also present in LTHSCs from patients with CML, and patient LTHSCs with high MPL expression had reduced sensitivity to BCR-ABL tyrosine kinase inhibitor treatment but increased sensitivity to JAK inhibitors. Together, our studies identify MPL expression levels as a key determinant of heterogeneous leukemia-initiating capacity and drug sensitivity of CML LTHSCs and suggest that high MPL–expressing CML stem cells are potential targets for therapy. PMID:26878174

[Effects on survival of shRNA mediated APE/Ref1 gene silencing in rat spiral ganglion cells in oxidative stress].

PubMed

Jiang, Zhendong; Zhong, Cheng; Li, Taijun; Xiang, Zhaolan; Zhang, Xueyuan

2014-02-01

To investigate the effects of reducing APE/Ref1 expression in the cultures of rat spiral ganglion cells with oxidative damage induced by H(2)O(2). Primary cultured rat spiral ganglion cells were infected with small interfering RNA to APE/Ref1 (Ape1siRNA) for 72 h, followed by treating with H(2)O(2) (0, 10, 25, 50, 100 and 300 µmol/L) for 1 h , and then cultured in normal medium for 24 h. Western blot were used to detect the level of APE/Ref1 protein and phosphorylation of histone protein H2AX in the infected cells. The caspase3 activation was tested by spectrophotometric method . The cell viability was determined by MTT and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick and labeling (TUNEL). Western blot showed that infection with Ape1siRNA resulted in APE/Ref1 reduced expression in the spiral ganglion cells. Exposing spiral ganglion cultures with reduced expression of APE/Ref1 to H(2)O(2) (50, 100, 300 µmol/L) for 1 h resulted in increasing in the phosphorylation of histone protein H2AX. The reduction in APE/Ref1 significantly reduced cell viability in cultures 24 h after 1 h expression to 50-300 µmol/L H(2)O(2). The apoptosis of cells and caspase 3 activity was detected significantly improved. The induced of APE/Ref1 results in significantly decrease in spiral ganglion cells viability in oxidative stress. The repairing function of APE/Ref1 is necessary for optimal levels of neuronal rat spiral ganglion cells survival.

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells.

PubMed

Raab, Monika; Sanhaji, Mourad; Matthess, Yves; Hörlin, Albrecht; Lorenz, Ioana; Dötsch, Christina; Habbe, Nils; Waidmann, Oliver; Kurunci-Csacsko, Elisabeth; Firestein, Ron; Becker, Sven; Strebhardt, Klaus

2018-03-16

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

PubMed

Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

2017-10-15

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

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.

Ranolazine inhibits NaV1.5-mediated breast cancer cell invasiveness and lung colonization.

PubMed

Driffort, Virginie; Gillet, Ludovic; Bon, Emeline; Marionneau-Lambot, Séverine; Oullier, Thibauld; Joulin, Virginie; Collin, Christine; Pagès, Jean-Christophe; Jourdan, Marie-Lise; Chevalier, Stéphan; Bougnoux, Philippe; Le Guennec, Jean-Yves; Besson, Pierre; Roger, Sébastien

2014-12-11

Na(V)1.5 voltage-gated sodium channels are abnormally expressed in breast tumours and their expression level is associated with metastatic occurrence and patients' death. In breast cancer cells, Na(V)1.5 activity promotes the proteolytic degradation of the extracellular matrix and enhances cell invasiveness. In this study, we showed that the extinction of Na(V)1.5 expression in human breast cancer cells almost completely abrogated lung colonisation in immunodepressed mice (NMRI nude). Furthermore, we demonstrated that ranolazine (50 μM) inhibited Na(V)1.5 currents in breast cancer cells and reduced Na(V)1.5-related cancer cell invasiveness in vitro. In vivo, the injection of ranolazine (50 mg/kg/day) significantly reduced lung colonisation by Na(V)1.5-expressing human breast cancer cells. Taken together, our results demonstrate the importance of Na(V)1.5 in the metastatic colonisation of organs by breast cancer cells and indicate that small molecules interfering with Na(V) activity, such as ranolazine, may represent powerful pharmacological tools to inhibit metastatic development and improve cancer treatments.

Sox2 promotes tamoxifen resistance in breast cancer cells

PubMed Central

Piva, Marco; Domenici, Giacomo; Iriondo, Oihana; Rábano, Miriam; Simões, Bruno M; Comaills, Valentine; Barredo, Inmaculada; López-Ruiz, Jose A; Zabalza, Ignacio; Kypta, Robert; Vivanco, Maria d M

2014-01-01

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo- and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen-resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2-expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2-dependent activation of Wnt signalling in cancer stem/progenitor cells. PMID:24178749

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

PubMed

Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

2010-10-01

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

TMC1 and TMC2 are components of the mechanotransduction channel in hair cells of the mammalian inner ear.

PubMed

Pan, Bifeng; Géléoc, Gwenaelle S; Asai, Yukako; Horwitz, Geoffrey C; Kurima, Kiyoto; Ishikawa, Kotaro; Kawashima, Yoshiyuki; Griffith, Andrew J; Holt, Jeffrey R

2013-08-07

Sensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea. Copyright © 2013 Elsevier Inc. All rights reserved.

Depletion of FAP+ cells reduces immunosuppressive cells and improves metabolism and functions CD8+T cells within tumors

PubMed Central

Zhang, Ying; Ertl, Hildegund C.J.

2016-01-01

The tumor stroma, which is essential to support growth and metastasis of malignant cells, provides targets for active immunotherapy of cancer. Previous studies have shown that depleting fibroblast activation protein (FAP)-expressing stromal cells reduces tumor progression and concomitantly increases tumor antigen (TA)-specific T cell responses. However the underlying pathways remain ill defined. Here we identify that immunosuppressive cells (ISCs) from tumor-bearing mice impose metabolic stress on CD8+T cells, which is associated with increased expression of the co-inhibitor PD-1. In two mouse melanoma models, depleting FAP+ stroma cells from the tumor microenvironment (TME) upon vaccination with an adenoviral-vector reduces frequencies and functions of ISCs. This is associated with changes in the cytokine/chemokine milieu in the TME and decreased activity of STAT6 signaling within ISCs. Decreases in ISCs upon FAP+stromal cell depletion is associated with reduced metabolic stress of vaccine-induced tumor infiltrating CD8+T cells and their delayed progression towards functional exhaustion, resulting in prolonged survival of tumor-bearing mice. PMID:26943036

Depletion of FAP+ cells reduces immunosuppressive cells and improves metabolism and functions CD8+T cells within tumors.

PubMed

Zhang, Ying; Ertl, Hildegund C J

2016-04-26

The tumor stroma, which is essential to support growth and metastasis of malignant cells, provides targets for active immunotherapy of cancer. Previous studies have shown that depleting fibroblast activation protein (FAP)-expressing stromal cells reduces tumor progression and concomitantly increases tumor antigen (TA)-specific T cell responses. However the underlying pathways remain ill defined. Here we identify that immunosuppressive cells (ISCs) from tumor-bearing mice impose metabolic stress on CD8+T cells, which is associated with increased expression of the co-inhibitor PD-1. In two mouse melanoma models, depleting FAP+ stroma cells from the tumor microenvironment (TME) upon vaccination with an adenoviral-vector reduces frequencies and functions of ISCs. This is associated with changes in the cytokine/chemokine milieu in the TME and decreased activity of STAT6 signaling within ISCs. Decreases in ISCs upon FAP+stromal cell depletion is associated with reduced metabolic stress of vaccine-induced tumor infiltrating CD8+T cells and their delayed progression towards functional exhaustion, resulting in prolonged survival of tumor-bearing mice.

Disruption of β-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

PubMed

Moheimani, Fatemeh; Roth, Hollis M; Cross, Jennifer; Reid, Andrew T; Shaheen, Furquan; Warner, Stephanie M; Hirota, Jeremy A; Kicic, Anthony; Hallstrand, Teal S; Kahn, Michael; Stick, Stephen M; Hansbro, Philip M; Hackett, Tillie-Louise; Knight, Darryl A

2015-11-01

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor β-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the β-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the β-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the β-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFβ1 in the presence or absence of the selective small molecule ICG-001 to inhibit β-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, β-catenin, fibronectin and ITGβ1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFβ1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFβ1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFβ1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGβ1 and fibronectin expression. These data support our hypothesis that modulating the β-catenin/CBP signaling axis plays a key role in epithelial plasticity and function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

PubMed

Chui, A; Murthi, P; Gunatillake, T; Brennecke, S P; Ignjatovic, V; Monagle, P T; Whitelock, J M; Said, J M

2014-08-01

Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Shikonin Inhibites Migration and Invasion of Thyroid Cancer Cells by Downregulating DNMT1

PubMed Central

Zhang, Yue; Sun, Bin; Huang, Zhi

2018-01-01

Background Shikonin is a component of Chinese herbal medicine. The aim of this study was to investigate the effects of shikonin on cell migration of papillary thyroid cancer cells of the TPC-1 cell line in vitro and expression levels of the phosphate and tensin homolog deleted on chromosome 10 (PTEN) and DNA methyltransferase 1 (DNMT1) genes. Material/Methods The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the proliferation of TPC-1 papillary thyroid cancer cells, and the normal thyroid cells, HTori-3, in vitro. A transwell motility assay was used to analyze the migration of TPC-1 cells. Western blot was performed to determine the expression levels of PTEN and DNMT1 genes. A methylation-specific polymerase chain reaction (PCR) (MSP) assay was used to evaluate the methylation of PTEN. Results Following treatment with shikonin, the cell survival rate of TPC-1 cells decreased in a dose-dependent manner; the inhibitory effects on HTori-3 cells were less marked. Shikonin inhibited TPC-1 cell migration and invasion in a dose-dependent manner. The methylation of PTEN was suppressed by shikonin, which also reduced the expression of DNMT1 in a dose-dependent manner, and increased the expression of PTEN. Overexpression of DNMT1 promoted the migration of TPC-1 cells and the methylation of PTEN. Levels of protein expression of PTEN in TPC-1 cells treated with shikonin decreased, and were increased by DNMT1 knockdown. Conclusions Shikonin suppressed the expression of DNMT1, reduced PTEN gene methylation, and increased PTEN protein expression, leading to the inhibition of TPC-1 cell migration. PMID:29389913

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

PubMed Central

Sheth, Sandeep; Jajoo, Sarvesh; Kaur, Tejbeer; Mukherjea, Debashree; Sheehan, Kelly; Rybak, Leonard P.; Ramkumar, Vickram

2012-01-01

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but independent of miR-21.These data suggest that resveratrol’s anti-tumor actions in prostate cancer could be explained, in part, through inhibition of Akt/miR-21 signaling pathway. PMID:23272133

MyD88 expression in the rat dental follicle: Implications for osteoclastogenesis and tooth eruption

PubMed Central

Liu, Dawen; Yao, Shaomian; Wise, Gary E.

2010-01-01

Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin-1 (IL-1) and IL-18 Toll-like receptor signaling pathway. Because it is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine gene expression of MyD88 in vivo in the DFs from the first mandibular molars of postnatal rats from days 1–11. The results showed that MyD88 was expressed maximally at day 3. Using siRNA to knock down MyD88 expression in the DF cells also reduced the gene expression of nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1). IL-1α up-regulated the expression of NFKB1, MCP-1 and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1α effect. Conditioned medium from DF cells with MyD88 knocked down reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis as opposed to controls. In conclusion, the maximal expression of MyD88 at day 3 in the DF may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression. PMID:20662905

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

PubMed

Takahashi, Yoichiro; Kubo, Rieko; Sano, Rie; Nakajima, Tamiko; Takahashi, Keiko; Kobayashi, Momoko; Handa, Hiroshi; Tsukada, Junichi; Kominato, Yoshihiko

2017-03-01

The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells. © 2016 AABB.

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

Petit, Chad M.; Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803; Chouljenko, Vladimir N.

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion wasmore » reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.« less

Effect of fluoride exposure on mRNA expression of cav1.2 and calcium signal pathway apoptosis regulators in PC12 cells.

PubMed

Liao, Qiuxia; Zhang, Rui; Wang, Xiaoyu; Nian, Weiwei; Ke, Lulu; Ouyang, Wei; Zhang, Zigui

2017-09-01

This study investigated the effects of fluoride exposure on the mRNA expression of Cav1.2 calcium signaling pathway and apoptosis regulatory molecules in PC12 cells. The viability of PC12 cell receiving high fluoride (5.0mM) and low fluoride (0.5mM) alone or fluoride combined with L-type calcium channel (LTCC) agonist/inhibitor (5umol/L FPL6417/2umol/L nifedipine) was detected using cell counting kit-8 at different time points (2, 4, 6, 8, 12, 10, and 24h). Changes in the cell configuration were observed after exposing the cells to fluoride for 24h. The expression levels of molecules related to the LTCC were examined, particularly, Cav1.2, c-fos, CAMK II, Bax, and Bcl-2. Fluoride poisoning induced severe cell injuries, such as decreased PC12 cell activity, enhanced cell apoptosis, high c-fos, CAMKII, and Bax mRNA expression levels. Bcl-2 expression level was also reduced. Meanwhile, high fluoride, high fluoride with FPL64176, and low fluoride with FPL64176 enhanced the Cav1.2 expression level. In contrast, low fluoride, high fluoride with nifedipine, and low fluoride with nifedipine reduced the Cav1.2 expression level. Thus, Cav1.2 may be an important molecular target for the fluorosis treatment, and the LTCC inhibitor nifedipine may be an effective drug for fluorosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Methylation of DACT2 accelerates esophageal cancer development by activating Wnt signaling

PubMed Central

Zhang, Meiying; Linghu, Enqiang; Zhan, Qimin; He, Tao; Cao, Baoping; Brock, Malcolm V.; Herman, James G.; Xiang, Rong; Guo, Mingzhou

2016-01-01

Esophageal cancer is one of the most common malignancies worldwide. DACT2 is frequently methylated in human lung, hepatic, gastric and thyroid cancers. The methylation status and function of DACT2 remain to be elucidated in human esophageal cancer. Ten esophageal cancer cell lines, 42 cases of dysplasia and 126 cases of primary esophageal cancer samples were analyzed in this study. The expression of DACT2 was detected in YES2 cells, while reduced DACT2 expression levels were found in TE8 and KYSE70 cells, and complete loss of DACT2 expression was found in KYSE30, KYSE140, KYSE150, KYSE410, KYSE450, TE3 and TE7 cells. Loss of expression or reduced expression of DACT2 correlated with promoter region hypermethylation in esophageal cancer cells. Restoration of DACT2 expression was induced by 5-aza-2′-deoxycytidine. In human primary esophageal squamous carcinoma, 69% (87/126) of samples were methylated. Methylation of DACT2 was significantly associated with tumor stage and metastasis (P < 0.01, P < 0.05). DACT2 suppressed colony formation, cell migration and invasion in esophageal cancer cells, and it also suppressed esophageal cancer cell xenograft growth. DACT2 inhibited Wnt signaling in human esophageal cancer cells. In conclusion, DACT2 is frequently methylated in human esophageal cancer and its expression is regulated by promoter region methylation. DACT2 suppresses esophageal cancer growth by inhibiting Wnt signaling. PMID:26919254

Matrix metalloproteinase-9 expression by Hodgkin-Reed-Sternberg cells is associated with reduced overall survival in young adult patients with classical Hodgkin lymphoma.

PubMed

Campos, Antonio Hugo; Vassallo, Jose; Soares, Fernando Augusto

2013-01-01

Previous studies have investigated the prognostic relevance of MMP9 in classical Hodgkin lymphoma (cHL), with negative results. However, we have found that MMP9 immunoistochemical expression by Hodgkin-Reed-Sternberg cells is associated with reduced overall survival in a subset of young adult Brazilian patients diagnosed with cHL. Additionally, we have observed that MMP9 expression by neoplastic cells in cHL is associated with EBV positivity. These results may support a rational basis for additional studies on the role of this metalloproteinase as a target for therapy in classical Hodgkin lymphoma.

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism

PubMed Central

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-01-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. PMID:26081516

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism.

PubMed

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-09-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE-/- mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling.

PubMed

Futrega, Kathryn; Yu, Jianshi; Jones, Jace W; Kane, Maureen A; Lott, William B; Atkinson, Kerry; Doran, Michael R

2016-04-21

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.

Valsartan independent of AT₁ receptor inhibits tissue factor, TLR-2 and -4 expression by regulation of Egr-1 through activation of AMPK in diabetic conditions.

PubMed

Ha, Yu Mi; Park, Eun Jung; Kang, Young Jin; Park, Sang Won; Kim, Hye Jung; Chang, Ki Churl

2014-10-01

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)-1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT1R) can reduce tissue factor (TF) and toll-like receptor (TLR)-2 and -4 by regulating Egr-1 in THP-1 cells and aorta in streptozotocin-induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr-1, TF, TLR-2 and -4 which were significantly reduced by valsartan. HG increased Egr-1 expression by activation of PKC and ERK1/2 in THP-1 cells. Valsartan increased AMPK phosphorylation in a concentration and time-dependent manner via activation of LKB1. Valsartan inhibited Egr-1 without activation of PKC or ERK1/2. The reduced expression of Egr-1 by valsartan was reversed by either silencing Egr-1, or compound C, or DN-AMPK-transfected cells. Valsartan inhibited binding of NF-κB and Egr-1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF-α, IL-6 and IL-1β) production and NF-κB activity in HG-activated THP-1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT1R in THP-1 cells or CHO cells, which were devoid of AT1R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr-1, TLR-2, -4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin-induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr-1 regulation. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Valsartan independent of AT1 receptor inhibits tissue factor, TLR-2 and-4 expression by regulation of Egr-1 through activation of AMPK in diabetic conditions

PubMed Central

Ha, Yu Mi; Park, Eun Jung; Kang, Young Jin; Park, Sang Won; Kim, Hye Jung; Chang, Ki Churl

2014-01-01

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)-1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT1R) can reduce tissue factor (TF) and toll-like receptor (TLR)-2 and-4 by regulating Egr-1 in THP-1 cells and aorta in streptozotocin-induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr-1, TF, TLR-2 and-4 which were significantly reduced by valsartan. HG increased Egr-1 expression by activation of PKC and ERK1/2 in THP-1 cells. Valsartan increased AMPK phosphorylation in a concentration and time-dependent manner via activation of LKB1. Valsartan inhibited Egr-1 without activation of PKC or ERK1/2. The reduced expression of Egr-1 by valsartan was reversed by either silencing Egr-1, or compound C, or DN-AMPK-transfected cells. Valsartan inhibited binding of NF-κB and Egr-1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF-α, IL-6 and IL-1β) production and NF-κB activity in HG-activated THP-1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT1R in THP-1 cells or CHO cells, which were devoid of AT1R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr-1, TLR-2,-4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin-induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr-1 regulation. PMID:25109475

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.

PubMed

Arwert, Esther N; Lal, Rohit; Quist, Sven; Rosewell, Ian; van Rooijen, Nico; Watt, Fiona M

2010-11-16

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

Sodium Butyrate Inhibits Colorectal Cancer Cell Migration by Downregulating Bmi-1 Through Enhanced miR-200c Expression.

PubMed

Xu, Zhiyao; Tao, Jingjing; Chen, Ping; Chen, Long; Sharma, Sherven; Wang, Guanyu; Dong, Qinghua

2018-03-01

Short-chain fatty acid sodium butyrate (NaB) is the byproduct of bacterial anaerobic fermentation of dietary fiber in the colon, and has been shown to have an antitumor effect on colorectal cancer (CRC). The miR-200 family is a key regulator of the epithelial-mesenchymal transition (EMT). We investigate the role of miR-200s expression on cell migration in NaB-treated CRC cells. HCT116 and LOVO CRC cells treated with NaB depicted reduced cell proliferation, enhanced apoptosis, and cell cycle arrest. NaB inhibited cell migration in the wound healing and transwell assays, and in spheriod cultures while regulating EMT-related protein expression. NaB reciprocally increased miR-200s but reduced expression of their target genes (Bmi-1, Zeb1, EZH2). Cells transfected with miR-200c shRNA displayed a significant blockade of NaB-induced anti-invasive activity. Upregulation of Bmi-1 expression partially reversed the effect of NaB. In addition to inhibition of tumor growth in vivo, qRT-PCR results showed that NaB increased miR-200c/200b/492 expression in the tumor tissues. Immunohistochemistry and Western blotting results demonstrated that NaB decreased Bmi-1 expression in vivo. NaB inhibits CRC cell migration by enhancing miR-200c expression-mediated downregulation of Bmi-1. These findings support the utility of NaB in colorectal cancer therapy. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

PubMed

Tao, Qi-Fei; Yuan, Sheng-Xian; Yang, Fu; Yang, Sen; Yang, Yuan; Yuan, Ji-Hang; Wang, Zhen-Guang; Xu, Qing-Guo; Lin, Kong-Ying; Cai, Jie; Yu, Jian; Huang, Wei-Long; Teng, Xiao-Lei; Zhou, Chuan-Chuan; Wang, Fang; Sun, Shu-Han; Zhou, Wei-Ping

2015-09-17

Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

MiR-199a-3p enhances cisplatin sensitivity of cholangiocarcinoma cells by inhibiting mTOR signaling pathway and expression of MDR1.

PubMed

Li, Qiang; Xia, Xuefeng; Ji, Jie; Ma, Jianghui; Tao, Liang; Mo, Linjun; Chen, Wei

2017-05-16

Several studies have reported reduced miRNA-199a-3p (miR-199a-3p) in different human malignancies, however, little is known about miR-199a-3p in cholangiocarcinoma cells. In this study, we demonstrate the essential role and mechanism of miR-199a-3p in regulating cisplatin sensitivity in cholangiocarcinoma cell lines. Using a CCK-8 cell counting assay we found that expression of miR-199a-3p was positively correlated with cisplatin sensitivity in cholangiocarcinoma cell lines. MiR-199a-3p overexpression could decrease the proliferation rate and increase apoptosis of cholangiocarcinoma cells in the presence of cisplatin, while miR-199a-3p inhibition had the opposite effect. Further study demonstrated that mTOR was the target gene of miR-199a-3p, and that miR-199a-3p mimics could inhibit expression of mTOR, which consequently reduced the phosphorylation of its downstream proteins 4EBP1 and p70s6k. Rescue experiments proved that miR-199a-3p could increase the cisplatin sensitivity of cholangiocarcinoma cell lines by regulating mTOR expression. Moreover, we also found that miR-199a-3p overexpression could reduce cisplatin induced MDR1 expression by decreasing the synthesis and increasing the degradation of MDR1, thus enhancing the effectiveness of cisplatin in cholangiocarcinoma. In conclusion, miR-199a-3p could increase cisplatin sensitivity of cholangiocarcinoma cell lines by inhibiting the activity of the mTOR signaling pathway and decreasing the expression of MDR1.

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro.

PubMed

Laihia, J K; Jansen, C T

2000-08-01

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.

TRAF2 regulates the cytoplasmic/nuclear distribution of TRAF4 and its biological function in breast cancer cells

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

Zhang, Xiaoli; Wen, Zhifeng; Sun, Limei

2013-06-28

Highlights: •TRAF2 appears to interact with TRAF4 in breast cancer cell lines. •TRAF2 affects the localization and function of TRAF4 in breast cancer cell lines. •TRAF4 may play an important role in the activation of NF-κB via TRAF2. -- Abstract: Although numerous studies have shown that tumor necrosis factor receptor-associated factor 4 (TRAF4) plays an important role in the carcinogenesis of many tumor types, its exact molecular mechanism remains elusive. In this study, we examined the regulation function of TRAF2 to the cytoplasmic/nuclear distribution of TRAF4 in the breast cancer cell line. Using cell immunofluorescent staining, we found that TRAF2more » and TRAF4 were co-localized to the cytoplasm in MCF-7 cells. Co-immunoprecipitation showed that TRAF2 could interact with TRAF4 in MCF-10A, MCF-7 and MDA-MB-231 cell lines. Western blotting showed TRAF2 depletion by targeted siRNA in MDA-MB-231 cells led to reduced TRAF4 expression in the cytoplasm and augmented TRAF4 expression in the nucleus. Cytoplasmic expression of TRAF4 was augmented and nuclear expression was reduced when MCF-7 cells were transfected with hTRAF2pLPCX-HA-Flag/P874. MCF-7 cells expressing hTRAF2pLPCX-HA-Flag/P874 had enhanced cell proliferation rates. The nuclear expression of NF-κB significantly increased after TNF-α treatment. When hTRAF2pLPCX-HA-Flag/P874 and the siRNA-TRAF4 plasmid were cotransfected, the nuclear expression of NF-κB was significantly reduced compared with cells transfected with hTRAF2pLPCX-HA-Flag/P874 only. In conclusion, TRAF2 appears to interact with TRAF4 and affect the localization of TRAF4 in breast cancer cell lines. The overexpression of TRAF2 augmented the cytoplasmic expression of TRAF4 which promoted cell proliferation and inhibited cell apoptosis by activating NF-κB nuclear transcription. TRAF4 may play an important role in the activation of NF-κB via TRAF2.« less

Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

PubMed

Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

2016-08-05

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

The combination of Bifidobacterium breve with non-digestible oligosaccharides suppresses airway inflammation in a murine model for chronic asthma.

PubMed

Sagar, Seil; Vos, Arjan P; Morgan, Mary E; Garssen, Johan; Georgiou, Niki A; Boon, Louis; Kraneveld, Aletta D; Folkerts, Gert

2014-04-01

Over the last decade, there has been a growing interest in the use of interventions that target the intestinal microbiota as a treatment approach for asthma. This study is aimed at exploring the therapeutic effects of long-term treatment with a combination of Bifidobacterium breve with non-digestible oligosaccharides on airway inflammation and remodeling. A murine ovalbumin-induced chronic asthma model was used. Pulmonary airway inflammation; mRNA expression of pattern recognition receptors, Th-specific cytokines and transcription factors in lung tissue; expression of Foxp3 in blood Th cells; in vitro T cell activation; mast cell degranulation; and airway remodeling were examined. The combination of B. breve with non-digestible oligosaccharides suppressed pulmonary airway inflammation; reduced T cell activation and mast cell degranulation; modulated expression of pattern recognition receptors, cytokines and transcription factors; and reduced airway remodeling. The treatment induced regulatory T cell responses, as shown by increased Il10 and Foxp3 transcription in lung tissue, and augmented Foxp3 protein expression in blood CD4+CD25+Foxp3+ T cells. This specific combination of beneficial bacteria with non-digestible oligosaccharides has strong anti-inflammatory properties, possibly via the induction of a regulatory T cell response, resulting in reduced airway remodeling and, therefore, may be beneficial in the treatment of chronic inflammation in allergic asthma. Copyright © 2014 Elsevier B.V. All rights reserved.

The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis.

PubMed

Nelson, Michaela; Yang, Ming; Dowle, Adam A; Thomas, Jerry R; Brackenbury, William J

2015-01-27

Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.

Curcumin sensitizes prostate cancer cells to radiation partly via epigenetic activation of miR-143 and miR-143 mediated autophagy inhibition.

PubMed

Liu, Jianbo; Li, Min; Wang, Yuewei; Luo, Jianchao

2017-08-01

Curcumin has been reported as a radiosensitizer in prostate cancer. But the underlying mechanism is not well understood. In this study, we firstly assessed how curcumin affects the expression of miR-143/miR-145 cluster. Then, we investigated whether miR-143 is involved in regulation of radiosensitivity and its association with autophagy in prostate cancer cells. Our data showed that PC3, DU145 and LNCaP cells treated with curcumin had significantly restored miR-143 and miR-145 expression. Curcumin showed similar effect as 5-AZA-dC on reducing methylation of CpG dinucleotides in miR-143 promoter. In addition, curcumin treatment reduced the expression of DNMT1 and DNMT3B, which contribute to promoter hypermethylation of the miR-143/miR-145 cluster. Therefore, we infer that curcumin can restore miR-143 and miR-145 expression via hypomethylation. MiR-143 overexpression and curcumin pretreatment enhanced radiation induced cancer cell growth inhibition and apoptosis. MiR-143 and curcumin remarkably reduced radiation-induced autophagy in PC3 and DU145 cells. MiR-143 overexpression alone also reduced the basal level of autophagy in DU145 cells. Mechanistically, miR-143 can suppress autophagy in prostate cancer cells at least via downregulating ATG2B. Based on these findings, we infer that curcumin sensitizes prostate cancer cells to radiation partly via epigenetic activation of miR-143 and miR-143 mediated autophagy inhibition.

Macrophage heterogeneity and cholesterol homeostasis: classically-activated macrophages are associated with reduced cholesterol accumulation following treatment with oxidized LDL.

PubMed

Chu, Eugene M; Tai, Daven C; Beer, Jennifer L; Hill, John S

2013-02-01

Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression. Copyright © 2012 Elsevier B.V. All rights reserved.

ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells.

PubMed

Lv, Wei; Sui, Linlin; Yan, Xiaona; Xie, Huaying; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Kong, Ying; Cao, Jun

2018-01-05

Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

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

Long, Cong; Wang, Jingchao; Guo, Wei

Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated thatmore » triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.« less

Inhibition of Wnt/beta-catenin signaling downregulates expression of aldehyde dehydrogenase isoform 3A1 (ALDH3A1) to reduce resistance against temozolomide in glioblastoma in vitro.

PubMed

Suwala, Abigail Kora; Koch, Katharina; Rios, Dayana Herrera; Aretz, Philippe; Uhlmann, Constanze; Ogorek, Isabella; Felsberg, Jörg; Reifenberger, Guido; Köhrer, Karl; Deenen, René; Steiger, Hans-Jakob; Kahlert, Ulf D; Maciaczyk, Jaroslaw

2018-04-27

Glioblastoma is the most aggressive type of glioma. The Wingless (Wnt) signaling pathway has been shown to promote stem cell properties and resistance to radio- and chemotherapy in glioblastoma. Here, we demonstrate that pharmacological Wnt pathway inhibition using the porcupine inhibitor LGK974 acts synergistically with temozolomide (TMZ), the chemotherapeutic drug currently used as standard treatment for glioblastoma, to suppress in vitro growth of glioma cells. Synergistic growth inhibition was independent of the O 6 -alkylguanine DNA alkyltransferase ( MGMT ) promoter methylation status. Transcriptomic analysis revealed that expression of aldehyde dehydrogenase 3A1 ( ALDH3A1 ) was significantly down-regulated when cells were treated with LGK974 and TMZ. Suppressing ALDH3A1 expression increased the efficacy of TMZ and reduced clonogenic potential accompanied by decreased expression of stem cell markers CD133, Nestin and Sox2. Taken together, our study suggests that previous observations concerning Wnt signaling blockade to reduce chemoresistance in glioblastoma is at least in part mediated by inhibition of ALDH3A1.

Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

PubMed Central

Tsai, Cheng-Fang; Yeh, Wei-Lan; Chen, Jia-Hong; Lin, Chingju; Huang, Shiang-Suo; Lu, Dah-Yuu

2014-01-01

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells. PMID:24599080

Helping Yourself by Offering Help: Mediators of Expressive Helping in Survivors of Hematopoietic Stem Cell Transplant.

PubMed

Williamson, Timothy J; Stanton, Annette L; Austin, Jane E; Valdimarsdottir, Heiddis B; Wu, Lisa M; Krull, Jennifer L; Rini, Christine M

2017-10-01

A randomized experiment by Rini et al. (Health Psychol. 33(12):1541-1551, 2014) demonstrated that expressive helping, which involves three expressive writing sessions regarding hematopoietic stem cell transplant, followed by one writing session directed toward helping other stem cell transplant recipients, reduced psychological distress and bothersome physical symptoms among stem cell transplant recipients with elevated survivorship problems, relative to a neutral writing control condition. The current study evaluated whether word use reflective of emotional expression, cognitive processing, and change in perspective mediates the effects of expressive helping. The essays of 67 stem cell transplant recipients with high survivorship problems were analyzed with Linguistic Inquiry and Word Count. Multiple mediation modeling was used to test the hypothesized mechanisms of expressive helping on distress and bothersome physical symptoms. Relative to the control condition, expressive helping produced significant reductions in psychological distress and marginal reductions in physical symptom bother in the analyzed subset of participants from the parent study. Results indicated that positive emotion word use significantly mediated effects of expressive helping on reduced distress, but only for participants who used average (compared to above or below average) rates of negative emotion words. Cognitive processing and change in perspective did not significantly mediate benefits of expressive helping. Expressive helping carried its positive effects on distress through participants' higher expression of positive emotions when coupled with moderate rates of negative emotions. Findings highlight the benefit of expressing both positive and negative emotions in stressful situations.

S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

PubMed Central

Khammanivong, Ali; Wang, Chengxing; Sorenson, Brent S.; Ross, Karen F.; Herzberg, Mark C.

2013-01-01

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches. PMID:23874958

Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains.

PubMed

Széliová, Diana; Krahulec, Ján; Šafránek, Martin; Lišková, Veronika; Turňa, Ján

2016-10-20

Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

PubMed

Woo, S M; Min, K-J; Seo, B R; Nam, J-O; Choi, K S; Yoo, Y H; Kwon, T K

2014-11-06

Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7.

PubMed

Sen, Triparna; Moulik, Shuvojit; Dutta, Anindita; Choudhury, Paromita Roy; Banerji, Aniruddha; Das, Shamik; Roy, Madhumita; Chatterjee, Amitava

2009-02-13

The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. MCF-7 was treated with EGCG (20 muM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors alpha5, beta1, alphav and beta3 due to EGCG treatment. Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Retinoic Acid Improves Morphology of Cultured Peritoneal Mesothelial Cells from Patients Undergoing Dialysis

PubMed Central

Retana, Carmen; Sanchez, Elsa I.; Gonzalez, Sirenia; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas-Munoz, Jesus; Alfaro-Cruz, Carmen; Vital-Flores, Socorro; Reyes, José L.

2013-01-01

Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and α-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor β1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor- β1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-β1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-β1expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures. PMID:24223992

Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice.

PubMed

He, Ping; Yang, Jong Won; Yang, Vincent W; Bialkowska, Agnieszka B

2018-04-01

Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. We performed studies in adult mice with conditional disruption of Klf5 (Klf5 fl/fl ) and/or expression of Kras G12D (LSL-Kras G12D ) via Cre ERTM recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of Kras G12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-Kras G12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-Cre ERTM ;LSL-Kras G12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-Cre ERTM ;LSL-Kras G12D ;Klf5 fl/fl mice formed fewer PanINs. After cerulein administration, Ptf1a-Cre ERTM ;LSL-Kras G12D mice formed more extensive ADM than Ptf1a-Cre ERTM ;LSL-Kras G12D ;Klf5 fl/fl mice. Pancreata from Ptf1a-Cre ERTM ;LSL-Kras G12D ;Klf5 fl/fl mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-Cre ERTM ;LSL-Kras G12D mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm 3 vs 121.44 ± 34.90 mm 3 from control cells) in flanks of mice. Levels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-Cre ERTM ;LSL-Kras G12D mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

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

Do, Minh Truong; Kim, Hyung Gyun; Khanal, Tilak

2013-09-01

Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancermore » A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells. The inactivation of AMPK by siRNA, DN-AMPK or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner. These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anticancer therapy in response to metformin treatment. - Highlights: • Metformin inhibits HO-1 expression in cancer cells. • Metformin attenuates Raf-ERK-Nrf2 signaling. • Suppression of HO-1 by metformin is independent of AMPK. • HO-1 inhibition contributes to anti-proliferative effects of metformin.« less

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Do antioxidants inhibit oxidative-stress-induced autophagy of tenofibroblasts?

PubMed

Kim, Ra-Jeong; Hah, Young-Sool; Sung, Chang-Meen; Kang, Jae-Ran; Park, Hyung Bin

2014-07-01

Recent research on tendinopathy has focused on its relationship to programmed cell death. Increased autophagy has been observed in ruptured rotator cuff tendon tissues, suggesting a causal relationship. We investigated whether autophagy occurs in human rotator cuff tenofibroblast death induced by oxidative stress and whether antioxidants protect against autophagic cell death. We used H2 O2 (0.75 mM) as oxidative stressor, cyanidin (100 µg/ml) as antioxidant, zVAD (20 µM) as apoptosis inhibitor, and 3-MA (10 mM) as autophagy inhibitor. We evaluated cell viability and known autophagic markers: LC3-II expression, GFP-LC3 puncta formation, autolysosomes, and Atg5-12 and Beclin 1 expression. H2 O2 exposure increased the rates of cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. After we induced apoptosis arrest using zVAD, H2 O2 exposure still induced cell death, LC3-II expression, and GFP-LC3 puncta formation. H2 O2 exposure also increased Atg5-12 and Beclin 1 expressions, indicating autophagic cell death. However, cyanidin treatment reduced H2 O2 -induced cell death, LC3-II expression, GFP-LC3 puncta formation, and autolysosomes. Cyanidin and 3-MA similarly reduced the cell-death rate, and Atg5-12 and Beclin 1 expression. This study demonstrated that H2 O2 , an oxidative stressor, induces autophagic cell death in rotator cuff tenofibroblasts, and that cyanidin, a natural antioxidant, inhibits autophagic cell death. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Reduction of Syndecan Transcript Levels in the Insulin-Producing Cells Affects Glucose Homeostasis in Adult Drosophila melanogaster.

PubMed

Warren, Jonathan L; Hoxha, Eneida; Jumbo-Lucioni, Patricia; De Luca, Maria

2017-11-01

Signaling by direct cell-matrix interactions has been shown to impact the transcription, secretion, and storage of insulin in mammalian β cells. However, more research is still needed in this area. Syndecans are transmembrane heparan sulfate proteoglycans that function independently and in synergy with integrin-mediated signaling to mediate cell adhesion to the extracellular matrix. In this study, we used the model organism Drosophila melanogaster to determine whether knockdown of the Syndecan (Sdc) gene expression specifically in the insulin-producing cells (IPCs) might affect insulin-like peptide (ILP) production and secretion. IPCs of adult flies produce three ILPs (ILP2, ILP3, and ILP5), which have significant homology to mammalian insulin. We report that flies with reduced Sdc expression in the IPCs did not show any difference in the expression of ilp genes compared to controls. However, they had significantly reduced levels of the circulating ILP2 protein, higher circulating carbohydrates, and were less glucose tolerant than control flies. Finally, we found that IPCs-specific Sdc knockdown led to reduced levels of head Glucose transporter1 gene expression, extracellular signal-regulated kinase phosphorylation, and reactive oxygen species. Taken together, our findings suggest a cell autonomous role for Sdc in insulin release in D. melanogaster.

Silencing of VEGF inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via VEGF/PI3K/AKT signaling pathway

PubMed Central

Peng, Ningning; Gao, Shuming; Guo, Xu; Wang, Guangya; Cheng, Cai; Li, Min; Liu, Kehun

2016-01-01

Background: Osteosarcoma is a kind of highly malignant tumor and the growth and metastasis is closely related to angiogenesis. Vascular endothelial growth factor (VEGF) is an important angiogenesis-promoting factor. In the current study, we investigated the effects of suppressed VEGF on osteosarcoma and its molecular mechanism provided for a basis by targeting angiogenesis. Material/Methods: We established bearing human osteosarcoma Wistar rats model by subcutaneous inoculation of human SaOS-2 cells and the adenovirus vector Ad-VEGF-siRNA was constructed for further study. We assessed the efficiency of VEGF silencing and its influence on SaOS-2 cells. The expression of mRNA and protein were detected by RT-PCR and western blotting, respectively. Intratumoral microvessel density (MVD), VEGF and CD31 were evaluated by immunohistochemistry. We detected the cell apoptotic rates by flow cytometry. Results: Our results indicated that Ad-VEGF-siRNA could effectively suppressed the expression of VEGF expression, inhibited the proliferation capability and promoted apoptosis of SaOS-2 cells in vitro. Silencing of VEGF expression also suppress osteosarcoma tumor growth and reduce osteosarcoma angiogenesis in the Wistar rats model in vivo. Furthermore, We found that phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation were considerably reduced while inhibition VEGF expression in SaOS-2 cells. Conclusion: Our data demonstrated that VEGF silencing could suppress cells proliferation, promote cells apoptosis and reduce osteosarcoma angiogenesis through inactivation of VEGF/PI3K/AKT signaling pathway. PMID:27158386

FLI1 levels impact CXCR3 expression and renal infiltration of T cells and renal glycosphingolipid metabolism in the MRL/lpr lupus mouse strain

PubMed Central

Sundararaj, Kamala P.; Thiyagarajan, Thirumagal; Molano, Ivan; Basher, Fahmin; Powers, Thomas W.; Drake, Richard R.; Nowling, Tamara K.

2015-01-01

The ETS factor FLI1 is a key modulator of lupus disease expression. Over-expressing FLI1 in healthy mice, results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1+/−) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1+/− lupus mice have reduced activation and IL-4 production, Neuraminidase1 (Neu1) expression, and the levels of the glycosphingolipid (GSL) lactosylceramide (LacCer). Here we demonstrate that MRL/lpr Fli1+/− mice have significantly decreased renal Neu1 and LacCer levels. This corresponds with a significant decrease in the number of total CD3+ cells, as well as CD4+ and CD44+CD62L− T cell subsets in the kidney of MRL/lpr Fli1+/− mice compared to the Fli1+/+ nephritic mice. We further demonstrate that the percentage of CXCR3+ T cells and Cxcr3 message levels in T cells are significantly decreased and corresponds with a decrease in renal CXCR3+ cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1+/− compared to the Fli1+/+ nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through down-regulation of CXCR3, reducing renal T cell infiltration and GSL levels. PMID:26538397

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer

PubMed Central

Burgess, Joshua T.; Bolderson, Emma; Saunus, Jodi M.; Zhang, Shu-Dong; Reid, Lynne E.; McNicol, Anne Marie; Lakhani, Sunil R.; Cuff, Katharine; Richard, Kerry; Richard, Derek J.; O'Byrne, Kenneth J.

2016-01-01

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation. PMID:27637080

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer.

PubMed

Burgess, Joshua T; Bolderson, Emma; Saunus, Jodi M; Zhang, Shu-Dong; Reid, Lynne E; McNicol, Anne Marie; Lakhani, Sunil R; Cuff, Katharine; Richard, Kerry; Richard, Derek J; O'Byrne, Kenneth J

2016-11-08

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation.

Prevention of Autoimmune Diabetes and Induction of β-Cell Proliferation in NOD Mice by Hyperbaric Oxygen Therapy

PubMed Central

Faleo, Gaetano; Fotino, Carmen; Bocca, Nicola; Molano, R. Damaris; Zahr-Akrawi, Elsie; Molina, Judith; Villate, Susana; Umland, Oliver; Skyler, Jay S.; Bayer, Allison L.; Ricordi, Camillo; Pileggi, Antonello

2012-01-01

We evaluated the effects of hyperbaric oxygen therapy (HOT) on autoimmune diabetes development in nonobese diabetic (NOD) mice. Animals received no treatment or daily 60-min HOT 100% oxygen (HOT-100%) at 2.0 atmospheres absolute and were monitored for diabetes onset, insulitis, infiltrating cells, immune cell function, and β-cell apoptosis and proliferation. Cyclophosphamide-induced diabetes onset was reduced from 85.3% in controls to 48% after HOT-100% (P < 0.005) and paralleled by lower insulitis. Spontaneous diabetes incidence reduced from 85% in controls to 65% in HOT-100% (P = 0.01). Prediabetic mice receiving HOT-100% showed lower insulitis scores, reduced T-cell proliferation upon stimulation in vitro (P < 0.03), increased CD62L expression in T cells (P < 0.04), reduced costimulation markers (CD40, DC80, and CD86), and reduced major histocompatibility complex class II expression in dendritic cells (DCs) (P < 0.025), compared with controls. After autoimmunity was established, HOT was less effective. HOT-100% yielded reduced apoptosis (transferase-mediated dUTP nick-end labeling-positive insulin-positive cells; P < 0.01) and increased proliferation (bromodeoxyuridine incorporation; P < 0.001) of insulin-positive cells compared with controls. HOT reduces autoimmune diabetes incidence in NOD mice via increased resting T cells and reduced activation of DCs with preservation of β-cell mass resulting from decreased apoptosis and increased proliferation. The safety profile and noninvasiveness makes HOT an appealing adjuvant therapy for diabetes prevention and intervention trials. PMID:22566533

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

Yamamoto, Tetsuya; Digumarthi, Hari; Aranbayeva, Zina

The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signalingmore » pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies.« less

Downregulated TIPE2 is associated with poor prognosis and promotes cell proliferation in non-small cell lung cancer

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

Li, Yuexia; Li, Xiaohui; Liu, Gang

2015-01-30

Highlights: • TIPE2 is down-regulated in NSCLC tissues. • TIPE2 inhibits NSCLC cell proliferation, colony formation and invasion. • TIPE2 reduces the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. - Abstract: The present study aims to investigate the expression pattern of TIPE2 protein and its clinical significance in human non-small cell lung cancer (NSCLC). We investigated the expression levels of TIPE2 in 96 NSCLC tumor samples by immunohistochemistry and then analyzed its clinical significance. Furthermore, the role of TIPE2 on the biological properties of the NSCLC cell line H1299 and A549 was experimentally tested in vitro and in vivo.more » We found that the expression level of TIPE2 was significantly higher in normal lung tissues compared with NSCLC tissues (P < 0.001), and TIPE2 downregulation was significantly correlated with advanced TNM stage (P = 0.006). TIPE2 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TIPE2 plasmid was performed in H1299 and A549 cells. TIPE2 overexpression inhibited lung cancer cell proliferation, colony formation and cell invasive in vitro, and prevented lung tumor growth in vivo. In addition, TIPE2 transfection reduced the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. Taken together, our results demonstrate that TIPE2 might serve as a tumor suppressor in NSCLC progression.« less

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1

PubMed Central

YANG, LIANG; LIU, REN; MA, HONG-BIN; YING, MING-ZHEN; WANG, YA-JIE

2015-01-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest. PMID:26622693

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1.

PubMed

Yang, Liang; Liu, Ren; Ma, Hong-Bin; Ying, Ming-Zhen; Wang, Ya-Jie

2015-09-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 ( GSTP1 ) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G 2 /M phase arrest in the GSTP1 -expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1 -expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G 2 /M phase arrest.

COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells

PubMed Central

Prima, Victor; Kaliberova, Lyudmila N.; Kaliberov, Sergey; Curiel, David T.; Kusmartsev, Sergei

2017-01-01

In recent years, it has been established that programmed cell death protein ligand 1 (PD-L1)–mediated inhibition of activated PD-1+ T lymphocytes plays a major role in tumor escape from immune system during cancer progression. Lately, the anti–PD-L1 and –PD-1 immune therapies have become an important tool for treatment of advanced human cancers, including bladder cancer. However, the underlying mechanisms of PD-L1 expression in cancer are not fully understood. We found that coculture of murine bone marrow cells with bladder tumor cells promoted strong expression of PD-L1 in bone marrow–derived myeloid cells. Tumor-induced expression of PD-L1 was limited to F4/80+ macrophages and Ly-6C+ myeloid-derived suppressor cells. These PD-L1–expressing cells were immunosuppressive and were capable of eliminating CD8 T cells in vitro. Tumor-infiltrating PD-L1+ cells isolated from tumor-bearing mice also exerted morphology of tumor-associated macrophages and expressed high levels of prostaglandin E2 (PGE2)-forming enzymes microsomal PGE2 synthase 1 (mPGES1) and COX2. Inhibition of PGE2 formation, using pharmacologic mPGES1 and COX2 inhibitors or genetic overexpression of PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), resulted in reduced PD-L1 expression. Together, our study demonstrates that the COX2/mPGES1/PGE2 pathway involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells and, therefore, reprogramming of PGE2 metabolism in tumor microenvironment provides an opportunity to reduce immune suppression in tumor host. PMID:28096371

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

PubMed

Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

2014-06-01

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors. Copyright © 2014 the American Physiological Society.

Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

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

Lee, Yea-Jin; Kim, Sung-Jo, E-mail: sungjo@hoseo.edu; Heo, Tae-Hwe, E-mail: thhur92@catholic.ac.kr

Highlights: {yields} Catechin reduces the expression level of ER stress marker protein in type I Gaucher disease cells. {yields} Catechin induces the proliferation rate of GD cells similar levels to normal cells. {yields} Catechin improves wound healing activity. {yields} Catechin-mediated reductions in ER stress may be associated with enhanced cell survival. {yields} We identified catechin as a protective agent against ER stress in GD cells. -- Abstract: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset inmore » adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.« less

Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

PubMed Central

Latif, Najma; Quillon, Alfred; Sarathchandra, Padmini; McCormack, Ann; Lozanoski, Alec; Yacoub, Magdi H.; Chester, Adrian H.

2015-01-01

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering. PMID:26042674

COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells

PubMed Central

Litchfield, Lacey M.; Appana, Savitri N.; Datta, Susmita; Klinge, Carolyn M.

2016-01-01

Reduced COUP-TFII expression contributes to endocrine resistance in breast cancer cells. Endocrine-resistant breast cancer cells have higher NFkappa B (NFκB) activity and target gene expression. The goal of this study was to determine if COUP-TFII modulates NFκB activity. Endocrine-resistant LCC9 cells with low endogenous COUP-TFII displayed ~5-fold higher basal NFκB activity than parental endocrine-sensitive MCF-7 breast cancer cells. Transient transfection of LCC9 cells with COUP-TFII inhibited NFκB activation and reduced NFκB target gene expression. COUP-TFII and NFκB were inversely correlated in breast cancer patient samples. Endogenous COUP-TFII coimmunoprecipitated with NFκB subunits RelB and NFκB1 in MCF-7 cells. COUP-TFII inhibited NFκB-DNA binding in vitro and impaired coactivator induced NFκB transactivation. LCC9 cells were growth-inhibited by an NFκB inhibitor and 4-hydroxytamoxifen compared to MCF-7 cells. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity and explain, in part, why decreased COUP-TFII expression results in an endocrine-resistant phenotype. PMID:24141032

A novel phenoxazine derivative suppresses surface IgM expression in DT40 B cell line

PubMed Central

Gao, Sanyang; Takano, Tomoko; Sada, Kiyonao; He, Jinsong; Noda, Chiseko; Hori-Tamura, Naoko; Tomoda, Akio; Yamamura, Hirohei

2002-01-01

2-amino-4, 4α-dihydro-4α, 7-dimethyl-3H-phenoxazine-3-one (Phx) has been demonstrated to be an actinomycin D-like phenoxazine, and to display anti-tumour activity. In this study, we report on the effect of Phx on B cell antigen receptor (BCR) and receptor-mediated signalling in DT40 B cells. Treatment of B cells with Phx for 12 h inhibited BCR-stimulated tyrosine phosphorylation of cellular proteins. B cells exposed to Phx exhibited down-regulation of surface IgM which is part of BCR. In contracts with actinomycin D, Phx rapidly reduced the expression of IgM without decreasing the expression of other signalling molecules. Analysis with confocal microscopy demonstrated that Phx treatment reduced IgM expression both at the cell surface and inside the cell. Treatment of B cells with Phx resulted in the reduction of IgM secretion. Since MG-132, a proteasomal inhibitor, restored IgM contents to the control levels, Phx has the specific effect of accelerating IgM degradation. These results suggest that Phx down-regulates the expression of IgM and inhibits BCR-mediated signalling and IgM secretion. Phx may be useful as an immunosuppressive agent for therapeutic purposes. PMID:12411404

Hedgehog Signaling Regulates the Survival of Gastric Cancer Cells by Regulating the Expression of Bcl-2

PubMed Central

Han, Myoung-Eun; Lee, Young-Suk; Baek, Sun-Yong; Kim, Bong-Seon; Kim, Jae-Bong; Oh, Sae-Ock

2009-01-01

Gastric cancer is the second most common cause of cancer deaths worldwide. The underlying molecular mechanisms of its carcinogenesis are relatively poorly characterized. Hedgehog (Hh) signaling, which is critical for development of various organs including the gastrointestinal tract, has been associated with gastric cancer. The present study was undertaken to reveal the underlying mechanism by which Hh signaling controls gastric cancer cell proliferation. Treatment of gastric cancer cells with cyclopamine, a specific inhibitor of Hh signaling pathway, reduced proliferation and induced apoptosis of gastric cancer cells. Cyclopamine treatment induced cytochrome c release from mitochondria and cleavage of caspase 9. Moreover, Bcl-2 expression was significantly reduced by cyclopamine treatment. These results suggest that Hh signaling regulates the survival of gastric cancer cells by regulating the expression of Bcl-2. PMID:19742123

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.

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

PubMed Central

Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

2016-01-01

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression. PMID:27043434

Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

PubMed Central

Hakkaart, Gerrit A J; Dassa, Emmanuel P; Jacobs, Howard T; Rustin, Pierre

2006-01-01

Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals. PMID:16322757

MiR-29b affects the secretion of PROG and promotes the proliferation of bovine corpus luteum cells

PubMed Central

Zhang, Li-Qun; Sun, Xu-Lei; Luo, Dan; Fu, Yao; Gao, Yan; Zhang, Jia-Bao

2018-01-01

The regulatory role of miRNAs has been explored in ovarian cells, and their effects on gonadal development, apoptosis, ovulation, steroid production and corpus luteum (CL) development have been revealed. In this study, we analyzed the expression of miR-29b at different stages of bovine CL development and predicted the target genes of miR-29b. We confirmed that miR-29b reduces the expression of the oxytocin receptor (OXTR), affects progesterone (PROG) secretion and regulates the function of the CL. RT-PCR showed that the expression of miR-29b was significantly higher in functional CL phases than in the regressed CL phase. Immunohistochemistry showed that OXTR was expressed in both large and small CL cells and was mainly located in the cell membrane and cytoplasm of these cells. We analyzed the expression levels of OXTR and found that transfection with a miR-29b mimic decreased OXTR expression, but transfection with the inhibitor had a limited effect on the expression of the OXTR protein. At the same time, the secretion of PROG was significantly increased in the miR-29b mimic-transfected group. We also analyzed the effect of miR-29b on the apoptosis of CL cells. Finally, we found that miR-29b could promote the proliferation of bovine CL cells. In conclusion, we found that miR-29b reduces the expression of OXTR and can promote PROG secretion and the proliferation of CL cells via OXTR. PMID:29617446

EMMPRIN regulates tumor growth and metastasis by recruiting bone marrow-derived cells through paracrine signaling of SDF-1 and VEGF

PubMed Central

Chen, Yanke; Gou, Xingchun; Kong, Derek Kai; Wang, Xiaofei; Wang, Jianhui; Chen, Zeming; Huang, Chen; Zhou, Jiangbing

2015-01-01

EMMPRIN, a cell adhesion molecule highly expressed in a variety of tumors, is associated with poor prognosis in cancer patients. Mechanistically, EMMPRIN has been characterized to contribute to tumor development and progression by controlling the expression of MMPs and VEGF. In the present study, by using fluorescently labeled bone marrow-derived cells (BMDCs), we found that the down-regulation of EMMPRIN expression in cancer cells reduces tumor growth and metastasis, and is associated with the reduced recruitment of BMDCs. Further protein profiling studies suggest that EMMPRIN controls BMDC recruitment through regulating the secretion of soluble factors, notably, VEGF and SDF-1. We demonstrate that the expression and secretion of SDF-1 in tumor cells are regulated by EMMPRIN. This study reveals a novel mechanism by which EMMPRIN promotes tumor growth and metastasis by recruitment of BMDCs through controlling secretion and paracrine signaling of SDF-1 and VEGF. PMID:26416452

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Systematic Analysis of the Functional Relevance of Nuclear Structure and Mechanics in Breast Cancer Progression

DTIC Science & Technology

2013-07-01

epithelial cells; MDA-MB-231 metastatic breast cancer cells) with systematic alterations in the expression of lamins A, B1, B2, C, and lamin B receptor...LBR). We then evaluated the effect of altered lamin expression on nuclear stiffness in these cell lines. While increased expression of lamin A...caused stiffer, less deformable nuclei, reduction of lamins A/C expression by shRNA reduced nuclear stiffness. The effect of alterations in other lamins

MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4

PubMed Central

Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Camacho, Carolina Espindola; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A. L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.; Dubinett, Steven M.; Critchlow, Susan E.; Kurdistani, Siavash K.; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G.; Christofk, Heather R.

2016-01-01

SUMMARY Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors further supporting their use as anti-cancer therapeutics. PMID:26876179

MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4.

PubMed

Hong, Candice Sun; Graham, Nicholas A; Gu, Wen; Espindola Camacho, Carolina; Mah, Vei; Maresh, Erin L; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A L; Gardner, Brian K; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K; Hurvitz, Sara A; Dubinett, Steven M; Critchlow, Susan E; Kurdistani, Siavash K; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G; Christofk, Heather R

2016-02-23

Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

PubMed Central

Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

2009-01-01

High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

Elevation of SHARPIN Protein Levels in Prostate Adenocarcinomas Promotes Metastasis and Impairs Patient Survivals.

PubMed

Huang, Hai; Du, Tao; Zhang, Yiming; Lai, Yiming; Li, Kaiwen; Fan, Xinxing; Zhu, Dingjun; Lin, Tianxin; Xu, Kewei; Huang, Jian; Liu, Leyuan; Guo, Zhenghui

2017-05-01

SHARPIN, SHANK-associated RH domain interacting protein, associates with a linear ubiquitin chain assembly complex (LUBAC) to regulate inflammation and immunity. It has been reported that SHARPIN is highly expressed in several human tumors including ovarian cancer and liver cancer. We found that SHARPIN is also highly expressed in prostate cancer cell lines of DU145, LNCAP, and PC-3. Suppression of SHARPIN caused an inhibition of NF-κB signal and decreases in tumorigenesis of cultured cells in NOD/SCID mouse model. Overexpression of SHARPIN in prostate cancer cells promoted cell growth and reduced apoptosis through NF-kB/ERK/Akt pathway and apoptosis-associated proteins. We analyzed the expression of SHARPIN in prostate cancer tissues from 95 patients and its relationship with other clinical characteristics associated with PCA malignancies and patient survivals, and examined the impacts of SHARPIN suppression with siRNA on proliferation, angiogenesis, invasion, and expression levels of MMP-9 of prostate cancer cells and metastasis to lung by these cells in nude mice. High levels of SHARPIN were associated with high malignancies of PCA and predicted shorter survivals of PCA patients. Suppression of SHARPIN impaired cell proliferation, angiogenesis, and invasion and reduced levels of MMP-9 in prostate cancer cells and reduced the size of metastatic lung tumors induced by these cells in mice. SHARPIN enhances the metastasis of prostate cancer and impair patient survivals. Prostate 77:718-728, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-01-01

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation. PMID:20133835

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-02-02

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.

A perisinusoidal niche for extramedullary haematopoiesis in the spleen.

PubMed

Inra, Christopher N; Zhou, Bo O; Acar, Melih; Murphy, Malea M; Richardson, James; Zhao, Zhiyu; Morrison, Sean J

2015-11-26

Haematopoietic stresses mobilize haematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary haematopoiesis (EMH). However, the cellular nature of the EMH niche is unknown. Here we assessed the sources of the key niche factors, SCF (also known as KITL) and CXCL12, in the mouse spleen after EMH induction by myeloablation, blood loss, or pregnancy. In each case, Scf was expressed by endothelial cells and Tcf21(+) stromal cells, primarily around sinusoids in the red pulp, while Cxcl12 was expressed by a subset of Tcf21(+) stromal cells. EMH induction markedly expanded the Scf-expressing endothelial cells and stromal cells by inducing proliferation. Most splenic HSCs were adjacent to Tcf21(+) stromal cells in red pulp. Conditional deletion of Scf from spleen endothelial cells, or of Scf or Cxcl12 from Tcf21+ stromal cells, severely reduced spleen EMH and reduced blood cell counts without affecting bone marrow haematopoiesis. Endothelial cells and Tcf21(+) stromal cells thus create a perisinusoidal EMH niche in the spleen, which is necessary for the physiological response to diverse haematopoietic stresses.

Pyruvate dehydrogenase expression is negatively associated with cell stemness and worse clinical outcome in prostate cancers

PubMed Central

Zhong, Yali; Li, Xiaoli; Ji, Yasai; Li, Xiaoran; Li, Yaqing; Yu, Dandan; Yuan, Yuan; Liu, Jian; Li, Huixiang; Zhang, Mingzhi; Ji, Zhenyu; Fan, Dandan; Wen, Jianguo; Goscinski, Mariusz Adam; Yuan, Long; Hao, Bin; Nesland, Jahn M; Suo, Zhenhe

2017-01-01

Cells generate adenosine-5′-triphosphate (ATP), the major currency for energy-consuming reactions, through mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. One of the remarkable features of cancer cells is aerobic glycolysis, also known as the “Warburg Effect”, in which cancer cells rely preferentially on glycolysis instead of mitochondrial OXPHOS as the main energy source even in the presence of high oxygen tension. One of the main players in controlling OXPHOS is the mitochondrial gatekeeperpyruvate dehydrogenase complex (PDHc) and its major subunit is E1α (PDHA1). To further analyze the function of PDHA1 in cancer cells, it was knock out (KO) in the human prostate cancer cell line LnCap and a stable KO cell line was established. We demonstrated that PDHA1 gene KO significantly decreased mitochondrial OXPHOS and promoted anaerobic glycolysis, accompanied with higher stemness phenotype including resistance to chemotherapy, enhanced migration ability and increased expression of cancer stem cell markers. We also examined PDHA1 protein expression in prostate cancer tissues by immunohistochemistry and observed that reduced PDHA1 protein expression in clinical prostate carcinomas was significantly correlated with poor prognosis. Collectively, our results show that negative PDHA1 gene expressionis associated with significantly higher cell stemness in prostate cancer cells and reduced protein expression of this gene is associated with shorter clinical outcome in prostate cancers. PMID:28076853

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

PubMed

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-12-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

PubMed

Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

2011-01-01

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

PubMed Central

Sotherton, A. G.; Peri, L. E.; Sanders, K. M.; Ward, S. M.; Keef, K. D.

2014-01-01

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα+ cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrαegfp/+, KitcopGFP/+, and smMHCCre-egfp mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα+ cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα+ cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα+ cells. The functional role of cGKI was investigated in cGKI−/− mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI−/− mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI+/+ and cGKI−/− mice although there was a small reduction in the cGKI−/− mouse. Nω-nitro-l-arginine (l-NNA) abolished responses during the first 20–30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI−/− mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway. PMID:25301187

Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

PubMed

Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

2010-10-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Decreased eIF3e/Int6 expression causes epithelial-to-mesenchymal transition in breast epithelial cells.

PubMed

Gillis, L D; Lewis, S M

2013-08-01

eIF3e/Int6 is a component of the multi-subunit eIF3 complex, which binds directly to the 40S ribosome to facilitate ribosome recruitment to mRNA and hence protein synthesis. Reduced expression of eIF3e/Int6 has been found in up to 37% of human breast cancers, and expression of a truncated mutant version of the mouse eIF3e/Int6 protein leads to malignant transformation of normal mammary cells. These findings suggest that eIF3e/Int6 is a tumor suppressor; however, a recent study has reported that a reduction of eIF3e/Int6 expression in breast cancer cells leads to reduced translation of oncogenes, suggesting that eIF3e/Int6 may in fact have an oncogenic role in breast cancer. To gain a better understanding of the role of eIF3e/Int6 in breast cancer, we have examined the effects of decreased eIF3e/Int6 expression in an immortalized breast epithelial cell line, MCF-10A. Surprisingly, we find that decreased expression of eIF3e/Int6 causes breast epithelial cells to undergo epithelial-to-mesenchymal transition (EMT). We show that EMT induced by a decrease in eIF3e/Int6 expression imparts invasive and migratory properties to breast epithelial cells, suggesting that regulation of EMT by eIF3e/Int6 may have an important role in breast cancer metastasis. Furthermore, we show that reduced eIF3e/Int6 expression in breast epithelial cells causes a specific increase in the expression of the key EMT regulators Snail1 and Zeb2, which occurs at both the transcriptional and post-transcriptional levels. Together, our data indicate a novel role of eIF3e/Int6 in the regulation of EMT in breast epithelial cells and support a tumor suppressor role of eIF3e/Int6.

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP.

PubMed

Waheed, Sana; Dorjbal, Batsukh; Hamilton, Chad A; Maxwell, G Larry; Rodriguez, Gustavo C; Syed, Viqar

2017-12-26

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP

PubMed Central

Waheed, Sana; Dorjbal, Batsukh; Hamilton, Chad A.; Maxwell, G. Larry; Rodriguez, Gustavo C.; Syed, Viqar

2017-01-01

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients. PMID:29371931

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

PubMed

Camp, Esther; Anderson, Peter J; Zannettino, Andrew C W; Glackin, Carlotta A; Gronthos, Stan

2018-09-01

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1 del/+ mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1 del/+ mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function. © 2018 Wiley Periodicals, Inc.

Pleiotropic effect of sigE over-expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803.

PubMed

Osanai, Takashi; Kuwahara, Ayuko; Iijima, Hiroko; Toyooka, Kiminori; Sato, Mayuko; Tanaka, Kan; Ikeuchi, Masahiko; Saito, Kazuki; Hirai, Masami Yokota

2013-11-01

Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells.

PubMed

Liu, Shikai; Song, Lili; Zeng, Saitian; Zhang, Liang

2016-01-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1) is a large, infrequently spliced non-coding RNA aberrantly expressed in cervical cancer. But the molecular mechanisms of its oncogenic role are still not quite clear. The present study explored whether there is a competing endogenous RNAs (ceRNAs) mechanism involved in the oncogenic effect of MALAT1. MALAT1 expression was firstly verified in high-risk human papillomavirus (HR-HPV)-positive tumor tissues and cell lines. Its regulation over miR-124 and the downstream target of miR-124 in regulation of growth, invasion, and apoptosis of the cancer cells are also studied. Findings of this study confirmed higher MALAT1 expression in HR-HPV (+) cervical cancer. Knockdown of endogenous MALAT1 significantly reduced cell growth rate and invasion and increased cell apoptosis of Hela and siHa cells. Besides, knockdown of MALAT1 increased the expression of miRNA-124, while ectopic expression of miR-124 decreased MALAT1 expression. In addition, we also verified a direct interaction between miR-124 and 3'UTR of GRB2. MALAT1 can indirectly modulate GRB2 expression via competing miR-124. Knockdown of GRB2 reduced cell invasion and increased cell apoptosis. In conclusion, MALAT1 can promote HR-HPV (+) cancer cell growth and invasion at least partially through the MALAT1-miR-124-RBG2 axis. This finding might provide some useful evidence about the lncRNA interaction regulatory network in tumorigenesis cervical cancer.

Anorexia Reduces GFAP+ Cell Density in the Rat Hippocampus.

PubMed

Reyes-Haro, Daniel; Labrada-Moncada, Francisco Emmanuel; Varman, Durairaj Ragu; Krüger, Janina; Morales, Teresa; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2016-01-01

Anorexia nervosa is an eating disorder observed primarily in young women. The neurobiology of the disorder is unknown but recently magnetic resonance imaging showed a volume reduction of the hippocampus in anorexic patients. Dehydration-induced anorexia (DIA) is a murine model that mimics core features of this disorder, including severe weight loss due to voluntary reduction in food intake. The energy supply to the brain is mediated by astrocytes, but whether their density is compromised by anorexia is unknown. Thus, the aim of this study was to estimate GFAP+ cell density in the main regions of the hippocampus (CA1, CA2, CA3, and dentate gyrus) in the DIA model. Our results showed that GFAP+ cell density was significantly reduced (~20%) in all regions of the hippocampus, except in CA1. Interestingly, DIA significantly reduced the GFAP+ cells/nuclei ratio in CA2 (-23%) and dentate gyrus (-48%). The reduction of GFAP+ cell density was in agreement with a lower expression of GFAP protein. Additionally, anorexia increased the expression of the intermediate filaments vimentin and nestin. Accordingly, anorexia increased the number of reactive astrocytes in CA2 and dentate gyrus more than twofold. We conclude that anorexia reduces the hippocampal GFAP+ cell density and increases vimentin and nestin expression.

Anorexia Reduces GFAP+ Cell Density in the Rat Hippocampus

PubMed Central

Labrada-Moncada, Francisco Emmanuel; Varman, Durairaj Ragu; Krüger, Janina; Morales, Teresa; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2016-01-01

Anorexia nervosa is an eating disorder observed primarily in young women. The neurobiology of the disorder is unknown but recently magnetic resonance imaging showed a volume reduction of the hippocampus in anorexic patients. Dehydration-induced anorexia (DIA) is a murine model that mimics core features of this disorder, including severe weight loss due to voluntary reduction in food intake. The energy supply to the brain is mediated by astrocytes, but whether their density is compromised by anorexia is unknown. Thus, the aim of this study was to estimate GFAP+ cell density in the main regions of the hippocampus (CA1, CA2, CA3, and dentate gyrus) in the DIA model. Our results showed that GFAP+ cell density was significantly reduced (~20%) in all regions of the hippocampus, except in CA1. Interestingly, DIA significantly reduced the GFAP+ cells/nuclei ratio in CA2 (−23%) and dentate gyrus (−48%). The reduction of GFAP+ cell density was in agreement with a lower expression of GFAP protein. Additionally, anorexia increased the expression of the intermediate filaments vimentin and nestin. Accordingly, anorexia increased the number of reactive astrocytes in CA2 and dentate gyrus more than twofold. We conclude that anorexia reduces the hippocampal GFAP+ cell density and increases vimentin and nestin expression. PMID:27579183

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

The expression of sphingosine-1 phosphate receptor-1 in chronic lymphocytic leukemia cells is impaired by tumor microenvironmental signals and enhanced by piceatannol and R406.

PubMed

Borge, Mercedes; Remes Lenicov, Federico; Nannini, Paula R; de los Ríos Alicandú, María M; Podaza, Enrique; Ceballos, Ana; Fernández Grecco, Horacio; Cabrejo, María; Bezares, Raimundo F; Morande, Pablo E; Oppezzo, Pablo; Giordano, Mirta; Gamberale, Romina

2014-09-15

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues. Copyright © 2014 by The American Association of Immunologists, Inc.

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

Ramos-Solano, Moisés, E-mail: mrsolano84@gmail.com; Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud; Meza-Canales, Ivan D., E-mail: imezacanales@ice.mpg.de

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviralmore » gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.« less

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

PubMed

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-09-09

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

PubMed

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-10-17

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells

PubMed Central

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-01-01

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. PMID:29156827

Characterization of GAB1 Expression Over the Menstrual Cycle in Women With and Without Polycystic Ovarian Syndrome Provides a New Insight Into Its Pathophysiology

PubMed Central

Roemer, K. L.; Young, S. L.

2014-01-01

Context: In a previous microarray analysis, GRB2-associated binding protein 1 (GAB1), a docking protein closely related to the insulin receptor substrate, was down-regulated in endometrium of women with polycystic ovary syndrome (PCOS). Objective: The objective of the study was to characterize the cyclic expression of endometrial GAB1 in vivo in normal women and those with PCOS as well as investigate the possible mechanisms of endometrial regulation of GAB1 expression and action in vitro. Design: This was an experimental and case-control study. Setting: The study was conducted at a tertiary university hospital. Patients: Normal proven fertile women (controls; n = 31) and women with PCOS (cases; n = 26) participated in the study. Interventions: Interventions included timed endometrial biopsies at different phases of the menstrual cycle. Ishikawa cells were cultured with β-estradiol (E2), medroxyprogesterone acetate, and E2 + medroxyprogesterone acetate. Transfection of small interfering RNA for GAB1 in Ishikawa cells incubated with or without insulin. Main Outcome Measures: GAB1 mRNA expression in Ishikawa cells and in endometrium of cases and controls was measured. Protein expression of phosphorylated MAPK by Western blot was also measured. Immunohistochemical localization and expression of phosphorylated GAB1 in endometrium was also measured, using a digital histological score. Results: In endometrial tissue, GAB1 mRNA was reduced in the proliferative phase of PCOS women, compared with controls (P = .003; ANOVA). When all the phases of the menstrual cycle were grouped, GAB1 protein expression was reduced in endometrium of PCOS women (P < .0001; Student t test). E2 increases GAB1 mRNA expression in Ishikawa cells (P = .001; ANOVA). Phosphorylated MAPK is reduced in cells transfected with small interfering RNA for GAB1 (P = .008; ANOVA) and incubated with insulin. Conclusions: GAB1 mRNA expression is positively modulated by E2. Endometrial GAB1 protein and mRNA expression are reduced in women with PCOS, suggesting that the endometrium of PCOS women have a defect in insulin signaling due to GAB1 down-regulation. PMID:25144631

Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

PubMed Central

Miccichè, Francesca; Da Riva, Luca; Fabbi, Marina; Pilotti, Silvana; Mondellini, Piera; Ferrini, Silvano; Canevari, Silvana; Pierotti, Marco A.; Bongarzone, Italia

2011-01-01

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology. PMID:21364949

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Protein Kinase C- ɛ Regulates the Apoptosis and Survival of Glioma Cells

PubMed Central

Okhrimenko, Hana; Lu, Wei; Xiang, Cunli; Hamburger, Nathan; Kazimirsky, Gila; Brodie, Chaya

2005-01-01

In this study, we examined the role of protein kinase C (PKC)-ɛ in the apoptosis and survival of glioma cells using tumor necrosis factor–related apoptosis inducing ligand (TRAIL)- stimulated cells and silencing of PKCɛ expression. Treatment of glioma cells with TRAIL induced activation, caspase-dependent cleavage, and down-regulation of PKCɛ within 3 to 5 hours of treatment. Overexpression of PKCɛ inhibited the apoptosis induced by TRAIL, acting downstream of caspase 8 and upstream of Bid cleavage and cytochrome c release from the mitochondria. A caspase-resistant PKCɛ mutant (D383A) was more protective than PKCɛ, suggesting that both the cleavage of PKCɛ and its down-regulation contributed to the apoptotic effect of TRAIL. To further study the role of PKCɛ in glioma cell apoptosis, we employed short interfering RNAs directed against the mRNA of PKCɛ and found that silencing of PKCɛ expression induced apoptosis of various glioma cell lines and primary glioma cultures. To delineate the molecular mechanisms involved in the apoptosis induced by silencing of PKCɛ, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that knockdown of PKCɛ did not affect the expression of Bcl2 and Bax or the phosphorylation and expression of Erk1/2, c-Jun-NH2-kinase, p38, or STAT, whereas it selectively reduced the expression of AKT. Similarly, TRAIL reduced the expression of AKT in glioma cells and this decrease was abolished in cells overexpressing PKCɛ. Our results suggest that the cleavage of PKCɛ and its down-regulation play important roles in the apoptotic effect of TRAIL. Moreover, PKCɛ regulates AKT expression and is essential for the survival of glioma cells. PMID:16103081

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

PubMed Central

2014-01-01

Background The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. Results Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5’-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. Conclusions We have identified and characterized a functional Pax8 binding site in the 5’-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype. PMID:25270402

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance

PubMed Central

Hjort, Magnus A.; Abdollahi, Pegah; Vandsemb, Esten N.; Fenstad, Mona H.; Lund, Bendik; Slørdahl, Tobias S.; Børset, Magne; Rø, Torstein B.

2018-01-01

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL. PMID:29423065

Differences in antiproliferative effect of STAT3 inhibition in HCC cells with versus without HBV expression

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

Hong, Yun; Zhou, Lin; Xie, Haiyang

2015-06-05

Chronic infection with hepatitis B virus (HBV) plays an important role in the etiology of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3) inactivation could inhibit the tumor growth of HCC. In this study, differential antiproliferative effect of STAT3 inhibition was observed with HBV-related HCC cells being more resistant than non-HBV-related HCC cells. Resistance of HBV-related HCC cells to STAT3 inhibition was positively correlated to the expression of HBV. Enhanced ERK activation after STAT3 blockade was detected in HBV-related HCC cells but not in non-HBV-related HCC cells. Combined ERK and STAT3 inhibition eliminates the discrepancy between themore » two types of HCC cells. Moderate reduced HBV expression was found after STAT3 inhibition. These findings disclose a discrepancy in cellular response to STAT3 inhibition between non-HBV-related and HBV-related HCC cells and underscore the complexity of antiproliferative effect of STAT3 inactivation in HBV-related HCC cells. - Highlights: • HBV endows HCC cells with resistance to STAT3 inactivation on proliferation. • Abnormal ERK activation after STAT3 inhibition in HBV-related HCC cells. • Combined ERK and STAT3 inhibition eliminates the discrepancy. • STAT3 inhibition moderately reduces HBV expression.« less

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

PubMed

Yang, Jie; Wang, Ya; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

2017-05-01

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and in vivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).

PubMed

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Lokensgard, James R

2018-06-01

Previous work from our laboratory has demonstrated in vivo persistence of CD103 + CD69 + brain resident memory CD8 + T-cells (bT RM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T RM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 + T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT RM . In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8 + T-cells promote development of CD103 + CD69 + CD8 + T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8 + T-cells obtained from either WT or PD-1 KO mice. We found that αCD3 Ab-stimulated CD8 + T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8 + T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69 + CD8 + T-cells, which promotes development of T RM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69 + CD8 + cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69 + CD8 + T-cells. Taken together, these results demonstrate a role for activated glia in promoting development of bT RM through the PD-1: PD-L1 pathway. © 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines

PubMed Central

Miura, Tanya A.; Wang, Jieru; Holmes, Kathryn V.; Mason, Robert J.

2007-01-01

We analyzed the ability of two rat coronavirus (RCoV) strains, sialodacryoadenitis virus (SDAV) and Parker’s RCoV (RCoV-P), to infect rat alveolar type I cells and induce chemokine expression. Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCoV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemoattractants. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation. PMID:17804032

Caveolin-1–mediated Suppression of Cyclooxygenase-2 via a β-catenin-Tcf/Lef–dependent Transcriptional Mechanism Reduced Prostaglandin E2 Production and Survivin Expression

PubMed Central

Rodriguez, Diego A.; Tapia, Julio C.; Fernandez, Jaime G.; Torres, Vicente A.; Muñoz, Nicolas; Galleguillos, Daniela; Leyton, Lisette

2009-01-01

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E2 (PGE2) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and β-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE2 and cell proliferation. Moreover, COX-2 overexpression or PGE2 supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE2 to the medium prevented effects attributed to caveolin-1–mediated inhibition of β-catenin-Tcf/Lef–dependent transcription. Finally, PGE2 reduced the coimmunoprecipitation of caveolin-1 with β-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE2-induced signaling events linked to β-catenin/Tcf/Lef–dependent transcription of tumor survival genes including cox-2 itself and survivin. PMID:19244345

BCOR regulates myeloid cell proliferation and differentiation

PubMed Central

Cao, Qi; Gearhart, Micah D.; Gery, Sigal; Shojaee, Seyedmehdi; Yang, Henry; Sun, Haibo; Lin, De-chen; Bai, Jing-wen; Mead, Monica; Zhao, Zhiqiang; Chen, Qi; Chien, Wen-wen; Alkan, Serhan; Alpermann, Tamara; Haferlach, Torsten; Müschen, Markus; Bardwell, Vivian J.; Koeffler, H. Phillip

2016-01-01

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukaemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes. PMID:26847029

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

PubMed

Doñate, Carmen; Ody, Christiane; McKee, Thomas; Ruault-Jungblut, Sylvie; Fischer, Nicolas; Ropraz, Patricia; Imhof, Beat A; Matthes, Thomas

2013-01-15

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

PubMed

Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa; Reynolds, Nicole; Fannous, Sanya; Kitagawa, Kouki; Marchand, James E

2006-04-01

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Interactions between insulin-like growth factor-I, estrogen receptor-α (ERα) and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

PubMed Central

Mendoza, Rhone A.; Enriquez, Marlene I; Mejia, Sylvia M; Moody, Emily E; Thordarson, Gudmundur

2011-01-01

Understanding of the interactions between estradiol (E2) and insulin-like growth factor-I (IGF-I) is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating non-interfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human growth hormone plus epidermal growth factor, but E2 did not cause increase in the number of the IGF-IR.low cells compared to controls. Proliferation rate of IGF-IR.low cells was only reduced in response to E2 compared to controls, whereas their basal and hormone stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E2, without affecting control cells. Further, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. Summary, suppressing the IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate. PMID:20974640

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells.

PubMed

Lucchetti, Donatella; Calapà, Federica; Palmieri, Valentina; Fanali, Caterina; Carbone, Federica; Papa, Alfredo; De Maria, Ruggero; De Spirito, Marco; Sgambato, Alessandro

2017-07-01

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Regulation of SFRP-1 expression in the rat dental follicle.

PubMed

Liu, Dawen; Yao, Shaomian; Wise, Gary E

2012-01-01

Tooth eruption requires osteoclastogenesis and subsequent bone resorption. Secreted frizzled-related protein-1 (SFRP-1) negatively regulates osteoclastogenesis. Our previous studies indicated that SFRP-1 is expressed in the rat dental follicle (DF), with reduced expression at days 3 and 9 close to the times for the major and minor bursts of osteoclastogenesis, respectively; but it remains unclear as to what molecules contribute to its reduced expression at these critical times. Thus, it was the aim of this study to determine which molecules regulate the expression of SFRP-1 in the DF. To that end, the DF cells were treated with cytokines that are maximally expressed at days 3 or 9, and SFRP-1 expression was determined. Our study indicated that colony-stimulating factor-1 (CSF-1), a molecule maximally expressed in the DF at day 3, down-regulated SFRP-1 expression. As to endothelial monocyte-activating polypeptide II (EMAP-II), a highly expressed molecule in the DF at day 3, it had no effect on the expression of SFRP-1. However, when EMAP-II was knocked down by siRNA, the expression of SFRP-1 was elevated, and this elevated SFRP-1 expression could be reduced by adding recombinant EMAP-II protein. This suggests that EMAP-II maintained a lower level of SFRP-1 in the DF. TNF-α is a molecule maximally expressed at day 9, and this study indicated that it also down-regulated the expression of SFRP-1 in the DF cells. In conclusion, CSF-1 and EMAP-II may contribute to the reduced SFRP-1 expression seen on day 3, while TNF-α may contribute to the reduced SFRP-1 expression at day 9.

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

PubMed Central

Holm, P. S.; Scanlon, K. J.; Dietel, M.

1994-01-01

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line. Images Figure 1 Figure 3 Figure 2 PMID:7914421

Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

PubMed

Anderson, Matthew Z; Gerstein, Aleeza C; Wigen, Lauren; Baller, Joshua A; Berman, Judith

2014-07-01

Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV-16 E6/E7 is Related to Reduced Macrophage Inflammatory Protein-3Alpha Production

DTIC Science & Technology

2005-01-01

high-risk human papillomavirus ( HPV ) types, particularly type 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal...been implicated in cervical/ anogenital cancer and oral squamous cell carcinomas (41). The mucosal lesions caused by HPVs often resolve over time, and a...Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV -16 E6/E7 is Related to Reduced Macrophage

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

NASA Astrophysics Data System (ADS)

Wang, Ze; Zhang, Hui

As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

PubMed

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

2011-01-01

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

PubMed Central

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H.; Rao, Jasti S.

2011-01-01

Background Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Methodology/Principal Findings Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Conclusion/Significance Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point. PMID:22022560

Perk Gene Dosage Regulates Glucose Homeostasis by Modulating Pancreatic β-Cell Functions

PubMed Central

Wang, Rong; Munoz, Elyse E.; Zhu, Siying; McGrath, Barbara C.; Cavener, Douglas R.

2014-01-01

Background Insulin synthesis and cell proliferation are under tight regulation in pancreatic β-cells to maintain glucose homeostasis. Dysfunction in either aspect leads to development of diabetes. PERK (EIF2AK3) loss of function mutations in humans and mice exhibit permanent neonatal diabetes that is characterized by insufficient β-cell mass and reduced proinsulin trafficking and insulin secretion. Unexpectedly, we found that Perk heterozygous mice displayed lower blood glucose levels. Methodology Longitudinal studies were conducted to assess serum glucose and insulin, intracellular insulin synthesis and storage, insulin secretion, and β-cell proliferation in Perk heterozygous mice. In addition, modulation of Perk dosage specifically in β-cells showed that the glucose homeostasis phenotype of Perk heterozygous mice is determined by reduced expression of PERK in the β-cells. Principal Findings We found that Perk heterozygous mice first exhibited enhanced insulin synthesis and secretion during neonatal and juvenile development followed by enhanced β-cell proliferation and a substantial increase in β-cell mass at the adult stage. These differences are not likely to entail the well-known function of PERK to regulate the ER stress response in cultured cells as several markers for ER stress were not differentially expressed in Perk heterozygous mice. Conclusions In addition to the essential functions of PERK in β-cells as revealed by severely diabetic phenotype in humans and mice completely deficient for PERK, reducing Perk gene expression by half showed that intermediate levels of PERK have a profound impact on β-cell functions and glucose homeostasis. These results suggest that an optimal level of PERK expression is necessary to balance several parameters of β-cell function and growth in order to achieve normoglycemia. PMID:24915520

Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.

PubMed

Thomas, Margaret Lois; de Antueno, Roberto; Coyle, Krysta Mila; Sultan, Mohammad; Cruickshank, Brianne Marie; Giacomantonio, Michael Anthony; Giacomantonio, Carman Anthony; Duncan, Roy; Marcato, Paola

2016-11-01

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti-cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti-ALDH1A3 activity. Twelve drugs (3-hydroxy-dl-kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2-associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient-derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA-MB-231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral-mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3-mediated colony formation and expression of ALDH1A3-inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3-mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high-ALDH1A3 CSCs is discussed. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

The Natural Compound Dansameum Reduces foam Cell Formation by Downregulating CD36 and Peroxisome Proliferator-activated Receptor-gamma; Expression

PubMed Central

Park, Kang-Seo; Ahn, Sang Hyun; Lee, Kang Pa; Park, Sun-Young; Cheon, Jin Hong; Choi, Jun-Yong; Kim, Kibong

2017-01-01

Background: Atherosclerosis-induced vascular disorders are major causes of death in most western countries. During the development of atherosclerotic lesions, foam cell formation is essential and formed through the expression of CD36 and the peroxisome proliferator-activated receptor gamma (PPAR-γ). Objective: To investigate whether dansameum extract (DSE) could show anti-atherosclerotic effect through down-regulating cellular redox state including CD36 and PARP-γ expression in oxidative low-density lipoprotein (oxLDL)-treated RAW264.7 cells and on differentiated foam cells in ApoE Knockout (ApoE-/-) mice. Materials and Methods: The Korean polyherbal medicine DSE was prepared from three plants in the following proportions: 40 g of Salvia miltiorrhiza root, 4 g of Amomumxanthioides fruit, and 4 g of Santalum album lignum. The immunohistochemistry and reverse transcription-polymerase chain reaction was used for analysis of protein and mRNA involved in foam cell formation. Results: We first showed that effects of DSE on foam cell formation in both oxLDL-induced RAW264.7 cells and in blood vessels from apolipoprotein E deficientApoE-/- mice with high fat diet-fed. DSE treatment significantly reduced the expression of CD36 and PPAR-γ in oxLDL-stimulated RAW264.7 cells and ApoE-/-mice, in the latter case by regulating heme oxygenase-1. Furthermore, DSE treatment also reduced cellular lipid content in vitro and in vivo experiments. Conclusion: Our data suggest that DSE may have anti-atherosclerotic properties through regulating foam cell formation. SUMMARY Dansameum extract (DSE) Regulates the expression of CD36 and peroxisome proliferator-activated receptor gamma in oxidative low-density lipoprotein-stimulated RAW264.7 Cells and ApoE Knockout (ApoE Knockout [ApoE-/-]) miceDSE Regulates Cholesterol Levels in the Serum of ApoE-deficient (ApoE-/-) miceDSE Reduced the Formation of Foam Cells by Regulating heme oxygenase-1 in ApoE-/- mice with high fat diet-fed. Abbreviations used: DSE: Dansameum extract, PPAR-γ: Peroxisome proliferator-activated receptor γ, HO-1: Heme oxygenase-1, CVD: Cardiovascular diseases PMID:29491646

The Natural Compound Dansameum Reduces foam Cell Formation by Downregulating CD36 and Peroxisome Proliferator-activated Receptor-gamma; Expression.

PubMed

Park, Kang-Seo; Ahn, Sang Hyun; Lee, Kang Pa; Park, Sun-Young; Cheon, Jin Hong; Choi, Jun-Yong; Kim, Kibong

2018-01-01

Atherosclerosis-induced vascular disorders are major causes of death in most western countries. During the development of atherosclerotic lesions, foam cell formation is essential and formed through the expression of CD36 and the peroxisome proliferator-activated receptor gamma (PPAR-γ). To investigate whether dansameum extract (DSE) could show anti-atherosclerotic effect through down-regulating cellular redox state including CD36 and PARP-γ expression in oxidative low-density lipoprotein (oxLDL)-treated RAW264.7 cells and on differentiated foam cells in ApoE Knockout (ApoE-/-) mice. The Korean polyherbal medicine DSE was prepared from three plants in the following proportions: 40 g of Salvia miltiorrhiza root, 4 g of Amomumxanthioides fruit, and 4 g of Santalum album lignum. The immunohistochemistry and reverse transcription-polymerase chain reaction was used for analysis of protein and mRNA involved in foam cell formation. We first showed that effects of DSE on foam cell formation in both oxLDL-induced RAW264.7 cells and in blood vessels from apolipoprotein E deficientApoE-/- mice with high fat diet-fed. DSE treatment significantly reduced the expression of CD36 and PPAR-γ in oxLDL-stimulated RAW264.7 cells and ApoE-/-mice, in the latter case by regulating heme oxygenase-1. Furthermore, DSE treatment also reduced cellular lipid content in vitro and in vivo experiments. Our data suggest that DSE may have anti-atherosclerotic properties through regulating foam cell formation. Dansameum extract (DSE) Regulates the expression of CD36 and peroxisome proliferator-activated receptor gamma in oxidative low-density lipoprotein-stimulated RAW264.7 Cells and ApoE Knockout (ApoE Knockout [ApoE-/-]) miceDSE Regulates Cholesterol Levels in the Serum of ApoE-deficient (ApoE-/-) miceDSE Reduced the Formation of Foam Cells by Regulating heme oxygenase-1 in ApoE-/- mice with high fat diet-fed. Abbreviations used: DSE: Dansameum extract, PPAR-γ: Peroxisome proliferator-activated receptor γ, HO-1: Heme oxygenase-1, CVD: Cardiovascular diseases.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

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

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

PubMed

Lee, Mi-Sook; Kim, Sudong; Kim, Baek Gil; Won, Cheolhee; Nam, Seo Hee; Kang, Suki; Kim, Hye-Jin; Kang, Minkyung; Ryu, Jihye; Song, Haeng Eun; Lee, Doohyung; Ye, Sang-Kyu; Jeon, Noo Li; Kim, Tai Young; Cho, Nam Hoon; Lee, Jung Weon

2014-09-01

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. Copyright © 2014 Elsevier B.V. All rights reserved.

Activation of cannabinoid CB2 receptor ameliorates atherosclerosis associated with suppression of adhesion molecules.

PubMed

Zhao, Yan; Yuan, Zuyi; Liu, Yan; Xue, Jiahong; Tian, Yuling; Liu, Weimin; Zhang, Weiping; Shen, Yan; Xu, Wei; Liang, Xiao; Chen, Tao

2010-03-01

Adhesion molecules have been implicated in the development and progression of atherosclerosis. Cannabinoids have been reported to modulate the migration and adhesion molecules expression of various cell types. Here we examined the effects of WIN55212-2, a cannabinoid receptor 1 (CB1-R)/cannabinoid receptor 2 (CB2-R) agonist on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. In the aorta of ApoE-/- mice, WIN55212-2 significantly reduced aortic root plaque area. The mechanism for this seemed to be reduced infiltration of macrophages into the atherosclerotic plaque which was also associated with reduced expression of vascular cellular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and P-selectin in the aorta. In vitro studies revealed reduced cell adhesion of a monocytic cell line (U937) to human umbilical vein endothelial cells after incubation with WIN55212-2. The reduction in macrophage adhesion also correlated with significant reductions in the expression of VCAM-1, ICAM-1, and P-selectin, indicating that reduced infiltration of macrophages in atherosclerotic plaques may occur as a result of the direct effect of WIN55212-2 on adhesion molecules in macrophages and endothelial cells. In conclusion, WIN55212-2 seems to have direct anti-atherosclerotic effects in an animal model of atherosclerosis. These effects were at least partly due to effects on the expression of VCAM-1, ICAM-1, and P-selectin, which led to reduced macrophage adhesion and infiltration. Furthermore, the protective effects completely blocked by the highly selective CB2 receptor antagonist AM630 suggest that these beneficial effects of WIN55212-2 may be mediated through the CB2 receptor.

Blood-derived small Dot cells reduce scar in wound healing

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

Kong, Wuyi; Li Shaowei; Longaker, Michael T.

2008-04-15

Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them 'Dot cells'. The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin {beta}1, CD184, CD34, CD13{sup low} and Sca1{sup low}, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 {mu}m diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherinmore » and integrin {beta}1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.« less

Knockdown of the schizophrenia susceptibility gene TCF4 alters gene expression and proliferation of progenitor cells from the developing human neocortex.

PubMed

Hill, Matthew J; Killick, Richard; Navarrete, Katherinne; Maruszak, Aleksandra; McLaughlin, Gemma M; Williams, Brenda P; Bray, Nicholas J

2017-05-01

Common variants in the TCF4 gene are among the most robustly supported genetic risk factors for schizophrenia. Rare TCF4 deletions and loss-of-function point mutations cause Pitt-Hopkins syndrome, a developmental disorder associated with severe intellectual disability. To explore molecular and cellular mechanisms by which TCF4 perturbation could interfere with human cortical development, we experimentally reduced the endogenous expression of TCF4 in a neural progenitor cell line derived from the developing human cerebral cortex using RNA interference. Effects on genome-wide gene expression were assessed by microarray, followed by Gene Ontology and pathway analysis of differentially expressed genes. We tested for genetic association between the set of differentially expressed genes and schizophrenia using genome-wide association study data from the Psychiatric Genomics Consortium and competitive gene set analysis (MAGMA). Effects on cell proliferation were assessed using high content imaging. Genes that were differentially expressed following TCF4 knockdown were highly enriched for involvement in the cell cycle. There was a nonsignificant trend for genetic association between the differentially expressed gene set and schizophrenia. Consistent with the gene expression data, TCF4 knockdown was associated with reduced proliferation of cortical progenitor cells in vitro. A detailed mechanistic explanation of how TCF4 knockdown alters human neural progenitor cell proliferation is not provided by this study. Our data indicate effects of TCF4 perturbation on human cortical progenitor cell proliferation, a process that could contribute to cognitive deficits in individuals with Pitt-Hopkins syndrome and risk for schizophrenia.

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf

PubMed Central

Hooper, Kirsten Mary; Kong, Weimin

2017-01-01

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2. PMID:28604806

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

Radiosensitization by inhibiting STAT1 in renal cell carcinoma.

PubMed

Hui, Zhouguang; Tretiakova, Maria; Zhang, Zhongfa; Li, Yan; Wang, Xiaozhen; Zhu, Julie Xiaohong; Gao, Yuanhong; Mai, Weiyuan; Furge, Kyle; Qian, Chao-Nan; Amato, Robert; Butler, E Brian; Teh, Bin Tean; Teh, Bin S

2009-01-01

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Expression of the voltage-gated potassium channel KCNQ1 in mammalian taste bud cells and the effect of its null-mutation on taste preferences.

PubMed

Wang, Hong; Iguchi, Naoko; Rong, Qi; Zhou, Minliang; Ogunkorode, Martina; Inoue, Masashi; Pribitkin, Edmund A; Bachmanov, Alexander A; Margolskee, Robert F; Pfeifer, Karl; Huang, Liquan

2009-01-20

Vertebrate taste buds undergo continual cell turnover. To understand how the gustatory progenitor cells in the stratified lingual epithelium migrate and differentiate into different types of mature taste cells, we sought to identify genes that were selectively expressed in taste cells at different maturation stages. Here we report the expression of the voltage-gated potassium channel KCNQ1 in mammalian taste buds of mouse, rat, and human. Immunohistochemistry and nuclear staining showed that nearly all rodent and human taste cells express this channel. Double immunostaining with antibodies against type II and III taste cell markers validated the presence of KCNQ1 in these two types of cells. Co-localization studies with cytokeratin 14 indicated that KCNQ1 is also expressed in type IV basal precursor cells. Null mutation of the kcnq1 gene in mouse, however, did not alter the gross structure of taste buds or the expression of taste signaling molecules. Behavioral assays showed that the mutant mice display reduced preference to some umami substances, but not to any other taste compounds tested. Gustatory nerve recordings, however, were unable to detect any significant change in the integrated nerve responses of the mutant mice to umami stimuli. These results suggest that although it is expressed in nearly all taste bud cells, the function of KCNQ1 is not required for gross taste bud development or peripheral taste transduction pathways, and the reduced preference of kcnq1-null mice in the behavioral assays may be attributable to the deficiency in the central nervous system or other organs.

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

Impact of C-rel inhibition of cord blood-derived B-, T-, and NK cells.

PubMed

Fallahi, Shirin; Mohammadi, Seyede Momeneh; Tayefi Nasrabadi, Hamid; Alihemmati, Alireza; Samadi, Naser; Gholami, Sanaz; Shanehbandi, Dariush; Nozad Charoudeh, Hojjatollah

2017-12-01

The c-Rel transcription factor is a unique member of the nuclear factor (NF)-κB family that has a role in curtailing the proliferation, differentiation, cytokine production, and overall activity of B- and T-cells. In addition, c-Rel is a key regulator of apoptosis in that it influences the expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL; conversely, inhibition of c-Rel increases cell apoptosis. To better understand the relationship between c-Rel expression and effects on B- and T-cell expansion, the current study evaluated c-Rel expression in cord blood mononuclear cells. This particular source was selected as cord blood is an important source of cells used for transplantation and immunotherapy, primarily in treating leukemias. As stem cell factor (SCF) and FLT3 are important agents for hematopoietic stem cell expansion, and cytokines like interleukin (IL)-2, -7, and -15 are essential for T- and B- (and also NK) cell development and proliferation, the current study evaluated c-Rel expression in cord blood mononuclear cells and CD34 +  cells, as well as effects on B-, T-, and NK cells associated with alterations in c-Rel expression, using flow cytometry and PCR. The results showed c-Rel expression increased among cells cultured in the presence of SCF and FLT3 but was reduced when IL-2, IL-7, and IL-15 were used all together. Further, inhibition of c-Rel expression by siRNA reduced cord blood-derived B-, T-, and NK cell differentiation and expansion. These results indicated that with cells isolated from cord blood, c-Rel has an important role in B-, T-, and NK cell differentiation and, further, that agents (select cytokines/growth factors) that could impact on its expression might not only affect immune cell profiles in a host but could potentially also limit apoptotic activities in (non-)immune cells in that host. In the context of cancer (immuno)therapy, in particular, when cord blood is used an important source in stem cell transplantation in leukemia patients, such down-regulating changes in c-Rel levels could be counter-productive.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells.

PubMed

Veiseh, Mandana; Leith, Sean J; Tolg, Cornelia; Elhayek, Sallie S; Bahrami, S Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B; Bissell, Mina J; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

PubMed Central

Veiseh, Mandana; Leith, Sean J.; Tolg, Cornelia; Elhayek, Sallie S.; Bahrami, S. Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B.; Bissell, Mina J.; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies. PMID:26528478

Human Prostatic Acid Phosphatase: Structure, Function and Regulation

PubMed Central

Muniyan, Sakthivel; Chaturvedi, Nagendra K.; Dwyer, Jennifer G.; LaGrange, Chad A.; Chaney, William G.; Lin, Ming-Fong

2013-01-01

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy. PMID:23698773

Ptpmt1 induced by HIF-2α regulates the proliferation and glucose metabolism in erythroleukemia cells

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

Xu, Qin-Qin; Qinghai Provincial People's Hospital, Xining; Xiao, Feng-Jun

Hypoxia provokes metabolism misbalance, mitochondrial dysfunction and oxidative stress in both human and animal cells. However, the mechanisms which hypoxia causes mitochondrial dysfunction and energy metabolism misbalance still remain unclear. In this study, we presented evidence that mitochondrial phosphatase Ptpmt1 is a hypoxia response molecule that regulates cell proliferation, survival and glucose metabolism in human erythroleukemia TF-1 cells. Exposure to hypoxia or DFO treatment results in upregulation of HIF1-α, HIF-2α and Ptpmt1. Only inhibition of HIF-2α by shRNA transduction reduces Ptpmt1 expression in TF-1 cells under hypoxia. Ptpmt1 inhibitor suppresses the growth and induces apoptosis of TF-1 cells. Furthermore, we demonstrated that Ptpmt1more » inhibition reduces the Glut1 and Glut3 expression and decreases the glucose consumption in TF-1 cells. In additional, Ptpmt1 knockdown also results in the mitochondrial dysfunction determined by JC1 staining. These results delineate a key role for HIF-2α-induced Ptpmt1 upregulation in proliferation, survival and glucose metabolism of erythroleukemia cells. It is indicated that Ptpmt1 plays important roles in hypoxia-induced cell metabolism and mitochondrial dysfunction. - Highlights: • Hypoxia induces upregulation of HIF-1α, HIF-2α and Ptpmt1; HIF-2a induces Ptpmt1 upregulation in TF-1 cells. • PTPMT-1 inhibition reduces growth and induces apoptosis of TF-1 cells. • PTPMT1 inhibition downregulates Glut-1, Glut-3 expression and reduces glucose consumption.« less

Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.

PubMed

Chen, Bin; Wei, Wei; Ma, Li; Yang, Bin; Gill, Ryan M; Chua, Mei-Sze; Butte, Atul J; So, Samuel

2017-06-01

Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-β-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

PubMed

Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

2017-08-15

Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation.

PubMed

Mehta, Jawahar L; Hu, Bo; Chen, Jiawei; Li, Dayuan

2003-12-01

LOX-1, a novel lectin-like receptor for oxidized LDL (ox-LDL), is expressed in response to ox-LDL, angiotensin II (Ang II), tumor necrosis factor (TNF)-alpha, and other stress stimuli. It is highly expressed in atherosclerotic tissues. Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, such as pioglitazone, exert antiatherosclerotic effects. This study examined the regulation of LOX-1 expression in human coronary artery endothelial cells (HCAECs) by pioglitazone. Fourth generation HCAECs were treated with ox-LDL, Ang II, or TNF-alpha with or without pioglitazone pretreatment. All 3 stimuli upregulated LOX-1 expression (mRNA and protein). Pioglitazone, in a concentration-dependent manner, reduced LOX-1 expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha alone). Ox-LDL, Ang II, and TNF-alpha each enhanced intracellular superoxide radical generation, and pioglitazone pretreatment reduced superoxide generation (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). Furthermore, all 3 stimuli upregulated the expression of the transcription factors nuclear factor-kappaB and activator protein-1 (determined by electrophoretic mobility shift assay), and pioglitazone pretreatment reduced this expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). To determine the biological significance of pioglitazone-mediated downregulation of LOX-1, we studied monocyte adhesion to ox-LDL-treated HCAECs. Pioglitazone reduced the adhesion of monocytes to activated HCAECs in a fashion similar to that produced by antisense to LOX-1 mRNA. These observations suggest that the PPAR-gamma ligand pioglitazone reduces intracellular superoxide radical generation and subsequently reduces the expression of transcription factors, expression of the LOX-1 gene, and monocyte adhesion to activated endothelium. The salutary effect of PPAR-gamma ligands in atherogenesis may involve the inhibition of LOX-1 and the adhesion of monocytes to endothelium.

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

PubMed

Che, Hui; Xiao, Guo-Sheng; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2016-06-01

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis

PubMed Central

Wu, Yuhsin V.; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O’Connor, Rachael; Geha, Rula C.; Somberg, C. Joy; Antonescu, Cristina; Singer, Samuel

2012-01-01

Well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n=84), WDLS (n=79), and normal fat (n=23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS compared to both WDLS and normal fat (15.2 fold and 27.8 fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for dedifferentiated liposarcomas. PMID:22170698

IL-33 activates tumor stroma to promote intestinal polyposis.

PubMed

Maywald, Rebecca L; Doerner, Stephanie K; Pastorelli, Luca; De Salvo, Carlo; Benton, Susan M; Dawson, Emily P; Lanza, Denise G; Berger, Nathan A; Markowitz, Sanford D; Lenz, Heinz-Josef; Nadeau, Joseph H; Pizarro, Theresa T; Heaney, Jason D

2015-05-12

Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

Bim regulates the survival and suppressive capability of CD8+ FOXP3+ regulatory T cells during murine GVHD.

PubMed

Agle, Kimberle; Vincent, Benjamin G; Piper, Clint; Belle, Ludovic; Zhou, Vivian; Shlomchik, Warren; Serody, Jonathan S; Drobyski, William R

2018-05-16

CD8 + Foxp3 + T cells (Tregs) are a potent regulatory population whose functional and ontological similarities to CD4 + Fox3 + T cells have not been well delineated. Using an experimental model of graft versus host disease (GVHD), we observed that CD8 + Tregs were significantly less potent than CD4 + Tregs for the suppression of GVHD. To define the mechanistic basis for this observation, we examined the T cell repertoire and the transcriptional profile of in vivo-derived CD4 + and CD8 + Tregs that emerged early during this disease. Polyclonal and alloantigen-induced CD8 + Tregs had repertoire diversity that was similar to that of conventional CD8 + T cells, indicating that a restricted repertoire was not the proximate cause of decreased suppression. Transcriptional profiling revealed that CD8 + Tregs possessed a canonical Treg transcriptional signature that was similar to that observed in CD4 + Tregs, yet distinct from conventional CD8 + T cells. Pathway analysis, however, demonstrated that CD8 + Tregs had differential gene expression in pathways involved in cell death and survival. This was further confirmed by detailed mRNA sequence analysis and protein expression studies which demonstrated that CD8 + Tregs had increased expression of Bim and reduced expression of Mcl-1. Transplantation with CD8 + Foxp3 + Bim -/- Tregs resulted in prolonged Treg survival and reduced GVHD lethality compared to wild type CD8 + Tregs, providing functional confirmation that increased expression of Bim was responsible for reduced in vivo efficacy. Thus, Bim regulates the survival and suppressive capability of CD8 + Tregs which may have implications for their use in regulatory T cell therapy. Copyright © 2018 American Society of Hematology.

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

Na+/H+ exchange regulatory factor 1 is required for ROMK1 K+ channel expression in the surface membrane of cultured M-1 cortical collecting duct cells.

PubMed

Suzuki, Takashi; Nakamura, Kazuyoshi; Mayanagi, Taira; Sobue, Kenji; Kubokawa, Manabu

2017-07-22

The ROMK1 K + channel, a member of the ROMK channel family, is the major candidate for the K + secretion pathway in the renal cortical collecting duct (CCD). ROMK1 possesses a PDZ domain-binding motif at its C-terminus that is considered a modulator of ROMK1 expression via interaction with Na + /H + exchange regulatory factor (NHERF) 1 and NHERF2 scaffold protein. Although NHERF1 is a potential binding partner of the ROMK1 K + channel, the interaction between NHERF1 and K + channel activity remains unclear. Therefore, in this study, we knocked down NHERF1 in cultured M-1 cells derived from mouse CCD and investigated the surface expression and K + channel current in these cells after exogenous transfection with EGFP-ROMK1. NHERF1 knockdown resulted in reduced surface expression of ROMK1 as indicated by a cell biotinylation assay. Using the patch-clamp technique, we further found that the number of active channels per patched membrane and the Ba 2+ -sensitive whole-cell K + current were decreased in the knockdown cells, suggesting that reduced K + current was accompanied by decreased surface expression of ROMK1 in the NHERF1 knockdown cells. Our results provide evidence that NHERF1 mediates K + current activity through acceleration of the surface expression of ROMK1 K + channels in M-1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Notch3-specific inhibition using siRNA knockdown or GSI sensitizes paclitaxel-resistant ovarian cancer cells.

PubMed

Kang, Haeyoun; Jeong, Ju-Yeon; Song, Ji-Ye; Kim, Tae Heon; Kim, Gwangil; Huh, Jin Hyung; Kwon, Ah-Young; Jung, Sang Geun; An, Hee Jung

2016-07-01

Notch signaling plays an important role in ovarian cancer chemoresistance, which is responsible for recurrence. Gamma-secretase inhibitor (GSI) is a broad-spectrum Notch inhibitor, but it has serious side effects. The efficacy of Notch3-specific inhibition in paclitaxel-resistant ovarian cancers was assessed in this study, which has not yet been evaluated relative to GSI. To analyze the effect of Notch3-specific inhibition on paclitaxel-resistant ovarian cancers, we compared cell viability, apoptosis, cell migration, angiogenesis, cell cycle, and spheroid formation after treatment with either Notch3 siRNA or GSI in paclitaxel-resistant SKpac cells and parental SKOV3 cells. Expression levels of survival, cell cycle, and apoptosis-related proteins were measured and compared between groups. Notch3 was significantly overexpressed in chemoresistant cancer tissues and cell lines relative to chemosensitive group. In paclitaxel-resistant cancer cells, Notch inhibition significantly reduced viability, migration, and angiogenesis and increased apoptosis, thereby boosting sensitivity to paclitaxel. Spheroid formation was also significantly reduced. Both Notch3 siRNA-treated cells and GSI-treated cells arrested in the G2/M phase of the cell cycle. Proteins of cell survival, cyclin D1 and cyclin D3 were reduced, whereas p21 and p27 were elevated. Both GSI and Notch3 siRNA treatment reduced expression of anti-apoptotic proteins (BCL-W, BCL2, and BCL-XL) and increased expression of pro-apoptotic proteins (Bad, Bak, Bim, Bid, and Bax). These results indicate that Notch3-specific inhibition sensitizes paclitaxel-resistant cancer cells to paclitaxel treatment, with an efficacy comparable to that of GSI. This approach would be likely to avoid the side effects of broad-spectrum GSI treatment. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Efficacy of a Cell-Cycle Decoying Killer Adenovirus on 3-D Gelfoam®-Histoculture and Tumor-Sphere Models of Chemo-Resistant Stomach Carcinomatosis Visualized by FUCCI Imaging

PubMed Central

Yano, Shuya; Takehara, Kiyoto; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2016-01-01

Stomach cancer carcinomatosis peritonitis (SCCP) is a recalcitrant disease. The goal of the present study was to establish an in vitro-in vivo-like imageable model of SCCP to develop cell-cycle-based therapeutics of SCCP. We established 3-D Gelfoam® histoculture and tumor-sphere models of SCCP. FUCCI-expressing MKN-45 stomach cancer cells were transferred to express the fluorescence ubiquinized cell-cycle indicator (FUCCI). FUCCI-expressing MKN-45 cells formed spheres on agarose or on Gelfoam® grew into tumor-like structures with G0/G1 cancer cells in the center and S/G2 cancer cells located in the surface as indicated by FUCCI imaging when the cells fluoresced red or green, respectively. We treated FUCCI-expressing cancer cells forming SCCP tumors in Gelfoam® histoculture with OBP-301, cisplatinum (CDDP), or paclitaxel. CDDP or paclitaxel killed only cycling cancer cells and were ineffective against G1/G2 MKN-45 cells in tumors growing on Gelfoam®. In contrast, the telomerase-dependent adenovirus OBP-301 decoyed the MKN-45 cells in tumors on Gelfoam® to cycle from G0/G1 phase to S/G2 phase and reduced their viability. CDDP- or paclitaxel-treated MKN-45 tumors remained quiescent and did not change in size. In contrast, OB-301 reduced the size of the MKN-45 tumors on Gelfoam®. We examined the cell cycle-related proteins using Western blotting. CDDP increased the expression of p53 and p21 indicating cell cycle arrest. In contrast, OBP-301 decreased the expression of p53 and p21 Furthermore, OBP-301 increased the expression of E2F and pAkt as further indication of cell cycle decoy. This 3-D Gelfoam® histoculture and FUCCI imaging are powerful tools to discover effective therapy of SCCP such as OBP-301. PMID:27673332

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Aging Periosteal Progenitor Cells have Reduced Regenerative Responsiveness to Bone Injury and to the Anabolic Actions of PTH 1-34 Treatment

PubMed Central

Yukata, Kiminori; Xie, Chao; Li, Tian-Fang; Takahata, Masahiko; Hoak, Donna; Kondabolu, Sirish; Zhang, Xinping; Awad, Hani A.; Schwarz, Edward M.; Beck, Christopher A.; Jonason, Jennifer H.; O’Keefe, Regis J.

2014-01-01

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects. PMID:24530870

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

Thrombopoietin inhibits murine mast cell differentiation

PubMed Central

Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

2009-01-01

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

PubMed Central

Merendino, Anna M.; Paul, Catherine; Vignola, Antonio M.; Costa, Maria A.; Melis, Mario; Chiappara, Giuseppina; Izzo, V.; Bousquet, J.; Arrigo, André-Patrick

2002-01-01

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27. PMID:12482203

TNF{alpha} and IL-1{beta} are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

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

Zheng, Wenwen; Zheng, Xuexing; Department of Anesthesiology, University of Miami Miller School of Medicine, Miami, FL 33136

2012-04-20

Highlights: Black-Right-Pointing-Pointer LPS induces proinflammatory cytokine release in HAPI cells. Black-Right-Pointing-Pointer JNK pathway is dependent on TLR4 signaling to release cytokines. Black-Right-Pointing-Pointer NF-{kappa}B pathway is dependent on Nod1 signaling to release cytokines. -- Abstract: A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathwaysmore » using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-{kappa}B (pNF-{kappa}B), TNF{alpha} and IL-1{beta}. Silencing TLR4 with siRNA reduced the expression of pJNK, TNF{alpha} and IL-1{beta}, but not pNF-{kappa}B in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNF{alpha} and IL-1{beta}. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-{kappa}B. Inhibition of NF-{kappa}B also reduced the expression of TNF{alpha} and IL-1{beta}. Nod1 ligand, DAP induced the upregulation of pNF-{kappa}B which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-{kappa}B is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-{kappa}B pathways is involved in the expression of TNF{alpha} and IL-1{beta}.« less

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

PubMed

Lee, Hye Rin; Shin, Hwa Kyoung; Park, So Youn; Kim, Hye Young; Bae, Sun Sik; Lee, Won Suk; Rhim, Byung Yong; Hong, Ki Whan; Kim, Chi Dae

2015-01-01

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer's disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM) or resveratrol (20 μM) prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

Reduced GNG2 expression levels in mouse malignant melanomas and human melanoma cell lines

PubMed Central

Yajima, Ichiro; Kumasaka, Mayuko Y; Naito, Yuji; Yoshikawa, Toshikazu; Takahashi, Hiro; Funasaka, Yoko; Suzuki, Tamio; Kato, Masashi

2012-01-01

Heterotrimeric G protein is composed of a Gα-subunit and a Gβγ-dimer. Previous studies have revealed that Gβγ-dimers including the Gγ2 subunit (Gng2/GNG2) are associated with cell proliferation, differentiation, invasion and angiogenesis. At present, however, there is no information on the expression level of Gng2/GNG2 alone in any kind of tumor. In this study, we performed DNA microarray analysis in a benign melanocytic tumor and a malignant melanoma from RET-transgenic mice (RET-mice). Gng2 transcript expression levels in a malignant melanoma were less than 1/10 of the level in a benign tumor. The difference in Gng2 transcript expression levels between benign tumors and malignant melanomas was greatest among all of the G protein γ subunits examined in this study. Moreover, protein expression levels of Gng2 were decreased in malignant melanomas compared with those in benign melanocytic tumors in RET-mice. Analysis of human malignant melanomas also showed reduced GNG2 protein expression levels in five human malignant melanoma cell lines compared with the expression levels in normal human epithelial melanocytes (NHEM). Thus, we demonstrated for the first time that Gng2/GNG2 expression levels are reduced in malignant melanoma, suggesting that GNG2 could be a novel biomarker for malignant melanoma. PMID:22679562

G protein-coupled estrogen receptor (GPER) expression in endometrial adenocarcinoma and effect of agonist G-1 on growth of endometrial adenocarcinoma cell lines.

PubMed

Skrzypczak, Maciej; Schüler, Susanne; Lattrich, Claus; Ignatov, Atanas; Ortmann, Olaf; Treeck, Oliver

2013-11-01

The G protein-coupled estrogen receptor (GPER, GPR30) is suggested to be involved in non-nuclear estrogen signaling and is expressed in a variety of hormone dependent cancer entities. This study was performed to further elucidate the role of this receptor in endometrial adenocarcinoma. We first analyzed GPER expression at the mRNA level in 88 endometrial cancer or normal endometrial tissue samples and compared it to those of nuclear steroid hormone receptors. GPER transcript levels were found to be about 6-fold reduced, but still present in endometrial cancer. Expression of this receptor was decreased in all grading subgroups when compared to pre- or postmenopausal endometrium. GPER mRNA expression was associated with PR mRNA levels (Spearman's rho 0.4610, p<0.001). We then tested the effect of the GPER ligand G-1 on growth of three endometrial cancer cell lines with different GPER expression. GPER protein levels were highest in RL95-2 cells, moderate in HEC-1A cells and not detectable in HEC-1B cells. The moderate expression level in HEC-1A cells was similar to average tumor tissue expression. Treatment with G-1 significantly inhibited growth of the GPER-positive cell lines RL95-2 and HEC-1A in a dose-dependent manner, whereas the GPER-negative line HEC-1B was not affected. Though GPER transcript levels were found to be reduced in endometrial cancer, our in vitro data suggest that moderate GPER expression might be sufficient to mediate growth-inhibitory effects triggered by its agonist G-1. Copyright © 2013 Elsevier Inc. All rights reserved.

Protective role of Smad6 in inflammation-induced valvular cell calcification

PubMed Central

Li, Xin; Lim, Jina J.; Lu, Jinxiu; Pedego, Taylor M.; Demer, Linda; Tintut, Yin

2016-01-01

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-a) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle alpha actin, and negative for an endothelial cell marker. TNF-a upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-a significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-a-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-a further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-a induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6. PMID:25864564

Protective Role of Smad6 in Inflammation-Induced Valvular Cell Calcification.

PubMed

Li, Xin; Lim, Jina; Lu, Jinxiu; Pedego, Taylor M; Demer, Linda; Tintut, Yin

2015-10-01

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I-Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I-Smads regulate inflammation-induced calcific vasculopathy is not clear. Therefore, we investigated the role of I-Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF-α) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle α actin, and negative for an endothelial cell marker. TNF-α upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF-α significantly upregulated bone morphogenetic protein 2 (BMP-2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF-α-induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF-α further augmented ALP activity, expression of BMP-2, Wnt- and redox-regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF-α induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP-2 signaling inhibitor, Smad6. © 2015 Wiley Periodicals, Inc.

High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy.

PubMed

Liu, Han; Du, Li; Wang, Ru; Wei, Chao; Liu, Bo; Zhu, Lei; Liu, Pixu; Liu, Qiang; Li, Jiang; Lu, Shi-Long; Xiao, Jing

2015-05-10

Salivary gland tumor (SGT) is one of the least studied cancers due to its rarity and heterogeneous histological types. Here, we reported that loss of PTEN expression was most frequently found in the poorly differentiated, high grade solid adenoid cystic carcinomas. Loss of PTEN expression correlated with activation of mTOR by increased phosphorylated S6 ribosome protein. We further functionally studied the role of PTEN in a pair of human SACC cell lines, SACC-83 and SACC-LM. Reduced PTEN level was correlated with the metastasis potential. When we knocked down PTEN in the SACC-83 cell line, we observed increased proliferation and enhanced migration/invasion in vitro, and increased tumor size in vivo. We further tested the therapeutical effect by applying a PI3K/mTOR inhibitor NVP-BEZ235 to both SACC cell lines. Decreased cell proliferation, increased apoptosis, as well as reduced cell migration/invasion were observed in both cell lines upon the NVP-BEZ235 treatment. Moreover, the NVP-BEZ235 treatment in a SGT xenograft mouse model significantly reduced primary tumor size and lung metastasis. Taken together, our results demonstrated that PTEN is a potent tumor suppressor in human SGTs, and targeting PI3K/mTOR pathway may be effective in the targeted therapy for human SGT patients with loss of PTEN expression.

The hematopoietic cell-specific transcription factor PU.1 is critical for expression of CD11c.

PubMed

Yashiro, Takuya; Kasakura, Kazumi; Oda, Yoshihito; Kitamura, Nao; Inoue, Akihito; Nakamura, Shusuke; Yokoyama, Hokuto; Fukuyama, Kanako; Hara, Mutsuko; Ogawa, Hideoki; Okumura, Ko; Nishiyama, Makoto; Nishiyama, Chiharu

2017-02-01

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene. © The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pyruvate kinase M2 interacts with DNA damage-binding protein 2 and reduces cell survival upon UV irradiation

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

Xie, Xiao; Wang, Mingsong; Mei, Ju, E-mail: jumei_xinhua@163.com

Pyruvate Kinase M2 (PKM2) is highly expressed in many solid tumors and associated with metabolism reprogramming and proliferation of tumors. Here, we report that PKM2 can bind to DNA Damage-Binding Protein 2 (DDB2), which is necessary for global nucleotide excision repair of UV induced DNA damage. The binding is promoted by UV irradiation and K433 acetylation of PKM2. Over expression of PKM2 facilitates phosphorylation of DDB2 and impairs DDB2-DDB1 binding. Furthermore, knocking down of PKM2 increases cell survival upon UV irradiation, while over expression of PKM2 reduces cell survival and over expression of DDB2-DDB1 reverts this effect. These results revealmore » a previously unknown regulation of PKM2 on DDB2 and provide a possible mechanism for UV induced tumorigenesis. - Highlights: • PKM2 interacts with DDB2. • UV irradiation increases PKM2-DDB2 binding via up regulation of PKM2 K433 acetylation. • PKM2 facilitates DDB2 phosphorylation and impairs DDB2-DDB1 binding. • PKM2 reduces cell survival upon UV irradiation.« less

Menadione (Vitamin K3) induces apoptosis of human oral cancer cells and reduces their metastatic potential by modulating the expression of epithelial to mesenchymal transition markers and inhibiting migration.

PubMed

Suresh, Shruthy; Raghu, Dinesh; Karunagaran, Devarajan

2013-01-01

Oral cancer is one of the most commonly occurring cancers worldwide, decreasing the patient's survival rate due to tumor recurrence and metastasis. Menadione (Vitamin K3) is known to exhibit cytotoxicity in various cancer cells but the present study focused on its effects on viability, apoptosis, epithelial to mesenchymal transition (EMT), anchorage independent growth and migration of oral cancer cells. The results show that menadione is more cytotoxic to SAS (oral squamous carcinoma) cells but not to non-tumorigenic HEK293 and HaCaT cells. Menadione treatment increased the expression of pro-apoptotic proteins, Bax and p53, with a concurrent decrease in anti-apoptotic proteins, Bcl-2 and p65. Menadione induced the expression of E-cadherin but reduced the expression of EMT markers, vimentin and fibronectin. Menadione also inhibited anchorage independent growth and migration in SAS cells. These findings reveal and confirm that menadione is a potential candidate in oral cancer therapy as it exhibits cytotoxic, antineoplastic and antimigratory effects besides effectively blocking EMT in oral cancer cells.

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

Zhang, Yi; Li, Dechun; Zhao, Xin

Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level ofmore » DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ{sup 2} = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells. - Highlights: • We investigated the role of DcR3 in FasL resistance in pancreatic cancer. • Knockdown of DcR3 in SW1990 cells reduced resistance to FasL-induced apoptosis. • DcR3 knockdown also elevated caspase expression, and reduced ERK1/2 phosphorylation. • Tumor and non-tumor tissues were collected from 66 pancreatic carcinoma patients. • 47 tumor tissue specimens, but only 15 matched non-tumor specimens contained DcR3.« less

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

PubMed Central

Gutierrez-Guerrero, Alejandra; Cobo, Marién; Muñoz, Pilar

2014-01-01

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. PMID:24400083

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

StearoylCoA Desaturase-5: A Novel Regulator of Neuronal Cell Proliferation and Differentiation

PubMed Central

Sinner, Debora I.; Kim, Gretchun J.; Henderson, Gregory C.; Igal, R. Ariel

2012-01-01

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and transduction signaling cascades, SCD5 participates in the regulation of neuronal cell growth and differentiation. PMID:22745828

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

PubMed

Nejidat, A; Saadi, I; Ronen, Z

2008-12-01

To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Micro-RNA-181a suppresses progestin-promoted breast cancer cell growth.

PubMed

Gu, Muqing; Wang, Lijuan; Yang, Chun; Li, Xue; Jia, Chanwei; Croteau, Stephane; Ruan, Xiangyan; Hardy, Pierre

2018-08-01

Recent investigations have indicated that hormone therapy may increase the risk of breast cancer (BC), and the addition of synthetic progestins may play a critical role in this. Several studies have pointed out the important role of progesterone receptor membrane component 1 (PGRMC1) in the development of BC, especially with hormone therapy using progestins. Although the deregulation of microRNA-181a (miR-181a) is often associated with human BC, the effect of miR-181a on PGRMC1 expression during hormone therapy has not been investigated. Cell viability assay and apoptosis assay were performed to investigate the pro-BC effect of progestin (norethisterone, NET) and anti-BC effect of miR-181a on MCF-7 cells. Quantitative RT-PCR and Western blot analysis were used to evaluate gene expressions in the NET-treated MCF-7 cells. NET dose-dependently increased BC cell viability and this effect was accompanied by increased expression of PGRMC1. Overexpression of miR-181a strongly reduced the cell viability of MCF-7 cells, mainly through increased apoptosis, which was evidenced by substantially increased gene expression of pro-apoptosis factors such as BAX and CASPASE 9 in miR-181a overexpressed cells. Importantly, miR-181a abrogated NET-stimulated cell viability and PGRMC1 expression. We provide evidence that miR-181a promotes MCF-7 cell apoptosis. Moreover, miR-181a suppressed NET-provoked cell viability and PGRMC1 expression in MCF-7 cells. These data may suggest a therapeutic strategy of using miR-181a to reduce BC risk in progestin hormone replacement therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Modulation of apoptosis by sulforaphane is associated with PGC-1α stimulation and decreased oxidative stress in cardiac myoblasts.

PubMed

Fernandes, Rafael O; Bonetto, Jéssica H P; Baregzay, Boran; de Castro, Alexandre L; Puukila, Stephanie; Forsyth, Heidi; Schenkel, Paulo C; Llesuy, Susana F; Brum, Ilma Simoni; Araujo, Alex Sander R; Khaper, Neelam; Belló-Klein, Adriane

2015-03-01

Sulforaphane is a naturally occurring isothiocyanate capable of stimulating cellular antioxidant defenses and inducing phase 2 detoxifying enzymes, which can protect cells against oxidative damage. Oxidative stress and apoptosis are intimately involved in the pathophysiology of cardiac diseases. Although sulforaphane is known for its anticancer benefits, its role in cardiac cells is just emerging. The aim of the present study was to investigate whether sulforaphane can modulate oxidative stress, apoptosis, and correlate with PGC-1α, a transcriptional cofactor involved in energy metabolism. H9c2 cardiac myoblasts were incubated with R-sulforaphane 5 µmol/L for 24 h. Cell viability, ANP gene expression, oxidative stress and apoptosis markers, and protein expression of PGC-1α were studied. In cells treated with sulforaphane, cellular viability increased (12 %) and ANP gene expression decreased (46 %) compared to control cells. Moreover, sulforaphane induced a significant increase in superoxide dismutase (103 %), catalase (101 %), and glutathione S-transferase (72 %) activity, reduced reactive oxygen species levels (15 %) and lipid peroxidation (65 %), as well as stimulated the expression of the cytoprotective enzyme heme oxygenase-1 (4-fold). Sulforaphane also promoted an increase in the expression of the anti-apoptotic protein Bcl-2 (60 %), decreasing the Bax/Bcl-2 ratio. Active Caspase 3\\7 and p-JNK/JNK were also reduced by sulforaphane, suggesting a reduction in apoptotic signaling. This was associated with an increased protein expression of PGC-1α (42 %). These results suggest that sulforaphane offers cytoprotection to cardiac cells by activating PGC1-α, reducing oxidative stress, and decreasing apoptosis signaling.

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.

NCAM polysialylation during adherence transitions: live cell monitoring using an antibody-mimetic EGFP-endosialidase and the viability dye DRAQ7.

PubMed

Smith, Paul J; Furon, Emeline; Wiltshire, Marie; Chappell, Sally; Patterson, Laurence H; Shnyder, Steven D; Falconer, Robert A; Errington, Rachel J

2013-07-01

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies. Copyright © 2013 International Society for Advancement of Cytometry.

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE−/− Mice

PubMed Central

Kyaw, Tin; Cui, Peng; Tay, Christopher; Kanellakis, Peter; Hosseini, Hamid; Liu, Edgar; Rolink, Antonius G.; Tipping, Peter

2013-01-01

Aims Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE−/− mouse. Methods and Results To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93− CD19+ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19+ B cells, CD4+ and CD8+ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93− CD19+ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment. Conclusion Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases. PMID:23560095

Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells.

PubMed

Noyan, Kajsa; Nguyen, Son; Betts, Michael R; Sönnerborg, Anders; Buggert, Marcus

2018-01-01

Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a "healthy" state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status.

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The antioxidant edaravone prevents cardiac dysfunction by suppressing oxidative stress in type 1 diabetic rats and in high-glucose-induced injured H9c2 cardiomyoblasts.

PubMed

Ji, Lei; Liu, Yingying; Zhang, Ying; Chang, Wenguang; Gong, Junli; Wei, Shengnan; Li, Xudong; Qin, Ling

2016-09-01

Edaravone, a radical scavenger, has been recognized as a potential protective agent for cardiovascular diseases. However, little is known about the effect of edaravone in cardiac complications associated with diabetes. Here, we have demonstrated that edaravone prevents cardiac dysfunction and apoptosis in the streptozotocin-induced type 1 diabetic rat heart. Mechanistic studies revealed that edaravone treatment improved cardiac function and restored superoxide dismutase levels. In addition, treatment of diabetic animals by edaravone increased protein expressions of sirtuin-1 (SIRT-1), peroxisome proliferator activated receptor γ coactivator α (PGC-1α), nuclear factor like-2 (NRF-2), and B cell lymphoma 2 (Bcl-2), and reduced protein expressions of Bax and Caspase-3 compared to the control group. High glucose incubation resulted in the production of reactive oxygen species (ROS) and cell death. Treatment of high-glucose-incubated H9c2 cells by edaravone reduced ROS production and cell death. In addition, the treatment of high-glucose-incubated H9c2 cells by edaravone increased the activity of antioxidative stress by increasing SIRT-1, PGC-1α, and NRF-2, and this treatment also reduced apoptosis by increasing Bcl-2 expression and reducing Bax and Caspase-3 expressions. Knockdown SIRT-1 with small interferer RNA abolished the effects of edaravone. Overall, our data demonstrated that edaravone may be an effective agent against the development of diabetic cardiomyopathy.

Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI*

PubMed Central

Wood, Peta; Mulay, Vishwaroop; Darabi, Masoud; Chan, Karen Cecilia; Heeren, Joerg; Pol, Albert; Lambert, Gilles; Rye, Kerry-Anne; Enrich, Carlos; Grewal, Thomas

2011-01-01

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes. PMID:21525007

Amyloid Precursor-like Protein 2 Association with HLA Class I Molecules

PubMed Central

Tuli, Amit; Sharma, Mahak; Wang, Xiaojian; Simone, Laura C.; Capek, Haley L.; Cate, Steven; Hildebrand, William H.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

2009-01-01

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2Kd and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression. PMID:19184004

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration.

PubMed

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration

PubMed Central

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species. PMID:26734024

Excessive fluoride reduces Foxo1 expression in dental epithelial cells of the rat incisor.

PubMed

Gao, Jianghong; Ruan, Jianping; Gao, Liping

2014-10-01

Enamel fluorosis is characterized by hypomineralization, and forkhead box O1 (Foxo1) is essential for mouse enamel biomineralization. This study investigated the effect of fluoride on Foxo1 expression and its implications for enamel fluorosis. Mandibular incisors were extracted from Sprague Dawley rats treated for 3 months with water containing 0, 50, or 100 p.p.m. F⁻. Immunohistochemistry was used to localize and quantify FOXO1 expression in dental epithelial layer cells of the incisors. The effect of fluoride on expression of Foxo1, kallikrein-4 (Klk4), and amelotin (Amtn) mRNAs was analyzed by real-time RT-PCR, and western blotting was used to measure total and nuclear FOXO1 protein levels in mature dental epithelial cells. The results revealed that nuclear FOXO1 was mainly localized in the transition and the mature ameloblasts and exhibited weaker expression in the rats exposed to fluoride. In addition to the reduced levels of Foxo1, Klk4, and AmtnmRNAs, the protein levels of total and nuclearFOXO1 were decreased in the mature dental epithelial cells exposed to fluoride. Thus, excessive fluoride may have an effect on the expression levels of Foxo1 in dental epithelial cells and thereby affect hypomineralization of the enamel during fluorosis. © 2014 Eur J Oral Sci.

Downregulation of FOXP2 promoter human hepatocellular carcinoma cell invasion.

PubMed

Yan, Xia; Zhou, Huiling; Zhang, Tingting; Xu, Pan; Zhang, Shusen; Huang, Wei; Yang, Linlin; Gu, Xingxing; Ni, Runzhou; Zhang, Tianyi

2015-12-01

Hepatocellular carcinoma (HCC) is a major health concern with a high morbidity and mortality rate worldwide. However, the mechanism underlying hepatocarcinogenesis remains unclear. Forkhead box P2 (FOXP2) has been implicated in various human cancer types. However, the role of FOXP2 in HCC remains unknown. Western blot and immunohistochemistry were used to measure the expression of FOXP2 protein in HCC and adjacent normal tissues in 50 patients. Wound healing and transwell assays were used to determine the cell invasion ability. We showed that the level of FOXP2 was significantly reduced in HCC compared with the adjacent non-tumorous tissue. There was statistical significance between the expression of FOXP2 and vein invasion (P = 0.017), number of tumor nodes (P = 0.028), and AFP (P = 0.033). Low expression of FOXP2 correlated with poor survival. Moreover, wound healing and transwell assays showed that FOXP2 could decrease cell invasion and affect the expression of vimentin and E-cadherin. Our results suggested that FOXP2 expression was downregulated in HCC tumor tissues, and reduced FOXP2 expression was associated with poor overall survival. In addition, downregulation of FOXP2 significantly enhanced cell invasiveness. These findings uncover that FOXP2 might be a new prognostic factor and be closely correlated with HCC cell invasion.

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs

PubMed Central

Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Bagami, Mohammed Al; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael

2016-01-01

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients. PMID:27081035

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

PubMed

Fouquet, Grégory; Debuysscher, Véronique; Ouled-Haddou, Hakim; Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Al Bagami, Mohammed; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael; Marcq, Ingrid; Bouhlal, Hicham

2016-05-31

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

Cytochrome P450 2J2, a new key enzyme in cyclophosphamide bioactivation and a potential biomarker for hematological malignancies.

PubMed

El-Serafi, I; Fares, M; Abedi-Valugerdi, M; Afsharian, P; Moshfegh, A; Terelius, Y; Potácová, Z; Hassan, M

2015-10-01

The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

PubMed

Jones, D L; Petty, J; Hoyle, D C; Hayes, A; Ragni, E; Popolo, L; Oliver, S G; Stateva, L I

2003-12-16

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.

PubMed

Raikwar, Sudhanshu P; Thangavel, Ramasamy; Dubova, Iuliia; Selvakumar, Govindhasamy Pushpavathi; Ahmed, Mohammad Ejaz; Kempuraj, Duraisamy; Zaheer, Smita A; Iyer, Shankar S; Zaheer, Asgar

2018-04-27

Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.

[Effect of the Industrial Nanoparticles TiO 2 , SiO 2 and ZnO on Cell Viability and Gene Expression in Red Bone Marrow of Mus Musculus].

PubMed

Zarria-Romero, Jacquelyne; Osorio, Ana; Pino, José; Shiga, Betty; Vivas-Ruiz, Dan

2017-01-01

To evaluate the effect of ZnO, TiO2 and SiO2 nanoparticles on cell viability and expression of the interleukin 7, interleukin 3, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes in Mus musculus. Red bone marrow was extracted from five Balb/c mice for the analysis of cell viability using the MTT test. The mice were divided into two groups of five each: one group was inoculated intraperitoneally with 0.5, 1.0, 2.5, 5.0, and 10 mg/kg of ZnO and SiO2 nanoparticles, respectively, and the other group was inoculated with 5.0, 10.0, 15.0, 20.0, and 25 mg/kg of TiO2 nanoparticles, respectively. Thirty hours later, RNA was extracted from the red bone marrow of the mice in both groups for gene expression analysis using quantitative PCR and RT-PCR. ZnO and SiO2 nanoparticles reduced cell viability in a dose-dependent manner by 37% and 26%, respectively, starting at a dose of 1 mg/kg. TiO2 nanoparticles at 5 mg/kg and 10 mg/kg reduced the gene expression of interleukins 7 and 3 by 55.3% and 70.2%, respectively, and SiO2 nanoparticles caused the greatest decrease (91%) in the expression of GM-CSF. ZnO nanoparticles reduced the expression of GM-CSF starting at doses of 20 mg/kg and 25 mg/kg. ZnO, SiO2 and TiO2 nanoparticles affect cell viability and gene expression in the mouse bone marrow.

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells

PubMed Central

Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y.; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-01-01

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas. PMID:28212568

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells.

PubMed

Islam, Md Soriful; Giampieri, Francesca; Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-04-04

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas.

CRACC-CRACC Interaction between Kupffer and NK Cells Contributes to Poly I:C/D-GalN Induced Hepatitis

PubMed Central

Li, Yangxi; Cao, Guoshuai; Zheng, Xiaodong; Wang, Jun; Wei, Haiming; Tian, Zhigang; Sun, Rui

2013-01-01

CD2-like receptor activating cytotoxic cells (CRACC) is known as a critical activating receptor of natural killer (NK) cells. We have previously reported that NK cells contribute to Poly I:C/D-galactosamine (D-GalN)-induced fulminant hepatitis. Since natural killer group 2, member D (NKG2D) is considered critical but not the only activating receptor for NK cells, we investigated the role of CRACC in this model. We found that CRACC was abundant on hepatic NK cells but with low expression levels on Kupffer cells under normal conditions. Expression of CRACC on NK cells and Kupffer cells was remarkably upregulated after poly I:C injection. Hepatic CRACC mRNA levels were also upregulated in Poly I:C/D-GalN-treated mice, and correlated positively with the serum alanine aminotransferase (ALT) levels. CRACC expression on Kupffer cells was specifically silenced by nano-particle encapsulated siRNA in vivo, which significantly reduced Poly I:C/D-GalN-induced liver injury. In co-culture experiments, it was further verified that silencing CRACC expression or blockade of CRACC activation by mAb reduced the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Collectively, our findings suggest that CRACC-CRACC interaction between NK cells and resident Kupffer cells contributes to Poly I:C/D-GalN-induced fulminant hepatitis. PMID:24098802

Immunoglobulin light chain allelic inclusion in systemic lupus erythematosus

PubMed Central

Fraser, Louise D.; Zhao, Yuan; Lutalo, Pamela M. K.; D'Cruz, David P.; Cason, John; Silva, Joselli S.; Dunn‐Walters, Deborah K.; Nayar, Saba; Cope, Andrew P.

2015-01-01

The principles of allelic exclusion state that each B cell expresses a single light and heavy chain pair. Here, we show that B cells with both kappa and lambda light chains (Igκ and Igλ) are enriched in some patients with the systemic autoimmune disease systemic lupus erythematosus (SLE), but not in the systemic autoimmune disease control granulomatosis with polyangiitis. Detection of dual Igκ and Igλ expression by flow cytometry could not be abolished by acid washing or by DNAse treatment to remove any bound polyclonal antibody or complexes, and was retained after two days in culture. Both surface and intracytoplasmic dual light chain expression was evident by flow cytometry and confocal microscopy. We observed reduced frequency of rearrangements of the kappa‐deleting element (KDE) in SLE and an inverse correlation between the frequency of KDE rearrangement and the frequency of dual light chain expressing B cells. We propose that dual expression of Igκ and Igλ by a single B cell may occur in some patients with SLE when this may be a consequence of reduced activity of the KDE. PMID:26036683

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

PubMed Central

Kim, Mihwa; Jung, Ji-Yeon; Choi, Seungho; Lee, Hyunseung; Morales, Liza D.; Koh, Jeong-Tae; Kim, Sun Hun; Choi, Yoo-Duk; Choi, Chan; Slaga, Thomas J.; Kim, Won Jae; Kim, Dae Joon

2017-01-01

ABSTRACT Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma. PMID:27754745

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy.

PubMed

Kim, Mihwa; Jung, Ji-Yeon; Choi, Seungho; Lee, Hyunseung; Morales, Liza D; Koh, Jeong-Tae; Kim, Sun Hun; Choi, Yoo-Duk; Choi, Chan; Slaga, Thomas J; Kim, Won Jae; Kim, Dae Joon

2017-01-02

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

PubMed

Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4 + T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4 + T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1 + CD4 + T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4 + T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation

PubMed Central

Celada, Lindsay J.; Rotsinger, Joseph E.; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = −0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression. PMID:27564547

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

PubMed

Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

2016-11-01

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

PubMed

Pace, E; Di Vincenzo, S; Ferraro, M; Bruno, A; Dino, P; Bonsignore, M R; Battaglia, S; Saibene, F; Lanata, L; Gjomarkaj, M

2016-08-01

Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

Dietary supplementation with zinc oxide decreases expression of the stem cell factor in the small intestine of weanling pigs.

PubMed

Ou, Deyuan; Li, Defa; Cao, Yunhe; Li, Xilong; Yin, Jingdong; Qiao, Shiyan; Wu, Guoyao

2007-12-01

Dietary supplementation with a high level of zinc oxide (ZnO) has been shown to reduce the incidence of diarrhea in weanling pigs, but the underlying mechanisms remain largely unknown. Intestinal-mucosal mast cells, whose maturation and proliferation is under the control of the stem cell factor (SCF), play an important role in the etiology of diarrhea by releasing histamine. The present study was conducted to test the novel hypothesis that supplementing ZnO to the diet for weanling piglets may inhibit SCF expression in the small intestine, thereby reducing the number of mast cells, histamine release, and diarrhea. In Experiment 1, 32 piglets (28 days of age) were weaned and fed diets containing 100 or 3000 mg zinc/kg (as ZnO) for 10 days (16 piglets per group). In Experiment 2, two groups of 28-day-old piglets (8 piglets per group) were fed the 100- or 3000-mg zinc/kg diet as in Experiment 1, except that they were pair-fed the same amounts of feed. Supplementation with a high level of ZnO reduced the incidence of diarrhea in weanling piglets. Dietary Zn supplementation reduced expression of the SCF gene at both mRNA and protein levels, the number of mast cells in the mucosa and submucosa of the small intestine and histamine release from mucosal mast cells. Collectively, our results indicate that dietary supplementation with ZnO inhibits SCF expression in the small intestine, leading to reductions in the number of mast cells and histamine release. These findings may have important implications for the prevention of weaning-associated diarrhea in piglets.

Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

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

Zhang, Kairui; Zhang, Sheng; Li, Qianqian

Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aimsmore » to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.« less

Deletion of RhoA in Progesterone Receptor-Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice.

PubMed

El Zowalaty, Ahmed E; Li, Rong; Zheng, Yi; Lydon, John P; DeMayo, Francesco J; Ye, Xiaoqin

2017-07-01

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/-). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized β-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.

ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease

PubMed Central

Zhang, Jilu; Ramadan, Abdulraouf M.; Griesenauer, Brad; Li, Wei; Turner, Matthew J.; Liu, Chen; Kapur, Reuben; Hanenberg, Helmut; Blazar, Bruce R.; Tawara, Isao; Paczesny, Sophie

2015-01-01

Graft-versus-host disease (GVHD) remains a devastating complication after allogeneic hematopoietic cell transplantation (HCT). We previously identified high plasma soluble suppression of tumorigenicity 2 (sST2) as a biomarker of the development of GVHD and death. sST2 sequesters interleukin (IL)-33, limiting its availability to T cells expressing membrane-bound ST2 (mST2) [Th2 cells and ST2+FoxP3+regulatory T cells]. Here, we report that blockade of sST2 in the peri-transplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17–producing T cells while maintaining protective mST2-expressing T cells, increasing the frequency of intestinal myeloid-derived suppressor cells, and decreasing frequency of intestinal CD103 dendritic cells. Finally, ST2 blockade preserved graft-versus-leukemia activity in a model of GFP+MLL-AF9 acute myeloid leukemia. Our findings suggest that ST2 is a therapeutic target for severe GVHD, and that the ST2/IL-33 pathway could be investigated in other T-cell mediated immune disorders with loss of tolerance. PMID:26446957

The effects of hyperthermia on the immunomodulatory properties of human umbilical cord vein mesenchymal stem cells (MSCs).

PubMed

Hesami, Shilan; Mohammadi, Mehdi; Rezaee, Mohamad Ali; Jalili, Ali; Rahmani, Mohammad Reza

2017-11-01

Hyperthermia can modulate inflammation and the immune response. Based on the recruitment of mesenchymal stem cells (MSCs) to inflamed tissues and the immunomodulatory properties of these cells, the aim of this study was to examine the effects of hyperthermia on the immunomodulatory properties of MSCs in a mixed lymphocyte reaction (MLR). Passages 4-6 of human umbilical cord vein mesenchymal stem cells were co-cultured in a two-way MLR. Cells in the hyperthermia groups were incubated at 41 °C for 45 min. A colorimetric assay was employed to examine the effects of MSCs on cell proliferation. The levels of IL-4 and TNF-α proteins in the cell culture supernatant were measured, and non-adherent cells were used for RNA extraction, which was then used for cDNA synthesis. RT-PCR was utilised to assess levels of IL-10, IL-17A, IL-4, TNF-α, TGF-β1, FOX P 3 , IFN-γ, CXCL12 and β-actin mRNA expression. UCV-MSCs co-cultured in an MLR reduced lymphocyte proliferation at 37 °C, whereas hyperthermia attenuated this effect. Hyperthermia increased expression of IL-10, TGF-β1 and FOXP3 mRNAs in co-culture; however, no effects on IL-17A and IFN-γ were observed, and it reduced CXCL12 expression. In co-culture, IL-4 mRNA and protein increased at 37 °C, an effect that was reduced by hyperthermia. No considerable change in TNF-α mRNA expression was found in hyperthermia-treated cells. Hyperthermia increases cell proliferation of the peripheral blood mononuclear cells and modifies the cytokine profile in the presence of UCV-MSCs.

miR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function.

PubMed

Lim, Shok Ping; Ioannou, Nikolaos; Ramsay, Alan G; Darling, David; Gäken, Joop; Mufti, Ghulam J

2018-05-01

MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3 + T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions. ©2018 Society for Leukocyte Biology.

Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

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

Warford, Jordan, E-mail: jordan.warford@dal.ca; Doucette, Carolyn D., E-mail: carolyn.doucette@dal.ca; Hoskin, David W., E-mail: d.w.hoskin@dal.ca

2014-01-10

Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycansmore » which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2 dependent cell line CTLL-2. The effect of surfen was antagonized dose-dependently by co-treatment with heparin sulfate. We conclude that surfen inhibits T cell proliferation in vitro and in vivo. When T cell receptor-driven activation is bypassed surfen had a neutral or stimulatory effect on T cell proliferation. The results imply that endogenous GAGs and proteoglycans play a complex role in promoting or inhibiting different aspects of T cell activation.« less

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.

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

PubMed

Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

2014-12-01

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo.

PubMed

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2017-10-27

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo , neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo

PubMed Central

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Avogadri-Connors, Francesca; Cutler, Richard E.; Lalani, Alshad S.; Dent, Paul

2017-01-01

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses. PMID:29163826

11β-HSD1 reduces metabolic efficacy and adiponectin synthesis in hypertrophic adipocytes.

PubMed

Koh, Eun Hee; Kim, Ah-Ram; Kim, Hyunshik; Kim, Jin Hee; Park, Hye-Sun; Ko, Myoung Seok; Kim, Mi-Ok; Kim, Hyuk-Joong; Kim, Bum Joong; Yoo, Hyun Ju; Kim, Su Jung; Oh, Jin Sun; Woo, Chang-Yun; Jang, Jung Eun; Leem, Jaechan; Cho, Myung Hwan; Lee, Ki-Up

2015-06-01

Mitochondrial dysfunction in hypertrophic adipocytes can reduce adiponectin synthesis. We investigated whether 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression is increased in hypertrophic adipocytes and whether this is responsible for mitochondrial dysfunction and reduced adiponectin synthesis. Differentiated 3T3L1 adipocytes were cultured for up to 21 days. The effect of AZD6925, a selective 11β-HSD1 inhibitor, on metabolism was examined. db/db mice were administered 600 mg/kg AZD6925 daily for 4 weeks via gastric lavage. Mitochondrial DNA (mtDNA) content, mRNA expression levels of 11 β -H sd1 and mitochondrial biogenesis factors, adiponectin synthesis, fatty acid oxidation (FAO), oxygen consumption rate and glycolysis were measured. Adipocyte hypertrophy in 3T3L1 cells exposed to a long duration of culture was associated with increased 11 β -Hsd1 mRNA expression and reduced mtDNA content, mitochondrial biogenesis factor expression and adiponectin synthesis. These cells displayed reduced mitochondrial respiration and increased glycolysis. Treatment of these cells with AZD6925 increased adiponectin synthesis and mitochondrial respiration. Inhibition of FAO by etomoxir blocked the AZD6925-induced increase in adiponectin synthesis, indicating that 11β-HSD1-mediated reductions in FAO are responsible for the reduction in adiponectin synthesis. The expression level of 11 β -Hsd1 was higher in adipose tissues of db/db mice. Administration of AZD6925 to db/db mice increased the plasma adiponectin level and adipose tissue FAO. In conclusion, increased 11β-HSD1 expression contributes to reduced mitochondrial respiration and adiponectin synthesis in hypertrophic adipocytes. © 2015 Society for Endocrinology.

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

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

Wang Hongmin; Monteiro, Mervyn J.

2007-08-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner.more » Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.« less

Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells

PubMed Central

Bauckman, Kyle; Haller, Edward; Taran, Nicholas; Rockfield, Stephanie; Ruiz-Rivera, Abigail; Nanjundan, Meera

2015-01-01

The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells. PMID:25697096

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

PubMed Central

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-01-01

Background: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Methods: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Results: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Conclusions: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients. PMID:25010864

Islet-specific monoamine oxidase A and B expression depends on MafA transcriptional activity and is compromised in type 2 diabetes.

PubMed

Ganic, Elvira; Johansson, Jenny K; Bennet, Hedvig; Fex, Malin; Artner, Isabella

2015-12-25

Lack or dysfunction of insulin producing β cells results in the development of type 1 and type 2 diabetes mellitus, respectively. Insulin secretion is controlled by metabolic stimuli (glucose, fatty acids), but also by monoamine neurotransmitters, like dopamine, serotonin, and norepinephrine. Intracellular monoamine levels are controlled by monoamine oxidases (Mao) A and B. Here we show that MaoA and MaoB are expressed in mouse islet β cells and that inhibition of Mao activity reduces insulin secretion in response to metabolic stimuli. Moreover, analysis of MaoA and MaoB protein expression in mouse and human type 2 diabetic islets shows a significant reduction of MaoB in type 2 diabetic β cells suggesting that loss of Mao contributes to β cell dysfunction. MaoB expression was also reduced in β cells of MafA-deficient mice, a mouse model for β cell dysfunction, and biochemical studies showed that MafA directly binds to and activates MaoA and MaoB transcriptional control sequences. Taken together, our results show that MaoA and MaoB expression in pancreatic islets is required for physiological insulin secretion and lost in type 2 diabetic mouse and human β cells. These findings demonstrate that regulation of monoamine levels by Mao activity in β cells is pivotal for physiological insulin secretion and that loss of MaoB expression may contribute to the β cell dysfunction in type 2 diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Apigenin suppresses GLUT-1 and p-AKT expression to enhance the chemosensitivity to cisplatin of laryngeal carcinoma Hep-2 cells: an in vitro study

PubMed Central

Xu, Ying-Ying; Wu, Ting-Ting; Zhou, Shui-Hong; Bao, Yang-Yang; Wang, Qin-Ying; Fan, Jun; Huang, Ya-Ping

2014-01-01

Glucose transporter-1 (GLUT-1) and PI3K/Akt are known to be closely involved in resistance to chemotherapy. Co-targeted therapy reducing GLUT-1 expression and PI3K/Akt pathway activity may overcome the chemoresistance of human cancers. Apigenin may inhibit the expression of GLUT-1 and the PI3K/Akt pathway. We hypothesized that over-expression of GLUT-1 and p-Akt was associated with the resistance to cisplatin of laryngeal carcinoma Hep-2 cells. We explored whether apigenin inhibited GLUT-1 and p-Akt, resulting in sensitization of laryngeal carcinoma Hep-2 cells to cisplatin. Real-time RT-PCR and Western blotting confirmed the presence of GLUT-1 mRNA, and GLUT-1 and p-Akt proteins in Hep-2 cells. We found that resistance or insensitivity of Hep-2 cells to cisplatin might be associated with such expression. Apigenin markedly enhanced the cisplatin-induced suppression of Hep-2 cell growth. This effect was concentration- and time-dependent. Thus apigenin may significantly reduce the levels of GLUT-1 mRNA, and GLUT-1 and p-Akt proteins, in cisplatin-treated Hep-2 cells, in a concentration- and time-dependent manner. To conclude, overexpression of GLUT-1 mRNA may be associated with the resistance to cisplatin of laryngeal carcinoma Hep-2 cells. Apigenin may enhance the sensitivity to cisplatin of laryngeal carcinoma cells via inhibition of GLUT-1 and p-Akt expression. PMID:25120770

Memory-Relevant Mushroom Body Output Synapses Are Cholinergic.

PubMed

Barnstedt, Oliver; Owald, David; Felsenberg, Johannes; Brain, Ruth; Moszynski, John-Paul; Talbot, Clifford B; Perrat, Paola N; Waddell, Scott

2016-03-16

Memories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. Here we demonstrate a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

ROLE OF GRB2-ASSOCIATED BINDER 1 (GAB1) IN EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)-INDUCED SIGNALING IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

PubMed Central

Hoeben, A.; Martin, D.; Clement, P. M.; Cools, J.; Gutkind, J. S.

2012-01-01

The Epidermal Growth Factor Receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents. PMID:22865653

Curcumin Significantly Enhances Dual PI3K/Akt and mTOR Inhibitor NVP-BEZ235-Induced Apoptosis in Human Renal Carcinoma Caki Cells through Down-Regulation of p53-Dependent Bcl-2 Expression and Inhibition of Mcl-1 Protein Stability

PubMed Central

Cho, Il Je; Kim, Sang Chan; Kwon, Taeg Kyu

2014-01-01

The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level. PMID:24743574

Rat Muc4 (sialomucin complex) reduces binding of anti-ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance.

PubMed

Price-Schiavi, Shari A; Jepson, Scott; Li, Peter; Arango, Maria; Rudland, Philip S; Yee, Lisa; Carraway, Kermit L

2002-06-20

Muc4 (also called sialomucin complex), the rat homolog of human MUC4, is a heterodimeric glycoprotein complex that consists of a peripheral O-glycosylated mucin subunit, ASGP-1, tightly but noncovalently linked to a N-glycosylated transmembrane subunit, ASGP-2. The complex is expressed in a number of normal, vulnerable epithelial tissues, including mammary gland, uterus, colon, cornea and trachea. Muc4/SMC is also overexpressed or aberrantly expressed on a number of human tumors including breast tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance and promote metastasis. In addition, as a ligand for ErbB2, Muc4/SMC can potentiate phosphorylation of ErbB2 and potentially alter signals generated from this receptor. Using A375 human melanoma cells and MCF7 human breast adenocarcinoma cells stably transfected with tetracycline regulatable Muc4, we have investigated whether overexpression of Muc4/SMC can repress antibody binding to cell surface-expressed ErbB2. Overexpression of Muc4/SMC does not affect the level of ErbB2 expression in either cell line, but it does reduce binding of a number of anti-ErbB2 antibodies, including Herceptin. Interestingly, overexpression of ErbB2 does not block binding of other unrelated antibodies of the same isotype, suggesting that the reduction in ErbB2 antibody binding is due to complex formation of Muc4/SMC and ErbB2. Furthermore, capping of Muc4/SMC with anti-Muc4/SMC antibodies reduces antibody binding to ErbB2 instead of increasing binding, again suggesting that reduced antibody binding to ErbB2 is due to steric hindrance from complex formation of Muc4/SMC and ErbB2. Thus, overexpression of Muc4/SMC on tumor cells may have both prognostic and therapeutic relevance. Copyright 2002 Wiley-Liss, Inc.

Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation

PubMed Central

Yang, Li-yun; He, Chang-yu; Chen, Xue-hua; Su, Li-ping; Liu, Bing-ya; Zhang, Hao

2016-01-01

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment. PMID:27356739

Prognostic significance of hepatocyte growth factor activator inhibitor type 1 (HAI-1) immunoreactivity in pancreatic ductal adenocarcinoma.

PubMed

Sakugawa, Chihiro; Haruyama, Yukihiro; Tanaka, Hiroyuki; Fukushima, Tsuyoshi; Kawaguchi, Makiko; Kataoka, Hiroaki

2017-12-04

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine protease inhibitor that is expressed on the surface of epithelial cells. Evidence has suggested that decreased cell surface HAI-1 in carcinoma cells results in enhanced invasiveness. However, little is known regarding the expression of HAI-1 in pancreatic ductal adenocarcinoma (PDAC). This study aimed to analyze HAI-1 expression in PDAC and its impact on patient prognosis. HAI-1 immunohistochemistry was performed on samples from 67 PDAC cases. HAI-1 expression was increased in intraepithelial neoplasia compared to the adjacent non-neoplastic ductal epithelium. Of the 67 samples tested, 58% (39/67) of PDAC cases showed diffuse (> 75%) immunoreactivity in PDAC cells. The remaining cases showed reduced HAI-1 immunoreactivity in a substantial number of cancer cells. Although there was no correlation between HAI-1 status and tumor size, histologic grade or lymph node metastasis, diffuse HAI-1 positive cases showed longer disease-free survival (DFS; p = 0.006, log-rank test). In conclusion, HAI-1 is upregulated in pancreatic intraepithelial neoplasia and broadly expressed in PDAC cells. However, PDAC cases having areas of reduced HAI-1 immunoreactivity may show shorter DFS.

The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

PubMed Central

McGarvey, Terry; Wang, Huiyi; Lal, Priti; Puthiyaveettil, Raghunath; Tomaszewski, John; Sepulveda, Jorge; Labelle, Ed; Weiss, Jayne S.; Nickerson, Michael L.; Kruth, Howard S.; Brandt, Wolfgang; Wessjohann, Ludger A.; Malkowicz, S. Bruce

2011-01-01

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression. PMID:21740188

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

PubMed

Danzer, Claudia; Koller, Anna; Baier, Julia; Arnold, Harald; Giessler, Claudia; Opoka, Robert; Schmidt, Stephanie; Willers, Maike; Mihai, Sidonia; Parsch, Hans; Wirtz, Stefan; Daniel, Christoph; Reinhold, Annegret; Engelmann, Swen; Kliche, Stefanie; Bogdan, Christian; Hoebe, Kasper; Mattner, Jochen

2016-09-01

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NHE8 plays important roles in gastric mucosal protection

PubMed Central

Xu, Hua; Li, Jing; Chen, Huacong; Wang, Chunhui

2013-01-01

Sodium/hydrogen exchanger (NHE) 8 is an apically expressed membrane protein in the intestinal epithelial cells. It plays important roles in sodium absorption and bicarbonate secretion in the intestine. Although NHE8 mRNA has been detected in the stomach, the precise location and physiological role of NHE8 in the gastric glands remain unclear. In the current study, we successfully detected the expression of NHE8 in the glandular region of the stomach by Western blotting and located NHE8 protein at the apical membrane in the surface mucous cells by a confocal microscopic method. We also identified the expression of downregulated-in-adenoma (DRA) in the surface mucous cells in the stomach. Using NHE8−/− mice, we found that NHE8 plays little or no role in basal gastric acid production, yet NHE8−/− mice have reduced gastric mucosal surface pH and higher incidence of developing gastric ulcer. DRA expression was reduced significantly in the stomach in NHE8−/− mice. The propensity for gastric ulcer, reduced mucosal surface pH, and low DRA expression suggest that NHE8 is indirectly involved in gastric bicarbonate secretion and gastric mucosal protection. PMID:23220221

miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor

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

Park, Jong-Kook; Henry, Jon C.; Jiang, Jinmai

2011-03-25

Research highlights: {yields} The expression of miR-132 and miR-212 are significantly increased in pancreatic cancer. {yields} miR-132 and miR-212 target the tumor suppressor pRb, resulting in enhanced proliferation. {yields} miR-132 and miR-212 expression is increased by a {beta}2 adrenergic receptor agonist, suggesting a novel mechanism for pancreatic cancer progression. -- Abstract: Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target themore » retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G{sub 2}/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the {beta}2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The {beta}2 adrenergic pathway may play an important role in this novel mechanism.« less

MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma.

PubMed

Guan, Yichun; Guo, Lankai; Zukerberg, Lawrence; Rueda, Bo R; Styer, Aaron K

2016-08-17

Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied. The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor. Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls. Our findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

Dimethyl Fumarate Selectively Reduces Memory T Cells and Shifts the Balance between Th1/Th17 and Th2 in Multiple Sclerosis Patients.

PubMed

Wu, Qi; Wang, Qin; Mao, Guangmei; Dowling, Catherine A; Lundy, Steven K; Mao-Draayer, Yang

2017-04-15

Dimethyl fumarate (DMF; trade name Tecfidera) is an oral formulation of the fumaric acid ester that is Food and Drug Administration approved for treatment of relapsing-remitting multiple sclerosis. To better understand the therapeutic effects of Tecfidera and its rare side effect of progressive multifocal leukoencephalopathy, we conducted cross-sectional and longitudinal studies by immunophenotyping cells from peripheral blood (particularly T lymphocytes) derived from untreated and 4-6 and 18-26 mo Tecfidera-treated stable relapsing-remitting multiple sclerosis patients using multiparametric flow cytometry. The absolute numbers of CD4 and CD8 T cells were significantly decreased and the CD4/CD8 ratio was increased with DMF treatment. The proportions of both effector memory T cells and central memory T cells were reduced, whereas naive T cells increased in treated patients. T cell activation was reduced with DMF treatment, especially among effector memory T cells and effector memory RA T cells. Th subsets Th1 (CXCR3 + ), Th17 (CCR6 + ), and particularly those expressing both CXCR3 and CD161 were reduced most significantly, whereas the anti-inflammatory Th2 subset (CCR3 + ) was increased after DMF treatment. A corresponding increase in IL-4 and decrease in IFN-γ and IL-17-expressing CD4 + T cells were observed in DMF-treated patients. DMF in vitro treatment also led to increased T cell apoptosis and decreased activation, proliferation, reactive oxygen species, and CCR7 expression. Our results suggest that DMF acts on specific memory and effector T cell subsets by limiting their survival, proliferation, activation, and cytokine production. Monitoring these subsets could help to evaluate the efficacy and safety of DMF treatment. Copyright © 2017 by The American Association of Immunologists, Inc.

Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains.

PubMed

Cafiso, Viviana; Bertuccio, Taschia; Spina, Daniela; Purrello, Simona; Campanile, Floriana; Di Pietro, Cinzia; Purrello, Michele; Stefani, Stefania

2012-01-01

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF.

The melanocortin receptor agonist NDP-MSH impairs the allostimulatory function of dendritic cells.

PubMed

Rennalls, La'Verne P; Seidl, Thomas; Larkin, James M G; Wellbrock, Claudia; Gore, Martin E; Eisen, Tim; Bruno, Ludovica

2010-04-01

As alpha-melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells and has potent immunosuppressive properties, it was determined whether human dendritic cells (DCs) express the receptor for this hormone. Reverse transcription-polymerase chain reaction detected messenger RNA specific for all of the known melanocortin receptors in DCs. Mixed lymphocyte reactions also revealed that treatment with [Nle(4), DPhe(7)]-alpha-MSH (NDP-MSH), a potent alpha-MSH analogue, significantly reduced the ability of DCs to stimulate allogeneic T cells. The expression of various cell surface adhesion, maturation and costimulatory molecules on DCs was also investigated. Although treatment with NDP-MSH did not alter the expression of CD83 and major histocompatibility complex class I and II, the surface expression of CD86 (B7.2), intercellular adhesion molecule (ICAM-1/CD54) and CD1a was reduced. In summary, our data indicate that NDP-MSH inhibits the functional activity of DCs, possibly by down-regulating antigen-presenting and adhesion molecules and that these events may be mediated via the extracellular signal-regulated kinase 1 and 2 pathway.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

PubMed

Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

2009-03-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

PubMed Central

Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

2009-01-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK1 cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R. PMID:19106211

Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

PubMed

Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

2015-12-15

Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of transforming growth factor-beta1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken.

PubMed

Li, X; Velleman, S G

2009-02-01

During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-beta1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared to a normal chicken line. The injection of TGF-beta1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-beta1 is likely due to different levels of endogenous TGF-beta1 where the LSN birds have increased TGF-beta1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-beta1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-beta1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-beta1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-beta1 treatment. Myogenin expression was reduced 29% by TGF-beta1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-beta1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-beta1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic and posthatch development. Furthermore, TGF-beta1 also reduced the expression of the cell adhesion receptor beta1 integrin subunit during embryonic and posthatch muscle growth in normal and LSN chickens. Therefore, the reduction of beta1 integrin in response to TGF-beta1 is also associated with decreased posthatch muscle growth. The results from this study indicate that TGF-beta1 inhibits skeletal muscle growth by regulating MyoD and myogenin expression. These data also suggest that a beta1 integrin-mediated alternative pathway is likely involved in the TGF-beta1-induced reduction of muscle growth.

Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

PubMed Central

McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

2013-01-01

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227

Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

PubMed Central

Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

1996-01-01

Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression.

PubMed

Heilig, C W; Kreisberg, J I; Freytag, S; Murakami, T; Ebina, Y; Guo, L; Heilig, K; Loberg, R; Qu, X; Jin, Y; Henry, D; Brosius, F C

2001-04-01

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent anti-tumor activity

PubMed Central

Caruso, Hillary G.; Hurton, Lenka V.; Najjar, Amer; Rushworth, David; Ang, Sonny; Olivares, Simon; Mi, Tiejuan; Switzer, Kirsten; Singh, Harjeet; Huls, Helen; Lee, Dean A.; Heimberger, Amy B.; Champlin, Richard E.; Cooper, Laurence J. N.

2015-01-01

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells. PMID:26330164

Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

PubMed

Natsuga, Ken; Cipolat, Sara; Watt, Fiona M

2016-01-01

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Viewing a humorous film decreases IgE production by seminal B cells from patients with atopic eczema.

PubMed

Kimata, Hajime

2009-02-01

Sperms induced IgE production by seminal B cells from patients with atopic eczema via interaction of B cells with galectin-3 on sperms. We studied the effect of viewing a humorous film on IgE production by seminal B cells cultured with sperms. Twenty-four male patients with atopic eczema viewed a humorous film (Modern Times, featuring Charlie Chaplin). Just before and immediately after viewing, semen was collected, and seminal B cells and sperms were purified. Seminal B cells were cultured with sperms and IgE production was measured, while expression of galectin-3 on sperms was assessed. After viewing the humorous film, IgE production by B cells cultured with sperms was significantly decreased. Moreover, expression of galectin-3 on sperms was reduced. Viewing a humorous film reduced galectin-3 expression on sperms, which in turn decreased IgE production by seminal B cells cultured with sperms. These results indicate that viewing a humorous film may be helpful for the study and treatment of local IgE production and allergy in the reproductive tract.

Inhibitory effect of calcium on non-heme iron absorption may be related to translocation of DMT-1 at the apical membrane of enterocytes.

PubMed

Thompson, Ben A V; Sharp, Paul A; Elliott, Ruan; Fairweather-Tait, Susan J

2010-07-28

Many studies show that calcium reduces iron absorption from single meals, but the underlying mechanism is not known. We tested the hypothesis that calcium alters the expression and/or functionality of iron transport proteins. Differentiated Caco-2 cells were treated with ferric ammonium citrate and calcium chloride, and ferritin, DMT-1, and ferroportin were quantified in whole-cell lysate and cell-membrane fractions. Calcium attenuated the iron-induced increase in cell ferritin levels in a dose-dependent manner; a significant decrease was seen at calcium concentrations of 1.25 and 2.5 mM but was only evident after a 16-24 h incubation period. Calcium and iron treatments decreased DMT-1 protein in Caco-2 cell membranes, although total DMT-1 in whole cell lysates was unchanged by either iron or calcium. No change was seen in ferroportin expression. Our data suggest that calcium reduces iron bioavailability by decreasing DMT-1 expression at the apical cell membrane, thereby downregulating iron transport into the cell.

Combination of ADMSCs and chondrocytes reduces hypertrophy and improves the functional properties of osteoarthritic cartilage.

PubMed

Ahmed, M R; Mehmood, A; Bhatti, F-ur-R; Khan, S N; Riazuddin, S

2014-11-01

To evaluate the therapeutic efficacy of Adipose derived MSCs (ADMSCs) in combination with chondrocytes in counteracting oxidative stress in chondrocytes in vitro and in rat model of osteoarthritis (OA). Cultured chondrocytes were exposed to oxidative stress with 200 μM Hydrogen peroxide (H2O2), followed by co-culture with ADMSCs or chondrocytes or combination of both cell types in a transwell culture system for 36 h. The cytoprotective effect was assessed by immunocytochemistry and gene expression analysis. In vivo study evaluated therapeutic effect of the above mentioned three treatments after transplantation in OA rats. The Combination of ADMSCs + Chondrocytes decreased the extent of oxidative stress-induced damage of chondrocytes. Enhanced expression level of Acan and Collagen type-II alpha (Col2a1) with a correspondingly decreased expression of Collagen type-I alpha (Col1a1) and Matrix metallopeptidase 13 (Mmp13) was maximally observed in this group. Moreover, reduced count of annexin-V positive cells, Caspase (Casp3) gene expression and Lactate dehydrogenase (LDH) release with concomitantly enhanced viability and expression of proliferating cell nuclear antigen (PCNA) gene was observed. In vivo study showed that homing of cells and proteoglycan contents of knee joints were significantly better in ADMSCs + Chondrocytes transplanted rats. Increased expression of Acan and Col2a1 along with decreased expression of Col1a1 and Mmp13 indicated formation of hyaline cartilage in this group. These rats also demonstrated significantly reduced expression of Casp3 while increased expression of PCNA genes than the other cell transplanted groups. Our results demonstrated that a combination of ADMSCs and chondrocytes may be a more effective therapeutic strategy against OA than the use of ADMSCs or chondrocytes separately. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Resveratrol Induces Cancer Cell Apoptosis through MiR-326/PKM2-Mediated ER Stress and Mitochondrial Fission.

PubMed

Wu, Haili; Wang, Yingying; Wu, Changxin; Yang, Peng; Li, Hanqing; Li, Zhuoyu

2016-12-14

Resveratrol (Res), a natural phytoalexin found in a variety of plants, has significant antitumor activity. Pyruvate kinase M2 (PKM2) has abnormally high expression in various tumor cells, and it has been implicated in the survival of tumors. However, whether and how Res inhibits PKM2 expression is poorly understood. In the present study, we found that treatment with Res inhibited cell proliferation and induced cell apoptosis. The IC 50 values of Res against DLD1, HeLa, and MCF-7 cells were 75 ± 4.54, 50 ± 3.65, and 50 ± 3.32 μM, respectively. To elucidate mechanisms underlying its antitumor activities, serial experiments were performed. Results showed that reduction of PKM2 expression in tumor cells by Res treatment increased the expression of ER stress and mitochondrial fission proteins but reduced cell viability and the levels of fusion proteins. These phenomena were reversed by artificial overexpression of PKM2. Quantitative analyses showed that the expression of microRNA-326 (miR-326) was increased upon Res treatment. Treatment with the miR-326 mimic reduced PKM2 expression, promoting recovery from ER stress and mitochondrial fission. Overall, these results demonstrate that miR-326/PKM2-mediated ER stress and mitochondrial dysfunction participate in apoptosis induced by Res. These results provide novel insight into the molecular mechanisms by which Res suppresses tumors and further support for the use of Res as an antitumor drug.

Silencing PRDM14 expression by an innovative RNAi therapy inhibits stemness, tumorigenicity, and metastasis of breast cancer

PubMed Central

Taniguchi, Hiroaki; Hoshino, Daisuke; Moriya, Chiharu; Zembutsu, Hitoshi; Nishiyama, Nobuhiro; Yamamoto, Hiroyuki; Kataoka, Kazunori; Imai, Kohzoh

2017-01-01

PR domain zinc finger protein 14 (PRDM14) maintains stemness in embryonic stem cells via epigenetic mechanisms. Although PRDM14 is elevated in several cancers, it is unclear if and how PRDM14 confers stem cell-like properties and epigenetic changes to cancer cells. Here, we examined the phenotypic characteristics and epigenetic and gene expression profiles of cancer cells that differentially express PRDM14, and assessed the potential of PRDM14-targeted cancer therapy. PRDM14 expression was markedly increased in many different cancer types and correlated with poor survival of breast cancer patients. PRDM14 conferred stem cell-like phenotypes to cancer cells and regulated the expression of genes involved in cancer stemness, metastasis, and chemoresistance. PRDM14 also reduced the methylation of proto-oncogene and stemness gene promoters and PRDM14-binding regions were primarily occupied by histone H3 Lys-4 trimethylation (H3K4me3), both of which are positively correlated with gene expression. Moreover, strong PRDM14 binding sites coincided with promoters containing both H3K4me3 and H3K27me3 histone marks. Using calcium phosphate hybrid micelles as an RNAi delivery system, silencing of PRDM14 expression by chimera RNAi reduced tumor size and metastasis in vivo without causing adverse effects. Conditional loss of PRDM14 function also improved survival of MMTV-Wnt-1 transgenic mice, a spontaneous model of murine breast cancer. Our findings suggest that PRDM14 inhibition may be an effective and novel therapy for cancer stem cells. PMID:28423353

Tyrosine Kinase Btk Is Required for NK Cell Activation

PubMed Central

Bao, Yan; Zheng, Jian; Han, Chaofeng; Jin, Jing; Han, Huanxing; Liu, Yinping; Lau, Yu-Lung; Tu, Wenwei; Cao, Xuetao

2012-01-01

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk−/− NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk−/− mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk−/− mice after the adoptive transfer of Btk+/+ NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response. PMID:22589540

Tyrosine kinase Btk is required for NK cell activation.

PubMed

Bao, Yan; Zheng, Jian; Han, Chaofeng; Jin, Jing; Han, Huanxing; Liu, Yinping; Lau, Yu-Lung; Tu, Wenwei; Cao, Xuetao

2012-07-06

Bruton tyrosine kinase (Btk) is not only critical for B cell development and differentiation but is also involved in the regulation of Toll-like receptor-triggered innate response of macrophages. However, whether Btk is involved in the regulation of natural killer (NK) cell innate function remains unknown. Here, we show that Btk expression is up-regulated during maturation and activation of mouse NK cells. Murine Btk(-/-) NK cells have decreased innate immune responses to the TLR3 ligand, with reduced expressions of IFN-γ, perforin, and granzyme-B and decreased cytotoxic activity. Furthermore, Btk is found to promote TLR3-triggered NK cell activation mainly by activating the NF-κB pathway. Poly(I:C)-induced NK cell-mediated acute hepatitis was observed to be attenuated in Btk(-/-) mice or the mice with in vivo administration of the Btk inhibitor. Correspondingly, liver damage was aggravated in Btk(-/-) mice after the adoptive transfer of Btk(+/+) NK cells, further indicating that Btk-mediated NK cell activation contributes to TLR3-triggered acute liver injury. Importantly, reduced TLR3-triggered activation of human NK cells was observed in Btk-deficient patients with X-linked agammaglobulinemia, as evidenced by the reduced IFN-γ, CD69, and CD107a expression and cytotoxic activity. These results indicate that Btk is required for activation of NK cells, thus providing insight into the physiological significance of Btk in the regulation of immune cell functions and innate inflammatory response.

A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts

PubMed Central

Liang, Yayun; Mafuvadze, Benford; Besch-Williford, Cynthia; Hyder, Salman M

2018-01-01

Background Between 30 and 40% of human breast cancers express a defective tumor suppressor p53 gene. Wild-type p53 tumor suppressor protein promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor–dependent angiogenesis, whereas mutant p53 protein (mtp53) lacks these functions, resulting in tumor cell survival and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for combating mtp53-expressing breast cancer. Methods In this study, we sought to determine whether administration of APR-246, a small-molecule drug that restores p53 function, in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits advanced hormone-dependent breast cancer tumor growth. Results APR-246 reduced cell viability in mtp53-expressing BT-474 and T47-D human breast cancer cells in vitro, and significantly induced apoptosis in a dose-dependent manner. However, APR-246 did not reduce cell viability in MCF-7 breast cancer cells, which express wild-type p53. We next examined APR-246’s anti-tumor effects in vivo using BT-474 and T47-D tumor xenografts established in female nude mice. Tumor-bearing mice were treated with APR-246 and/or 2aG4 and tumor volume followed over time. Tumor growth was more effectively suppressed by combination treatment than by either agent alone, and combination therapy completely eradicated some tumors. Immunohistochemistry analysis of tumor tissue sections demonstrated that combination therapy more effectively induced apoptosis and reduced cell proliferation in tumor xenografts than either agent alone. Importantly, combination therapy dramatically reduced the density of blood vessels, which serve as the major route for tumor metastasis, in tumor xenografts compared with either agent alone. Conclusion Based on our findings, we contend that breast tumor growth might effectively be controlled by simultaneous targeting of mtp53 protein and tumor blood vessels in mtp53-expressing cancers. PMID:29606888

Emodin from Polygonum multiflorum ameliorates oxidative toxicity in HT22 cells and deficits in photothrombotic ischemia.

PubMed

Ahn, Sung Min; Kim, Ha Neui; Kim, Yu Ri; Choi, Young Whan; Kim, Cheol Min; Shin, Hwa Kyoung; Choi, Byung Tae

2016-07-21

Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. Emodin, an active component from Polygonum multiflorum Thunb., provides benefits for brain disturbances induced by severe cerebral injury. We investigated the neuroprotective effect of emodin from Polygonum multiflorum Thunb. against glutamate-induced oxidative toxicity and cerebral ischemia. For examination of neuroprotective effects of emodin, cell viability, cytotoxicity, flow cytometry, and Western blot were performed in HT22 cells and infarct volume, behavioral tests and Western blot in a mouse model of photothrombotic ischemic stroke. Pretreatment with emodin resulted in significantly reduced glutamate-induced apoptotic cell death in HT22 cells. However, blocking of phosphatidylinositol-3 kinase (PI3K) activity with LY294002 resulted in significantly inhibited cell survival by emodin. Exposure of glutamate-treated cells to emodin induced an increase in the level of Bcl-2 expression, whereas the expression of Bax and active caspase-3 proteins was significantly reduced. In addition, treatment with emodin resulted in increased phosphorylation of Akt and cAMP response element binding protein (CREB), and expression of mature brain-derived neurotrophic factor (BDNF). This expression by emodin was also significantly inhibited by blocking of PI3K activity. In a photothrombotic ischemic stroke model, treatment with emodin resulted in significantly reduced infarct volume and improved motor function. We confirmed the critical role of the expression levels of Bcl-2/Bax, active caspase-3, phosphorylated (p)Akt, p-CREB, and mature BDNF for potent neuroprotective effects of emodin in cerebral ischemia. These results suggest that emodin may afford a significant neuroprotective effect against glutamate-induced apoptosis through activation of the PI3K/Akt signaling pathway, and subsequently enhance behavioral function in cerebral ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The atypical chemokine receptor ACKR2 drives pulmonary fibrosis by tuning influx of CCR2+ and CCR5+ IFNγ-producing γδT cells in mice.

PubMed

Russo, Remo Castro; Savino, Benedetta; Mirolo, Massimiliano; Buracchi, Chiara; Germano, Giovanni; Anselmo, Achille; Zammataro, Luca; Pasqualini, Fabio; Mantovani, Alberto; Locati, Massimo; Teixeira, Mauro M

2018-02-22

Chemokines coordinate lung inflammation and fibrosis by acting on chemokine receptors expressed on leukocytes and other cell types. Atypical chemokine receptors (ACKRs) bind, internalize and degrade chemokines, tuning homeostasis and immune responses. ACKR2 recognizes and decreases levels of inflammatory CC chemokines. The role of ACKR2 in fibrogenesis is unknown. Investigate the role of ACKR2 in the context of pulmonary fibrosis. The effects of ACKR2 expression and deficiency during inflammation and fibrosis were analyzed using a bleomycin-model of fibrosis, ACKR2-deficient mice, bone marrow chimeras and antibody-mediated leukocyte depletion. ACKR2 was up-regulated acutely in response to bleomycin and normalized over time. ACKR2-/- mice showed reduced lethality and lung fibrosis. Bone marrow chimeras showed that lethality and fibrosis depended on ACKR2 expression in pulmonary resident (non-hematopoietic) cells but not on leukocytes. ACKR2-/- mice exhibited decreased expression of tissue remodeling genes, reduced leukocyte influx, pulmonary injury, and dysfunction. ACKR2-/- mice had early-increased levels of CCL5, CCL12, CCL17 and IFNγ, and increased number of CCR2+ and CCR5+ IFNγ-producing γδT cells in the airways counterbalanced by low Th17 lymphocyte influx. There was reduced accumulation of IFNγ-producing γδT cells in CCR2-/- and CCR5-/- mice. Moreover, depletion of γδT cells worsened the clinical symptoms induced by bleomycin and reversed the phenotype of ACKR2-/- mice exposed to bleomycin. ACKR2 controls the CC chemokine expression that drives the influx of CCR2+ and CCR5+ IFNγ-producing γδT cells tuning the Th17 response that mediate pulmonary fibrosis triggered by bleomycin instillation.

The roles of ERAS during cell lineage specification of mouse early embryonic development.

PubMed

Zhao, Zhen-Ao; Yu, Yang; Ma, Huai-Xiao; Wang, Xiao-Xiao; Lu, Xukun; Zhai, Yanhua; Zhang, Xiaoxin; Wang, Haibin; Li, Lei

2015-08-01

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development. © 2015 The Authors.

TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells

PubMed Central

Sutton, Selina K.; Koach, Jessica; Tan, Owen; Liu, Bing; Carter, Daniel R.; Wilmott, James S.; Yosufi, Benafsha; Haydu, Lauren E.; Mann, Graham J.; Thompson, John F.; Long, Georgina V.; Liu, Tao; McArthur, Grant; Zhang, Xu Dong; Scolyer, Richard A.; Cheung, Belamy B.; Marshall, Glenn M.

2014-01-01

High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. PMID:25333256

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

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

Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp; Naganuma, Kaori; Kato, Kenichi

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histonemore » H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.« less

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

PubMed

Carvell, Melanie J; Marsh, Phil J; Persaud, Shanta J; Jones, Peter M

2007-01-01

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation. Copyright (c) 2007 S. Karger AG, Basel.

Vitamin K2 and cotylenin A synergistically induce monocytic differentiation and growth arrest along with the suppression of c-MYC expression and induction of cyclin G2 expression in human leukemia HL-60 cells.

PubMed

Maniwa, Yasuhisa; Kasukabe, Takashi; Kumakura, Shunichi

2015-08-01

Although all-trans retinoic acid (ATRA) is a standard and effective drug used for differentiation therapy in acute promyelocytic leukemia, ATRA-resistant leukemia cells ultimately emerge during this treatment. Therefore, the development of new drugs or effective combination therapy is urgently needed. We demonstrate that the combined treatment of vitamin K2 and cotylenin A synergistically induced monocytic differentiation in HL-60 cells. This combined treatment also synergistically induced NBT-reducing activity and non-specific esterase-positive cells as well as morphological changes to monocyte/macrophage-like cells. Vitamin K2 and cotylenin A cooperatively inhibited the proliferation of HL-60 cells in short-term and long-term cultures. This treatment also induced growth arrest at the G1 phase. Although 5 µg/ml cotylenin A or 5 µM vitamin K2 alone reduced c-MYC gene expression in HL-60 cells to approximately 45% or 80% that of control cells, respectively, the combined treatment almost completely suppressed c-MYC gene expression. We also demonstrated that the combined treatment of vitamin K2 and cotylenin A synergistically induced the expression of cyclin G2, which had a positive effect on the promotion and maintenance of cell cycle arrest. These results suggest that the combination of vitamin K2 and cotylenin A has therapeutic value in the treatment of acute myeloid leukemia.

Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Offidani, Massimo; Amantini, Consuelo; Gentili, Silvia; Soriani, Alessandra; Cardinali, Claudio; Leoni, Pietro; Santoni, Giorgio

2016-11-22

Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities.

Mitochondrial β-Carotene 9',10' Oxygenase Modulates Prostate Cancer Growth via NF-κB Inhibition: A Lycopene-Independent Function.

PubMed

Gong, Xiaoming; Marisiddaiah, Raju; Zaripheh, Susan; Wiener, Doris; Rubin, Lewis P

2016-10-01

Despite numerous inquiries into protective roles of lycopene in prostate cancer prevention or therapy, little is known about mechanisms by which lycopene or its metabolites inhibit prostate cancer. The enzyme β-carotene 9',10'-oxygenase (BCO2), which catalyzes asymmetric cleavage of several carotenoids, is the principal regulator of lycopene metabolism, but the range of BCO2 biological functions is incompletely understood. This study investigated expression and functional roles of BCO2 in human prostate cancer. Expression of the bco2 gene is dramatically decreased in prostate cancer tissue and in a range of prostate cancer cell lines as compared with nonneoplastic prostate tissue and normal prostatic epithelial cells, respectively. Inhibition of DNA methyltransferase activity restored bco2 expression in prostate cancer cell lines tested. Treatment with lycopene or its metabolite, apo-10-lycopenal, also increased bco2 expression and reduced cell proliferation in androgen-sensitive cell lines, but lycopene neither altered bco2 expression nor cell growth in androgen-resistant cells. Notably, restoring bco2 expression in prostate cancer cells inhibited cell proliferation and colony formation, irrespective of lycopene exposure. Exogenous expression of either wild-type BCO2 or a mutant (enzymatically inactive) BCO2 in prostate cancer cells reduced NF-κB activity and decreased NF-κB nuclear translocation and DNA binding. Together, these results indicate epigenetic loss of BCO2 expression is associated with prostate cancer progression. Moreover, these findings describe previously unanticipated functions of BCO2 that are independent of its enzymatic role in lycopene metabolism. This study identifies BCO2 as a tumor suppressor in prostate cancer. BCO2-mediated inhibition of NF-κB signaling implies BCO2 status is important in prostate cancer progression. Mol Cancer Res; 14(10); 966-75. ©2016 AACR. ©2016 American Association for Cancer Research.

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

Peippo, Minna, E-mail: minna.peippo@utu.fi; MediCity Research Laboratory, University of Turku; Gardberg, Maria, E-mail: maria.gardberg@utu.fi

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanomamore » cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.« less

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

PubMed

Behjati, Mohaddeseh; Hashemi, Mohammad; Kazemi, Mohammad; Salehi, Mansoor; Javanmard, Shaghayegh Haghjooy

2017-01-01

Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels ( P = 0.09) and vWF ( P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Monocarboxylate transporters MCT1 and MCT4 are independent prognostic biomarkers for the survival of patients with clear cell renal cell carcinoma and those receiving therapy targeting angiogenesis.

PubMed

Cao, Yan-Wei; Liu, Yong; Dong, Zhen; Guo, Lei; Kang, En-Hao; Wang, Yong-Hua; Zhang, Wei; Niu, Hai-Tao

2018-04-12

Prognostic biomarkers for patients with clear cell renal cell carcinoma (ccRCC), particularly those receiving therapy targeting angiogenesis, are not well established. In this study, we examined the correlations of monocarboxylate transporter 1 (MCT1) and MCT4, 2 critical transporters for glycolytic metabolism, with various clinicopathological parameters as well as survival of patients with ccRCC and those treated with vascular endothelial growth factor receptor (VEGFR) inhibitors. A cohort of 150 ccRCC patients were recruited into this study. All patients underwent radical or partial nephrectomy as the first-line treatment, and 38 received targeted therapy (sorafenib or sunitinib) after the surgery. Expression levels of MCT1, MCT4, and CD34 were examined by immunohistochemistry. Correlations between MCT1 or MCT4 expression and different clinicopathological parameters or patient survival were analyzed among all as well as patients receiving targeted therapy. MCT1 or MCT4 expression did not significantly correlate with sex, age, tumor diameter, microvascular density, tumor staging, pathological Furmann grade, or MSKCC (P>0.05). High expression of either MCT1 or MCT4 significantly correlated with reduced overall survival (OS) and progression-free survival (PFS) among the total cohort of ccRCC patients. For patients receiving targeted therapy, high expression of either MCT1 or MCT4 significantly correlated with reduced PFS, but not OS. Both conditions were independent prognostic biomarkers for reduced PFS among all patients or those receiving targeted therapy. MCT1 and MCT4 are prognostic biomarkers for patients with ccRCC or those receiving targeted therapy. High expression of these 2 proteins predicts reduced PFS in these patients. Copyright © 2018 Elsevier Inc. All rights reserved.

Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis

PubMed Central

Xiong, Si-qi; Jiang, Hai-bo; Li, Yan-xiu; Li, Hai-bo; Xu, Hui-zhuo; Wu, Zhen-kai; Zheng, Wei

2016-01-01

Objective To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. Methods siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor–reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. Results The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. Conclusions Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF. PMID:27994436

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

PubMed Central

Knight, B L; Patel, D D; Soutar, A K

1983-01-01

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow. PMID:6305342

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon

PubMed Central

Kon, Risako; Tsubota, Yuika; Minami, Moe; Kato, Saki; Matsunaga, Yukari; Kimura, Hiroshi; Murakami, Yuta; Fujikawa, Tetsuya; Sakurai, Ryoya; Tomimoto, Rei; Machida, Yoshiaki; Ikarashi, Nobutomo; Sugiyama, Kiyoshi

2018-01-01

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea. PMID:29316651

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon.

PubMed

Kon, Risako; Tsubota, Yuika; Minami, Moe; Kato, Saki; Matsunaga, Yukari; Kimura, Hiroshi; Murakami, Yuta; Fujikawa, Tetsuya; Sakurai, Ryoya; Tomimoto, Rei; Machida, Yoshiaki; Ikarashi, Nobutomo; Sugiyama, Kiyoshi

2018-01-06

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea.

Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoidinduced leucine zipper

PubMed Central

Lim, Wonchung; Park, Choa; Shim, Myeong Kuk; Lee, Yong Hee; Lee, You Mie; Lee, YoungJoo

2014-01-01

Background and Purpose The COX-2/PGE2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumourigenesis. However, the mechanism by which glucocorticoid receptors (GRs) inhibit COX-2 during hypoxia has not been elucidated. Hence, we explored the mechanisms underlying glucocorticoid-mediated inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. Experimental Approach The expressions of COX-2 and glucocorticoid-induced leucine zipper (GILZ) in A549 cells were determined by Western blot and/or quantitative real time-PCR respectively. The anti-invasive effect of GILZ on A549 cells was evaluated using the matrigel invasion assay. Key Results The hypoxia-induced increase in COX-2 protein and mRNA levels and promoter activity were suppressed by dexamethasone, and this effect of dexamethasone was antagonized by the GR antagonist RU486. Overexpression of GILZ in A549 cells also inhibited hypoxia-induced COX-2 expression levels and knockdown of GILZ reduced the glucocorticoid-mediated inhibition of hypoxia-induced COX-2 expression, indicating that the inhibitory effects of dexamethasone on hypoxia-induced COX-2 are mediated by GILZ. GILZ suppressed the expression of hypoxia inducible factor (HIF)-1α at the protein level and affected its signalling pathway. Hypoxia-induced cell invasion was also dramatically reduced by GILZ expression. Conclusion and Implications Dexamethasone-induced upregulation of GILZ not only inhibits the hypoxic-evoked induction of COX-2 expression and cell invasion but further blocks the HIF-1 pathway by destabilizing HIF-1α expression. Taken together, these findings suggest that the suppression of hypoxia-induced COX-2 by glucocorticoids is mediated by GILZ. Hence, GILZ is a potential key therapeutic target for suppression of inflammation under hypoxia. PMID:24172143

4-Phenylbutyrate modulates ubiquitination of hepatocanalicular MRP2 and reduces serum total bilirubin concentration.

PubMed

Hayashi, Hisamitsu; Mizuno, Tadahaya; Horikawa, Reiko; Nagasaka, Hironori; Yabuki, Takashi; Takikawa, Hajime; Sugiyama, Yuichi

2012-05-01

Multidrug resistance-associated protein 2 (in humans, MRP2; in rodents, Mrp2) mediates biliary excretion of bilirubin glucuronides. Therefore, upregulation of MRP2/Mrp2 expression may improve hyperbilirubinemia. We investigated the effects of 4-phenylbutyrate (4PBA), a drug used to treat ornithine transcarbamylase deficiency (OTCD), on the cell surface expression and transport function of MRP2/Mrp2 and serum T-Bil concentration. MRP2-expressing MDCKII (MRP2-MDCKII) cells and rats were studied to explore the change induced by 4PBA treatment in the cell surface expression and transport function of MRP2/Mrp2 and its underlying mechanism. Serum and liver specimens from OTCD patients were analyzed to examine the effect of 4PBA on hepatic MRP2 expression and serum T-Bil concentration in humans. In MRP2-MDCKII cells and the rat liver, 4PBA increased the cell surface expression and transport function of MRP2/Mrp2. In patients with OTCD, hepatic MRP2 expression increased and serum T-Bil concentration decreased significantly after 4PBA treatment. In vitro studies designed to explore the mechanism underlying this drug action suggested that cell surface-resident MRP2/Mrp2 is degraded via ubiquitination-mediated targeting to the endosomal/lysosomal degradation pathway and that 4PBA inhibits the degradation of cell surface-resident MRP2/Mrp2 by reducing its susceptibility to ubiquitination. 4PBA activates MRP2/Mrp2 function through increased expression of MRP2/Mrp2 at the hepatocanalicular membrane by modulating its ubiquitination, and thereby decreases serum T-Bil concentration. 4PBA has thus therapeutic potential for improving hyperbilirubinemia. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology.

PubMed

Mullen, Brian R; Ross, Brennan; Chou, Joan Wang; Khankan, Rana; Khialeeva, Elvira; Bui, Kimberly; Carpenter, Ellen M

2016-06-01

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors-reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon-gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology. © The Author(s) 2016.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

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

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain.

PubMed

Sundararaj, Kamala P; Thiyagarajan, Thirumagal; Molano, Ivan; Basher, Fahmin; Powers, Thomas W; Drake, Richard R; Nowling, Tamara K

2015-12-15

The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels. Copyright © 2015 by The American Association of Immunologists, Inc.

The inhibition of 45A ncRNA expression reduces tumor formation, affecting tumor nodules compactness and metastatic potential in neuroblastoma cells

PubMed Central

Russo, Debora; Poggi, Alessandro; Villa, Federico; Brizzolara, Antonella; Canale, Claudio; Mescola, Andrea; Daga, Antonio; Russo, Claudio; Nizzari, Mario; Florio, Tullio; Menichini, Paola; Pagano, Aldo

2017-01-01

We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA. 45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach. PMID:28029658

The inhibition of 45A ncRNA expression reduces tumor formation, affecting tumor nodules compactness and metastatic potential in neuroblastoma cells.

PubMed

Penna, Ilaria; Gigoni, Arianna; Costa, Delfina; Vella, Serena; Russo, Debora; Poggi, Alessandro; Villa, Federico; Brizzolara, Antonella; Canale, Claudio; Mescola, Andrea; Daga, Antonio; Russo, Claudio; Nizzari, Mario; Florio, Tullio; Menichini, Paola; Pagano, Aldo

2017-01-31

We recently reported the in vitro over-expression of 45A, a RNA polymerase III-transcribed non-coding (nc)RNA, that perturbs the intracellular content of FE65L1 affecting cell proliferation rate, short-term response to genotoxic stress, substrate adhesion capacity and, ultimately, increasing the tumorigenic potential of human neuroblastoma cells. In this work, to deeply explore the mechanism by which 45A ncRNA contributes to cancer development, we targeted in vitro and in vivo 45A levels by the stable overexpression of antisense 45A RNA.45A downregulation leads to deep modifications of cytoskeleton organization, adhesion and migration of neuroblastoma cells. These effects are correlated with alterations in the expression of several genes including GTSE1 (G2 and S phase-expressed-1), a crucial regulator of tumor cell migration and metastatic potential. Interestingly, the downregulation of 45A ncRNA strongly affects the in vivo tumorigenic potential of SKNBE2 neuroblastoma cells, increasing tumor nodule compactness and reducing GTSE1 protein expression in a subcutaneous neuroblastoma mouse model. Moreover, intracardiac injection of neuroblastoma cells showed that downregulation of 45A ncRNA also influences tumor metastatic ability. In conclusion, our data highlight a key role of 45A ncRNA in cancer development and suggest that its modulation might represent a possible novel anticancer therapeutic approach.

The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

PubMed

Wang, Hong-Xing; Gillio-Meina, Carolina; Chen, Shuli; Gong, Xiang-Qun; Li, Tony Y; Bai, Donglin; Kidder, Gerald M

2013-08-01

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

Silibinin-induced glioma cell apoptosis by PI3K-mediated but Akt-independent downregulation of FoxM1 expression.

PubMed

Zhang, Mingjie; Liu, Yunhui; Gao, Yun; Li, Shaoyi

2015-10-15

The oncogenic transcription factor Forkhead box M1 (FoxM1) is overexpressed in many human tumors, including glioma. As a critical regulator of the cell cycle and apoptosis-related genes, FoxM1 is a potential therapeutic target against human malignant glioma. Silibinin, a flavonoid isolated from Silybum marianum, dose-dependently reduced glioma cell proliferation, promoted apoptosis, and downregulated FoxM1 expression. Knockdown of FoxM1 by small hairpin RNA (shRNA) transfection also promoted glioma cell apoptosis and augmented the antiproliferative and pro-apoptotic properties of silibinin. Moreover, silibinin increased caspase-3 activation, upregulated pro-apoptotic Bax, and suppressed anti-apoptotic Bcl-2 expression, effects enhanced by FoxM1 knockdown. Silibinin treatment suppressed U87 cell PI3K phospho-activation, and simultaneous silibinin exposure, FoxM1 knockdown, and PI3K inhibition additively increased U87 cell apoptosis. Furthermore, PI3K inhibition reduced FoxM1 expression. Akt activity was also suppressed by FoxM1 downregulation but Akt inhibition did not alter FoxM1 expression. Thus, silibinin likely inhibited glioma cell proliferation and induced apoptosis through inactivation of PI3K and FoxM1, leading to activation of the mitochondrial apoptotic pathway. FoxM1 may be a novel target for chemotherapy against human glioma. Copyright © 2015 Elsevier B.V. All rights reserved.

Overexpression of c-jun, junB, or junD affects cell growth differently.

PubMed

Castellazzi, M; Spyrou, G; La Vista, N; Dangy, J P; Piu, F; Yaniv, M; Brun, G

1991-10-15

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.

Overexpression of c-jun, junB, or junD affects cell growth differently.

PubMed Central

Castellazzi, M; Spyrou, G; La Vista, N; Dangy, J P; Piu, F; Yaniv, M; Brun, G

1991-01-01

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth. Images PMID:1924349

[Effects of moxibustion with seed-sized moxa cone on apoptosis of myocardial cells after sport fatigue in mice].

PubMed

Xu, Huiqian; Hu, Yin; Gu, Yihuang; Zhang, Hongru

2015-03-01

To observe the effects of moxibustion on factors related with apoptosis of myocardial cells after sports fatigue in mice as well as the relationship among histone acetyltransferases p300 (p300), CREB binding protein (CBP) and cell apoptosis to discuss the role of p300 and CBP in moxibustion against apoptosis of myocardial cells. Sixty clean-grade male Kunming mice were randomly divided into a control group, a sport group and a moxibustion group, 20 cases in each one. Mice in all group received identical feeding environment. Mice in the control group did not received sport nor moxibustion; mice in the sport group and moxibustion group received non-weight swimming training which lasted from 30 min per day to 90 min per day gradually for 21 days; 1 h after swimming training, mice in the moxibustion group received moxibustion with seed-sized moxa cone at "Zusanli" (ST 36) and "Guanyuan" (CV 4), 5 cones at each acupoint, once a day for 21 days. 24 h after the final swimming training, cardiac muscle tissue was collected to test factor associated suicide (Fas), B cell lymphoma/lewkmia-2 (Bcl-2) by immunohistochemical method and expression of p300 and CBP. Compared with the control group, the apoptosis rate of myocardial cells in the sport group was significantly increased (P<0.01), and apoptosis body with dense distribution and deep coloring can be seen in the field of microscope; the expression of Fas protein was significantly increased (P<0.01), and expression of Bcl-2, p300 and CBP was reduced (all P<0.01). The equally distributed apoptosis body with slight coloring was seen in the moxibustion group. Compared with the sport group, the apoptosis rate of myocardial cells in the moxibustion group was significantly reduced (P<0.05); the expression of Fas protein was significantly reduced (P<0.05), and expression of Bcl-2, p300 and CBP was increased (all P<0.05). Moxibustion could promote the expression of p300 and CBP in myocardial cells after sports fatigue in mice to inhibit the starting of apoptotic process, therefore reducing the apoptosis of myocardial cells after heavy exercise and protecting heart function.

A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells

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

Kojima, Keitaro; Department of Longevity and Aging Research, Gifu International Institute of Biotechnology, 1-1 Naka-fudogaoka, Kakamigahara, Gifu 504-0838; Ohhashi, Riyako

2008-08-29

SIRT1, which belongs to the family of type III histone deacetylase, is implicated in diverse cellular processes. We have determined the expression levels of SIRT1 in human prostate cancer cell lines and have examined the roles of SIRT1 in cell growth and chemoresistance. SIRT1 expression was markedly up-regulated in androgen-refractory PC3 and DU145 cells compared with androgen-sensitive LNCaP cells and its expression level was correlated with cell growth in PC3 cells. Treatment with a SIRT1 inhibitor, sirtinol, inhibited cell growth and increased sensitivity to camptothecin and cisplatin. Silencing of SIRT1 expression by siRNA also suppressed cell proliferation and reduced camptothecinmore » resistance in PC3 cells, mimicking the chemosensitizing effect caused by sirtinol. Also in DU145 cells, sirtinol treatment enhanced sensitivity to camptothecin and cisplatin. These results suggest that up-regulation of SIRT1 expression may play an important role in promoting cell growth and chemoresistance in androgen-refractory PC3 and DU145 cells.« less