Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

PubMed

Zhang, Lixia; Li, Yan; Li, Xiaoyan; Zhang, Qing; Qiu, Shaowei; Zhang, Qi; Wang, Min; Xing, Haiyan; Rao, Qing; Tian, Zheng; Tang, Kejing; Wang, Jianxiang; Mi, Yingchang

2017-09-01

The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryl iodine]-1, 3-diphenyl-2-thiobarbituric acid (UCF-101) treatment was detected with reverse transcription-quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF-101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF-101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF-101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF-101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF-101, resulting in the upregulation of the WT1 protein level. However; UCF-101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF-101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal-related kinase (ERK1/2) pathway markedly and persistently, but UCF-101

The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

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

Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming

2012-11-15

Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catecholmore » enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.« less

Electrochemical K-562 cells sensor based on origami paper device for point-of-care testing.

PubMed

Ge, Shenguang; Zhang, Lina; Zhang, Yan; Liu, Haiyun; Huang, Jiadong; Yan, Mei; Yu, Jinghua

2015-12-01

A low-cost, simple, portable and sensitive paper-based electrochemical sensor was established for the detection of K-562 cell in point-of-care testing. The hybrid material of 3D Au nanoparticles/graphene (3D Au NPs/GN) with high specific surface area and ionic liquid (IL) with widened electrochemical windows improved the good biocompatibility and high conductivity was modified on paper working electrode (PWE) by the classic assembly method and then employed as the sensing surface. IL could not only enhance the electron transfer ability but also provide sensing recognition interface for the conjugation of Con A with cells, with the cell capture efficiency and the sensitivity of biosensor strengthened simultaneously. Concanavalin A (Con A) immobilization matrix was used to capture cells. As proof-of-concept, the paper-based electrochemical sensor for the detection of K-562 cells was developed. With such sandwich-type assay format, K-562 cells as model cells were captured on the surface of Con A/IL/3D AuNPs@GN/PWE. Con A-labeled dendritic PdAg NPs were captured on the surface of K-562 cells. Such dendritic PdAg NPs worked as catalysts promoting the oxidation of thionine (TH) by H2O2 which was released from K-562 cells via the stimulation of phorbol 12-myristate-13-acetate (PMA). Therefore, the current signal response was dependent on the amount of PdAg NPs and the concentration of H2O2, the latter of which corresponded with the releasing amount from cells. So, the detection method of K-562 cell was also developed. Under optimized experimental conditions, 1.5×10(-14) mol of H2O2 releasing from each cell was calculated. The linear range and the detection limit for K-562 cells were determined to be 1.0×10(3)-5.0×10(6) cells/mL and 200 cells/mL, respectively. Such as-prepared sensor showed excellent analytical performance with good fabrication reproducibility, acceptable precision and satisfied accuracy, providing a novel protocol in point-of-care testing of cells

[Effects of Aptamer-siRNA Nucleic Acid Compound on Growth and Apoptosis in Myeloid Leukemia Cell Line K562].

PubMed

Ping, Juan; Shen, Zhi-Hui; Wang, Bao-Quan; Zhao, Na; Li, Rui; Li, Mian; Pang, Xiao-Bin; Chen, Chuan-Bo

2015-04-01

To explore the effects of aptamer-siRNA nucleic acid compound on growth and apoptosis in myeloid leukemia cell line K562. the changes of cellular morphology and structure were observed by using fluorescence microscope, laser confocal microscope, JEM-4000EX transmission electron microscopy; MTT assay were performed to evaluate the sensibility of K562 cells to aptamer-siRNA compound, the apoptosis was detected by DNA gel electro-phoresis. The remarkably changes of morphology and structure of K562 cells treated with 200 µmol/L aptamer-siRNA were observed under fluorescence microscopy and electromicroscopy. As compared with control, the aptamer-siRNA compound showed more inhibitory effect on K562 cells and there was significant difference (P<0.05). The MTT assay showed that the IC50 value of aptamer-siRNA compound for K562 cells was 150 µmol/L. According to agarose gel electrophoresis observation, when the aptamer-siRNA compound showed effect on K562 cells, the typical DNA lader could be observed. The aptamer-siRNA compound can significantly induce K562 cell apoptosis, and provide reference for gene therapy of patients with chronic myelocytic lenkemia.

Research on the effect of formononetin on photodynamic therapy in K562 cells.

PubMed

Sun, Dan; Lu, Yao; Zhang, Su-Juan; Wang, Kai-Ge; Sun, Zhe

2017-10-01

At the present time, many cancer patients combine some forms of complementary and alternative medicine therapies with their conventional therapies. The most common choice of these therapies is the use of antioxidants. Formononetin is presented in different foods. It has a variety of biological activities including antioxidant and anti-cancer properties. On account of its antioxidant activity, formononetin might protect cancer cells from free radical damage in photodynamic therapy (PDT) during which reactive oxygen species (ROS) production was stimulated leading to irreversible tumor cell injury. In this study, the influence of formononetin on K562 cells in PDT was demonstrated. The results showed that formononetin supplementation alone did not affect the lipid peroxidation, DNA damage and apoptosis in K562 cells. It increases the lipid peroxidation, DNA damage and apoptosis in K562 cells induced by PDT. The singlet oxygen quencher sodium azide suppresses the apoptosis induced by PDT with formononetin. In conclusion, formononetin consumption during PDT increases the effectiveness of cancer therapy on malignant cells. The effect of antioxidants on PDT maybe was determined by its sensitization ability to singlet oxygen.

Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

PubMed

Tsutsumi, T; Tokumura, A; Kitazawa, S

1998-02-05

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between

Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

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

Yao, Jingyun; Wei, Xing; Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai

Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressedmore » MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line. - Highlights: • Chaetominine enhanced chemosensitivity of ADR against K562/Adr cells. • Chaetominine increased intracellular ADR levels via inhibiting MRP1. • Chaetominine induced apoptosis of K562/Adr cells through upregulation of ROS and modulation of Bax/Bcl-2. • Inhibition of MRP1 and Nrf2 by chaetominine treatment was correlative with blockade of PI3K/Akt signaling.« less

3'-Geranyl-mono-substituted chalcone Xanthoangelovl induces apoptosis in human leukemia K562 cells via activation of mitochondrial pathway.

PubMed

Teng, Yuou; Wang, Lixin; Liu, Huan; Yuan, Yuan; Zhang, Qian; Wu, Meng; Wang, Luyao; Wang, Haomeng; Liu, Zhen; Yu, Peng

2017-01-05

3'-Geranyl-mono-substituted chalcone Xanthoangelol (1b), a chalcone derivative, was previously reported to show selective cytotoxicity against human chronic myelogenous leukemia K562 cells with a half-maximal inhibitory concentration (IC 50 ) of 3.98 μM. In the present study, we investigated the molecular mechanism underlying the cytotoxicity of 1b in K562 cells. Treatment with compound 1b caused K562 cells to adopt a typical apoptotic morphology. Flow cytometric analysis also confirmed the presence of an apoptotic cell population following treatment of Annexin-V-FITC and propidium iodide (PI) double-labeled K562 cells with 1b. Furthermore, we observed dissipation of the mitochondrial membrane potential, caspase-3 activation, and a reduction of the Bcl-2/Bax ratio in these cells, which suggest that the mitochondrial apoptotic pathway is induced by 1b in K562 cells. Collectively, our findings demonstrate that compound 1b notably induces mitochondrial-mediated apoptosis in K562 cells, which might have a potential anticancer activity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Small Molecule TH-39 Potentially Targets Hec1/Nek2 Interaction and Exhibits Antitumor Efficacy in K562 Cells via G0/G1 Cell Cycle Arrest and Apoptosis Induction.

PubMed

Zhu, Yongxia; Wei, Wei; Ye, Tinghong; Liu, Zhihao; Liu, Li; Luo, Yong; Zhang, Lidan; Gao, Chao; Wang, Ningyu; Yu, Luoting

2016-01-01

Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia. © 2016 The Author(s) Published by S. Karger AG, Basel.

Glycometabolic adaptation mediates the insensitivity of drug-resistant K562/ADM leukaemia cells to adriamycin via the AKT-mTOR/c-Myc signalling pathway.

PubMed

Zhang, Xueyan; Ai, Ziying; Chen, Jing; Yi, Juan; Liu, Zhuan; Zhao, Huaishun; Wei, Hulai

2017-04-01

In human leukaemia, resistance to chemotherapy leads to treatment ineffectiveness or failure. Previous studies have indicated that cancers with increased levels of aerobic glycolysis are insensitive to numerous forms of chemotherapy and respond poorly to radiotherapy. Whether glycolysis serves a key role in drug resistance of leukaemia cells remains unclear. The present study systematically investigated aerobic glycolytic alterations and regulation in K562/adriamycin (ADM) multidrug‑resistant (MDR) and ADM‑sensitive K562 leukaemia cells in normoxia, and the association between drug resistance and improper glycometabolism. The cell proliferating activity was assessed with an MTT colorimetric assay, glycolysis, including glucose consumption, lactate export and key‑enzyme activity was determined by corresponding commercial testing kits. The expression levels of hexokinase‑II (HK‑II), lactate dehydrogenase A (LDHA), glucose transporter‑4 (GLUT‑4), AKT, p‑AKT473/308, mammalian target of rapamycin (mTOR), p‑mTOR, c‑Myc and hypoxia‑inducible factor‑1α (HIF‑1α) were analyzed by western blot or reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). K562/ADM cells exhibited increased glucose consumption and lactate accumulation, increased lactate dehydrogenase, hexokinase and pyruvate kinase activities, and reduced phosphofructokinase activity. In addition, K562/ADM cells expressed significantly more HK‑II and GLUT‑4. Notably, inhibition of glycolysis effectively killed sensitive and resistant leukaemia cells and potently restored the sensitivity of MDR cells to the anticancer agent ADM. The AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (mTOR) signalling pathway, a crucial regulator of glycometabolic homeostasis, mediated over‑activation and upregulation of c‑Myc expression levels in K562/ADM cells, which directly stimulated glucose consumption and enhanced glycolysis. In conclusion, the present

Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth

PubMed Central

Wang, Xiaozhong; Zeng, Jianming; Shi, Mei; Zhao, Shiqiao; Bai, Weijun; Cao, Weixi; Tu, Zhiguang; Huang, Zonggan

2011-01-01

The protein signal transducer and activator of transcription 5 (STAT5) of the JAK/STAT pathway is constitutively activated because of its phosphorylation by tyrosine kinase activity of fusion protein BCR-ABL in chronic myelogenous leukemia (CML) cells. This study investigated the potential therapeutic effect of STAT5 decoy oligodeoxynucleotides (ODN) using leukemia K562 cells as a model. Our results showed that transfection of 21-mer-long STAT5 decoy ODN into K562 cells effectively inhibited cell proliferation and induced cell apoptosis. Further, STAT5 decoy ODN downregulated STAT5 targets bcl-xL, cyclinD1, and c-myc at both mRNA and protein levels in a sequence-specific manner. Collectively, these data demonstrate the therapeutic effect of blocking the STAT5 signal pathway by cis-element decoy for cancer characterized by constitutive STAT5 activation. Thus, our study provides support for STAT5 as a potential target downstream of BCR-ABL for CML treatment and helps establish the concept of targeting STAT5 by decoy ODN as a novel therapy approach for imatinib-resistant CML. PMID:21091189

Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells.

PubMed

Shi, Yan-Qiu; Qu, Xian-Jun; Liao, Yong-Xiang; Xie, Chun-Feng; Cheng, Yan-Na; Li, Song; Lou, Hong-Xiang

2008-04-14

Plagiochin E is a new macrocyclic bisbibenzyl compound isolated from Marchantia polymorpha. In the previous studies, we reported that when combined with fluconazole, plagiochin E had synergetic effects against the resistant strain of Candida albicans. Herein, we examined the reversal effect of plagiochin E on multidrug resistance in adriamycin-induced resistant K562/A02 cells and the parental K562 cells. Its cytotoxicity and reversal effects on multidrug resistance were assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining. The effects of plagiochin E on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity and P-glycoprotein expression on the basis of the flow cytometric technology, respectively. The results showed that plagiochin E ranging from 2 to 12 mug/ml had little cytotoxicity against K562/A02 cells. When combined with adriamycin, it significantly promoted the sensitivity of K562/A02 cells toward adriamycin through increasing intracellular accumulation of adriamycin in a dose-dependent manner. Further study demonstrated that the inhibitory effect of plagiochin E on P-glycoprotein activity was the major cause of increased stagnation of adriamycin inside K562/A02 cells, indicating that plagiochin E, as a new class of mutidrug resistance inhibitor, may effectively reverse the multidrug resistance in K562/A02 cells via inhibiting expression and drug-transport function of P-glycoprotein.

Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

PubMed Central

Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Park, Nam Gyu; Chang, Young-Chae; Lee, Young-Choon; Chung, Tae-Wook; Ha, Ki-Tae; Son, Jong-Keun

2017-01-01

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells. PMID:28133573

Anticancer activity of Pupalia lappacea on chronic myeloid leukemia K562 cells.

PubMed

Ravi, Alvala; Alvala, Mallika; Sama, Venkatesh; Kalle, Arunasree M; Irlapati, Vamshi K; Reddy, B Madhava

2012-12-05

Cancer is one of the most prominent human diseases which has enthused scientific and commercial interest in the discovery of newer anticancer agents from natural sources. Here we demonstrated the anticancer activity of ethanolic extract of aerial parts of Pupalia lappacea (L) Juss (Amaranthaceae) (EAPL) on Chronic Myeloid Leukemia K562 cells. Antiproliferative activity of EAPL was determined by MTT assay using carvacrol as a positive control. Induction of apoptosis was studied by annexin V, mitochondrial membrane potential, caspase activation and cell cycle analysis using flow cytometer and modulation in protein levels of p53, PCNA, Bax and Bcl2 ratio, cytochrome c and cleavage of PARP were studied by Western blot analysis. The standardization of the extract was performed through reverse phase-HPLC using Rutin as biomarker. The results showed dose dependent decrease in growth of K562 cells with an IC50 of 40 ± 0.01 μg/ml by EAPL. Induction of apoptosis by EAPL was dose dependent with the activation of p53, inhibition of PCNA, decrease in Bcl2/Bax ratio, decrease in the mitochondrial membrane potential resulting in release of cytochrome c, activation of multicaspase and cleavage of PARP. Further HPLC standardization of EAPL showed presence 0.024% of Rutin. Present study significantly demonstrates anticancer activity of EAPL on Chronic Myeloid Leukemia (K562) cells which can lead to potential therapeutic agent in treating cancer. Rutin, a known anti cancer compound is being reported and quantified for the first time from EAPL.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

PubMed

Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

2015-08-01

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells.

PubMed

Kim, Eunbi; Na, Sunghun; An, Borim; Yang, Se-Ran; Kim, Woo Jin; Ha, Kwon-Soo; Han, Eun-Taek; Park, Won Sun; Lee, Chang-Min; Lee, Ji Yoon; Lee, Seung-Joon; Hong, Seok-Ho

2017-03-01

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562.

PubMed

Li, Nianyi; Chen, Qinfen; Gu, Jingwen; Li, Shuang; Zhao, Guangjie; Wang, Wei; Wang, Zhicheng; Wang, Xiaoqin

2017-05-30

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV.

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562

PubMed Central

Li, Nianyi; Chen, Qinfen; Gu, Jingwen; Li, Shuang; Zhao, Guangjie; Wang, Wei; Wang, Zhicheng; Wang, Xiaoqin

2017-01-01

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV. PMID:28388554

Low‑dose radiation‑induced apoptosis in human leukemia K562 cells through mitochondrial pathways.

PubMed

Xin, Yong; Zhang, Hai-Bin; Tang, Tian-You; Liu, Gui-Hong; Wang, Jian-She; Jiang, Guan; Zhang, Long-Zhen

2014-09-01

High‑dose total body irradiation (TBI) has an established role as preparative regimen for bone‑marrow transplantation in the treatment of chronic myelogenous leukemia (CML), but this regimen still has a relatively high rate of acute and late toxicity. Low‑dose radiation (LDR) induces apoptosis of tumor cells and has numerous beneficial effects on normal tissues, including radiation homeostasis and adaptive response. Based on the previous evidence, in the present study, K562 cells were exposed to LDR, high‑dose radiation (HDR), and LDR in combination with HDR to investigate the possible mechanism of the apoptotic effect and hypersensitivity induced by LDR. The apoptotic rate increased in all radiation groups in a time‑dependent manner. An upregulation of Bax protein expression and a downregulation of Bcl‑xl in a dose‑dependent manner in human leukemia K562 cells was observed. However, the expression of p53 protein did not change in all of the radiation cell groups. The mitochondrial membrane potential (ΔΨm) in K562 cells decreased in all of the radiation cell groups in a dose‑dependent manner. Furthermore, the decrease of ΔΨm was enhanced in the LDR/HDR group compared with that in the LDR or HDR groups. The activity of caspase‑3 was enhanced in all of the radiation groups. In the LDR/HDR group, the activity of caspase‑3 was higher than that in the HDR or LDR groups. The present study provided preliminary experimental evidence of LDR being beneficial in combination with TBI in the treatment of CML.

Differentiation of K562 cells under ELF-EMF applied at different time courses.

PubMed

Ayşe, Inhan-Garip; Zafer, Akan; Sule, Oncul; Işil, Işal-Turgut; Kalkan, Tunaya

2010-08-01

The time-course of ELF-EMF application to biological systems is thought to be an important parameter determining the physiological outcome. This study investigated the effect of ELF-EMF on the differentiation of K562 cells at different time courses. ELF-EMF (50 Hz, 5 mT, 1 h) was applied at two different time-courses; first at the onset of hemin induction for 1 h, and second, daily 1 h for four days. While single exposure to ELF-EMF resulted in a decrease in differentiation, ELF-EMF applied everyday for 1 h caused an increase in differentiation. The effect of co-stressors, magnesium, and heat-shock was also determined and similar results were obtained. ELF-EMF increased ROS levels in K562 cells not treated with hemin, however did not change ROS levels of hemin treated cells indicating that ROS was not the cause. Overall, these results imply that the time-course of application is an important parameter determining the physiological response of cells to ELF-EMF.

RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

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

Wang, Houcai; Yu, Jing; Zhang, Lixia

2014-04-18

Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL)more » patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.« less

Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

PubMed

Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

2014-02-01

The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.

Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

PubMed

Ayello, Janet; Hochberg, Jessica; Flower, Allyson; Chu, Yaya; Baxi, Laxmi V; Quish, William; van de Ven, Carmella; Cairo, Mitchell S

2017-02-01

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p < 0.05); LAMP-1 (p < 0.05), granzyme B, and perforin expression (p < 0.001); and in vitro cytotoxicity against B-NHL (p < 0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p = 0.0086) and B-NHL cell numbers (p < 0.01) at 5 weeks and significantly increased survival (p < 0.001) at 10 weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting in vitro and in vivo. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

PubMed Central

Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

2013-01-01

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. PMID:23770036

The fusarin analogue NG-391 impairs nucleic acid formation in K-562 leukemia cells

USDA-ARS?s Scientific Manuscript database

The clavicipitaceous fungus Metarhizium robertsii produces the fusarin-like mycotoxin NG-391. We report on the biological effects of NG-391 on K-562 human cancer cells, obtained with radionuclide incorporation assays, along with nucleosome release and caspase assays, respectively. Our data suggests ...

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

PubMed

Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Huang, Qingchun

2017-06-01

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca 2+ ] i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia. Copyright © 2017 Elsevier B.V. All rights reserved.

A new disposable electrode for electrochemical study of leukemia K562 cells and anticancer drug sensitivity test.

PubMed

Yu, Chunmei; Zhu, Zhenkun; Wang, Li; Wang, Qiuhong; Bao, Ning; Gu, Haiying

2014-03-15

Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. In this work, a new disposable electrochemical cell sensor with low cost and simple fabrication was proposed to study the electrochemical behavior of leukemia K562 cells and the effect of anticancer drugs on cell viability. The analytical device was integrated by using ITO glass as the substrate of working electrodes and paper as the electrolytic cell. The cyclic voltammetry of the K562 cells at the disposable electrode exhibited an irreversible anodic peak and the peak current is proportional to the cell number. This anodic peak is attributed to the oxidation of guanine in cells involving two protons per transfer of two electrons. For the drug sensitivity tests, arsenic trioxide and cyclophosphamide were added to cell culture media. As a result, the electrochemical responses of the K562 cells decreased significantly. The cytotoxicity curves and results obtained corresponded well with the results of CCK-8 assays. In comparison to conventional methods, the proposed method is simple, rapid and inexpensive. More importantly, the developed sensor is supposed to be a single-use disposable device and electrodes were prepared "as new" for each experiment. We think that such disposable electrodes with these characteristics are suitable for experimental study with cancer cells or other types of pathogens for disease diagnosis, drug selection and on-site monitoring. © 2013 Elsevier B.V. All rights reserved.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells.

PubMed

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na + ,K + -ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na + ,K + -ATPase α1 in K562 cells, and significantly arrested cells in G 2 /M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells

PubMed Central

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na+,K+-ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na+,K+-ATPase α1 in K562 cells, and significantly arrested cells in G2/M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML. PMID:29118925

Analysis of the Effects of δ-Tocopherol on RAW264.7 and K562 Cells Based on 1H NMR Metabonomics.

PubMed

Lu, Yang; Li, Hui; Geng, Yue

2018-01-31

δ-Tocopherol (δ-TOH) is a form of vitamin E with higher bioactivity. In this study, we studied the bioactivity of δ-TOH using the IC 50 of δ-TOH on RAW264.7 (80 μM) and K562 (110 μM) cells. We compared the differential metabolites from the cell lines with and without δ-TOH treatment by 1 H NMR metabonomics analysis. It was found that δ-TOH affected the protein biosynthesis, betaine metabolism, and urea cycle in various ways in both cell lines. Metabolic levels of the cell lines were changed after treatment with δ-TOH as differential metabolites were produced. The betaine level in RAW264.7 cells was reduced significantly, while the l-lactic acid level in K562 cells was significantly enhanced. The metabolic changes might contribute to the switch of the respiration pattern from aerobic respiration to anaerobic respiration in K562 cells. These results are helpful in further understanding the subtoxicity of δ-TOH.

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols

PubMed Central

2010-01-01

Background Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFκB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFκB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFκB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents. Results Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFκB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism. Conclusions This demonstrates that different classes of natural NFκB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis. PMID:20438634

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

PubMed

Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

2013-08-15

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

PubMed Central

Liu, Fu-Hwa; Huang, Yu-Wen; Huang, Huei-Mei

2013-01-01

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and

Aclacinomycin A sensitizes K562 chronic myeloid leukemia cells to imatinib through p38MAPK-mediated erythroid differentiation.

PubMed

Lee, Yueh-Lun; Chen, Chih-Wei; Liu, Fu-Hwa; Huang, Yu-Wen; Huang, Huei-Mei

2013-01-01

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and

Distinct Hypericum perforatum L. total extracts exert different antitumour activity on erythroleukemic K562 cells.

PubMed

Valletta, Elena; Rinaldi, Annamaria; Marini, Mario; Franzese, Ornella; Roscetti, Gianna

2018-05-22

Total flower extracts of Hypericum perforatum L. obtained with 3 different solvent systems were tested on tumour cell line cultures by comparing two groups of plants harvested in different times and places. The extracts, characterized according to the spectroscopic profile and the hypericin content, were tested on the growth and apoptotic death of K562 cells, a human erythroleukemic cell line. Growth and apoptosis were analysed by viable cell count, flow cytometry, and fluorescence microscopy at 6, 24, and 48 hr of culture following 1 hr exposure to the extracts under investigation. Here, we show that Hypericum extracts are able to reduce the growth of K562 cells and induce different degrees and kinetics of apoptosis according to the group of plants of origin. Also, we highlighted interesting differences in terms of efficacy among the extracts, with some samples losing their effectiveness along the culture time and others able to maintain or even increase their efficacy. Furthermore, the data herein obtained confirm the role of non hypericin compounds that are present in different proportions in the two plant groups and in the extracts analysed. Copyright © 2018 John Wiley & Sons, Ltd.

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

PubMed

Liu, Jin-Yun; Liu, Zhong; Wang, Dong-Mei; Li, Man-Mei; Wang, Shao-Xiang; Wang, Rui; Chen, Jian-Ping; Wang, Yi-Fei; Yang, De-Po

2011-04-25

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

PubMed Central

Pessoto, Felipe S.; Yokomizo, Cesar H.; Prieto, Tatiana; Fernandes, Cleverton S.; Silva, Alan P.; Kaiser, Carlos R.; Basso, Ernani A.; Nantes, Iseli L.

2015-01-01

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics. PMID:26075034

Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines

PubMed Central

Tayarani-Najaran, Zahra; Makki, Farideh-Sadat; Alamolhodaei, Nafiseh-Sadat; Mojarrab, Mahdi; Emami, Seyed Ahmad

2017-01-01

Objective(s): Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies. Materials and Methods: In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol: water (1:1 v/v) extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60) and J774 as normal cells. Results: CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells. Conclusion: The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines. PMID:28293393

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

PubMed

Ren, Xueyan; Xie, Wei; Wang, Youfu; Xu, Menghuai; Liu, Fang; Tang, Mingying; Li, Chenchen; Wang, Min; Zhang, Juan

2016-08-01

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Bcr-Abl-independent mechanism of resistance to imatinib in K562 cells: Induction of cyclooxygenase-2 (COX-2) by histone deacetylases (HDACs).

PubMed

Kalle, Arunasree M; Sachchidanand, Sachchidanand; Pallu, Reddanna

2010-09-01

Our previous studies have shown that overexpression of MDR1 and cyclooygenase-2 (COX-2) resulted in resistance development to imatinib in chronic myelogenous leukemia (CML) K562 (IR-K562) cells. In the present study, the regulatory mechanism of MDR1 induction by COX-2 was investigated. A gradual overexpression of MDR1 and COX-2 during the process of development was observed. Furthermore, down regulation of MDR1 upon COX-2 knockdown by siRNA showed a decrease in the PKC levels and activation of PKC by addition of PGE(2) to K562 cells, suggesting a role for PKC in the COX-2 mediated induction of MDR1. The present study demonstrates COX-2 induction by HDACs and MDR1 induction by COX-2 via PGE(2)-cAMP-PKC-mediated pathway. Copyright 2010 Elsevier Ltd. All rights reserved.

Time-series analysis in imatinib-resistant chronic myeloid leukemia K562-cells under different drug treatments.

PubMed

Zhao, Yan-Hong; Zhang, Xue-Fang; Zhao, Yan-Qiu; Bai, Fan; Qin, Fan; Sun, Jing; Dong, Ying

2017-08-01

Chronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein. Imatinib is the first-line treatment of CML; however, many patients are resistant to this drug. In this study, we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments. GSE24946 was downloaded from the GEO database, which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5). Three drug treatment groups were considered for this study: arsenic trioxide (ATO), AMN107, and ATO+AMN107. Each group had one sample at each time point (3, 12, 24, and 48 h). Time-series genes with a ratio of standard deviation/average (coefficient of variation) >0.15 were screened, and their expression patterns were revealed based on Short Time-series Expression Miner (STEM). Then, the functional enrichment analysis of time-series genes in each group was performed using DAVID, and the genes enriched in the top ten functional categories were extracted to detect their expression patterns. Different time-series genes were identified in the three groups, and most of them were enriched in the ribosome and oxidative phosphorylation pathways. Time-series genes in the three treatment groups had different expression patterns and functions. Time-series genes in the ATO group (e.g. CCNA2 and DAB2) were significantly associated with cell adhesion, those in the AMN107 group were related to cellular carbohydrate metabolic process, while those in the ATO+AMN107 group (e.g. AP2M1) were significantly related to cell proliferation and antigen processing. In imatinib-resistant CML cells, ATO could influence genes related to cell adhesion, AMN107 might affect genes involved in cellular carbohydrate metabolism, and the combination therapy might regulate genes involved in cell proliferation.

PaDef defensin from avocado (Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis.

PubMed

Flores-Alvarez, Luis José; Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2018-06-01

Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC 50  = 97.3 μg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin. Copyright © 2018 Elsevier Ltd. All rights reserved.

Involvement of PKC and ROS in the cytotoxic mechanism of anti-leukemic decursin and its derivatives and their structure-activity relationship in human K562 erythroleukemia and U937 myeloleukemia cells.

PubMed

Kim, Hyeon Ho; Sik Bang, Sung; Seok Choi, Jin; Han, Hogyu; Kim, Ik-Hwan

2005-06-08

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Recently, various PKC modulators were used as a chemotherapeutic agent of leukemia. Decursin (1), a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. For the development of more effective anticancer agents with PKC modulation activity, 11 decursin derivatives 2-12 were chemically synthesized and evaluated for their ability to act as a tumor-suppressing PKC activator and as an antagonist to phorbol 12-myristate 13-acetate (PMA), a tumor-promoting PKC activator. In the presence of phosphatidylserine (PS), all of 12 compounds 1-12 activated PKC (mainly alpha, beta, and gamma isozymes) but only three compounds 1-3 activated PKC even in the absence of PS. Six compounds 1-6 containing the coumarin structure were cytotoxic to human K562 erythroleukemia and U937 myeloleukemia cells. A cytotoxic mechanism of decursin and its derivatives was investigated using TUR cells, a PKC betaII-deficient variant of U937 cells. Among six compounds 1-6 with cytotoxicity to K562 and U937 leukemia cells, only three compounds 1-3 were cytotoxic to TUR cells. Therefore, compounds 1-3 and 4-6 inhibit the proliferation of leukemia cells in a PKC betaII-independent and dependent manner, respectively, indicating that the side chain of compounds determines the dependency of their cytotoxicity on PKC betaII. To further elucidate the cytotoxic mechanism of compounds 1 and 2, levels of PKC isozymes and generation of reactive oxygen species (ROS) were investigated. Compounds 1-2 induced the down-regulation of PKC alpha and betaII in K562 cells and the production of ROS in U937 cells. Thus, PKC and ROS are probably important factors in the cytotoxic mechanism of compounds 1-2. From these results, the structure-activity relationship of decursin and its derivatives

Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562.

PubMed

Kairane, Ceslava; Mahlapuu, Riina; Ehrlich, Kersti; Kilk, Kalle; Zilmer, Mihkel; Soomets, Ursel

2012-01-01

The main goal of the present paper was to examine the influence of the replacement of γ-Glu moiety to α-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting in α-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 and α-GSH decreased the activity of CuZnSOD in K562 cells, at peptide concentration of 10 μM by 42% and 38% or 35% and 24%, respectively. After three-hour incubation, UPF1 increased and UPF17 decreased the intracellular level of total GSH. Additionally, it was shown that UPF1 is not degraded by γ-glutamyltranspeptidase, which performs glutathione breakdown. These results indicate that effective antioxidative character of peptides does not depend only on the reactivity of the thiol group, but also of the other functional groups, and on the spatial structure of peptides.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

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

Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui

2015-04-01

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressedmore » c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.« less

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

PubMed

Verrax, Julien; Stockis, Julie; Tison, Aurélie; Taper, Henryk S; Calderon, Pedro Buc

2006-09-14

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

PubMed

Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

2014-01-01

Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

PubMed

Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

2010-06-01

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

PubMed Central

Majid, Amin Malik Shah Abdul; Kit-Lam, Chan; Abdullah, Wan Zaidah; Zaki, Abdelhamid; Jamal Din, Shah Kamal Khan; Yusoff, Narazah Mohd

2014-01-01

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 107 K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management. PMID:24409284

Comparison of different methods for erythroid differentiation in the K562 cell line.

PubMed

Shariati, Laleh; Modaress, Mehran; Khanahmad, Hossein; Hejazi, Zahra; Tabatabaiefar, Mohammad Amin; Salehi, Mansoor; Modarressi, Mohammad Hossein

2016-08-01

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

Comparative Study of Different Nano-Formulations of Curcumin for Reversal of Doxorubicin Resistance in K562R Cells.

PubMed

Dash, Tapan K; Konkimalla, V Badireenath

2017-02-01

Curcumin is very well established as a chemo-therapeutic, chemo-preventive and chemo-sensitizing agent in diverse disease conditions. As the isolated pure form has poor solubility and pharmacokinetic problems, therefore it is encapsulated in to several nano-formulations to improve its bioavailability. Here in the current study, we aim to compare different nano-formulations of curcumin for their chemo-sensitizing activity in doxorubicin (DOX) resistant K562 cells. Four different curcumin formulations were prepared namely DMSO assisted curcumin nano-dispersion (CurD, 260 nm), liposomal curcumin (CurL, 165 nm), MPEG-PCL micellar curcumin (CurM, 18 nm) and cyclodextrin encapsulated curcumin (CurN, 37 nm). The formulations were subjected to particle characterizations (size, zeta potential, release studies), followed by biological assays such as cellular uptake, P-gp inhibitory activity and reversal of DOX resistance by co-treatment with DOX. Curcumin uptake in K562N and K562R cells was mildly reduced when treated with CurL and CurM, while for CurD and CurN the uptake remained equivalent. However, CurL retained P-gp inhibitory activity of curcumin and with a considerable chemo-sensitizing effect but CurM showed no P-gp inhibitory activity. CurN retained above biological activities, but requires a secondary carrier under in vivo conditions. From the results, CurM was found to be most suitable for solubilization of curcumin where as CurL can be considered as most suitable nano-formulation for reversal of DOX resistance.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells.

PubMed

Estrella-Parra, Edgar A; Gomez-Verjan, Juan C; González-Sánchez, Ignacio; Vázquez-Martínez, Edgar Ricardo; Vergara-Castañeda, Edgar; Cerbón, Marco A; Alavez-Solano, Dagoberto; Reyes-Chilpa, Ricardo

2014-01-01

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 μM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

PubMed

Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

2010-06-15

CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation. 2010 Elsevier Ltd. All rights reserved.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line.

PubMed

Benfica, Polyana Lopes; Ávila, Renato Ivan de; Rodrigues, Bruna Dos Santos; Cortez, Alane Pereira; Batista, Aline Carvalho; Gaeti, Marilisa Pedroso Nogueira; Lima, Eliana Martins; Rezende, Kênnia Rocha; Valadares, Marize Campos

2017-12-01

4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. Cytotoxicity of 4-NRC (4.17-534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. IC 50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562, and HL-60.

PubMed

Deezagi, Abdolkhaleg; Manteghi, Sanaz; Khosravani, Pardis; Vaseli-Hagh, Neda; Soheili, Zahra-Soheila

2009-09-01

The purpose of this research was to understand the effect of hyperthermia on the telomerase activity in human leukemic cell lines (HL-60, K562, and TF-1). The cells were treated by hyperthermia at the range of 41-44 degrees C for 120 min and incubated for 96 h. Then telomerase activity, cell proliferation, and apoptosis were assessed. The results indicated that hyperthermia significantly induced apoptosis on the cells. The cells exhibited pre-apoptotic pattern at 41 and 42 degrees C at 60-120 min and apoptotic pattern at 43 and 44 degrees C over 30 min after hyperthermia. Telomerase activity (that was assayed immediately after hyperthermia) was stable at 41-42 degrees C for 60 min but decreased to 35-40% at 120 min. However, at severe hyperthermia (43-44 degrees C) telomerase activity was decreased in a time- and dose-dependent manner. Following hyperthermia (41-44 degrees C up to 120 min), the cells were incubated for 96 h. In these conditions, the telomerase activity was decreased by about 60-80% in comparison with that untreated control cells.

Cytotoxic and Apoptotic Effects of Different Extracts of Artemisia turanica Krasch. on K562 and HL-60 Cell Lines

PubMed Central

Tayarani-Najaran, Zahra; Sareban, Mahla; Gholami, Atefeh; Emami, Seyed Ahmad; Mojarrab, Mahdi

2013-01-01

Artemisia is an important genus of Iranian flora. Cytotoxic activities for some species of the genus have already been reported. In this study, we have investigated the cytotoxic effects of n-hexane, CH2Cl2, EtOAc, EtOH, and EtOH/H2O (1 : 1) extracts of A. turanica Krasch. on two human leukemic cancer cell lines (K562 and HL-60) and J774 as normal cells using alamarBlue (resazurin) assay. PI staining of the fragmented DNA and western blot analysis were used to evaluate the possible apoptotic effect of the extract. The CH2Cl2 extract of A. turanica showed the most antiproliferative effect on cancer cells among all tested extracts with IC50 values of 69 and 104 μg/mL on K562 and HL-60 cells, respectively, whereas the normal cells were not affected significantly by this extract. Sub-G1 peak in the flow cytometry histogram of the cells treated with CH2Cl2 extract of A. turanica and cleavage of PARP protein confirmed the induction of apoptosis with CH2Cl2 extract. Taken together, the findings of the present work suggest the anticancer potential of CH2Cl2 extract of A. turanica on human leukemic cancer cell lines. PMID:24288497

Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence

PubMed Central

Ianniciello, Angela; Dumas, Pierre-Yves; Drullion, Claire; Guitart, Amélie; Villacreces, Arnaud; Peytour, Yan; Chevaleyre, Jean; Brunet de la Grange, Philippe; Vigon, Isabelle; Desplat, Vanessa; Priault, Muriel; Sbarba, Persio Dello; Ivanovic, Zoran; Mahon, François-Xavier; Pasquet, Jean-Max

2017-01-01

Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells. PMID:29228587

Phenethyl isothiocyanate inhibits growth of human chronic myeloid leukemia K562 cells via reactive oxygen species generation and caspases.

PubMed

Wang, Yating; Wei, Sixi; Wang, Jishi; Fang, Qin; Chai, Qixiang

2014-07-01

Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562 cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochrome c as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrin IX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

PubMed

Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

2015-07-31

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

PubMed

Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

2016-05-01

Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

Vaccination with autologous myeloblasts admixed with GM-K562 cells in patients with advanced MDS or AML after allogeneic HSCT

PubMed Central

Kim, Haesook T.; Bavli, Natalie; Mihm, Martin; Pozdnyakova, Olga; Piesche, Matthias; Daley, Heather; Reynolds, Carol; Souders, Nicholas C.; Cutler, Corey; Koreth, John; Alyea, Edwin P.; Antin, Joseph H.; Ritz, Jerome; Dranoff, Glenn; Soiffer, Robert J.

2017-01-01

We report a clinical trial testing vaccination of autologous myeloblasts admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells after allogeneic hematopoietic stem cell transplantation (HSCT). Patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with ≥5% marrow blasts underwent myeloblast collection before HSCT. At approximately day +30, 6 vaccines composed of irradiated autologous myeloblasts mixed with GM-K562 were administered. Tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was not tapered until vaccine completion (∼day 100). Thirty-three patients with AML (25) and MDS (8) enrolled, 16 (48%) had ≥5% marrow blasts at transplantation. The most common vaccine toxicity was injection site reactions. One patient developed severe eosinophilia and died of eosinophilic myocarditis. With a median follow-up of 67 months, cumulative incidence of grade 2-4 acute and chronic GVHD were 24% and 33%, respectively. Relapse and nonrelapse mortality were 48% and 9%, respectively. Progression-free survival (PFS) and overall survival (OS) at 5 years were 39% and 39%. Vaccinated patients who were transplanted with active disease (≥5% marrow blasts) had similar OS and PFS at 5 years compared with vaccinated patients transplanted with <5% marrow blasts (OS, 44% vs 35%, respectively, P = .81; PFS, 44% vs 35%, respectively, P = .34). Postvaccination antibody responses to angiopoietin-2 was associated with superior OS (hazard ratio [HR], 0.43; P = .031) and PFS (HR, 0.5; P = .036). Patients transplanted with active disease had more frequent angiopoeitin-2 antibody responses (62.5% vs 20%, P = .029) than those transplanted in remission. GM-K562/leukemia cell vaccination induces biologic activity, even in patients transplanted with active MDS/AML. This study is registered at www.clinicaltrials.gov as #NCT 00809250. PMID:29296875

Osthole shows the potential to overcome P-glycoprotein‑mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway.

PubMed

Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Wang, Jian-Yong; Li, You-Jie

2016-06-01

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has been reported to play a pivotal role in tumor chemotherapy failure. Study after study has illustrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with P-gp expression in many human malignancies. In the present study, osthole, an O-methylated coumarin, exhibited potent reversal capability of MDR in myelogenous leukemia K562/ADM cells. Simultaneously, the uptake and efflux of Rhodamine-123 (Rh-123) and the accumulation of doxorubicin assays combined with flow cytometric analysis suggested that osthole could increase intracellular drug accumulation. Furthermore, osthole decreased the expression of multidrug resistance gene 1 (MDR1) at both the mRNA and protein levels. Further experiments elucidated that osthole could suppress P-gp expression by inhibiting the PI3K/Akt signaling pathway which might be the main mechanism accounting for the reversal potential of osthole in the MDR in K562/ADM cells. In conclusion, osthole combats MDR and could be a promising candidate for the development of novel MDR reversal modulators.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

PubMed Central

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-01-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

PubMed

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-12-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Kinetics of ultraweak light emission from human erythroleukemia K562 cells upon electroporation.

PubMed

Maccarrone, M; Fantini, C; Agrò, A F; Rosato, N

1998-11-11

Electroporation involves the application of an electric pulse that creates transient aqueous channels (electropores) across the lipid bilayer membranes. Here, we describe an instrument set up suitable to record ultraweak light emission from human erythroleukemia K562 cells during and immediately after delivery of electric pulses. Most of light was emitted in the first seconds after each pulse, following a complex decay which can be fitted by a double exponential equation characterized by two different time constants (T1 and T2), both in the order of seconds. T1 was approximately 10-fold shorter than T2 and both time constants were dependent on field strength of the electric pulse. The effect of various antioxidants on the amount of emitted photons and on T1 and T2 values was investigated, in order to shed some light on the chemical species responsible for cellular luminescence.

Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2 Pathways and the Abrogation of VEGF Secretion.

PubMed

Souid, Soumaya; Najjaa, Hanen; Riahi-Chebbi, Ichrak; Haoues, Meriam; Neffati, Mohamed; Arnault, Ingrid; Auger, Jacques; Karoui, Habib; Essafi, Makram; Essafi-Benkhadir, Khadija

2017-01-01

Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK 1/2 , Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways.

PubMed

Zhu, Jianhua; Yao, Jianfeng; Huang, Rongfu; Wang, Yueqin; Jia, Min; Huang, Yan

2018-04-06

Ghrelin is a gastric acyl-peptide that plays an important role in cell proliferation. In the present study, we explored the role of ghrelin in A549 cell proliferation and the possible molecular mechanisms. We found that ghrelin promotes A549 cell proliferation, knockdown of the growth hormone secretagogue receptor (GHSR) attenuated A549 cell proliferation caused by ghrelin. Ghrelin induced the rapid phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, ERK, mammalian target of rapamycin (mTOR) and P70S6K. PI3K inhibitor (LY 294002), ERK inhibitor (PD98059) and mTOR inhibitor (Rapamycin) inhibited ghrelin-induced A549 cell proliferation. Moreover, GHSR siRNA inhibited phosphorylation of PI3K, Akt, ERK, mTOR and P70S6K induced by ghrelin. Akt and mTOR/P70S6K phosphorylation was inhibited by LY 294002 but not by PD98059. These results indicate that ghrelin promotes A549 cell proliferation via GHSR-dependent PI3K/Akt/mTOR/P70S6K and ERK signaling pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis of a Novel Series of 2-Methylsulfanyl Fatty Acids and their Toxicity on the Human K-562 and U-937 Leukemia Cell Lines

PubMed Central

Carballeira, Néstor M.; Miranda, Carlos; Orellano, Elsie A.; González, Fernando A.

2006-01-01

The hitherto unknown 2-methylsulfanyldecanoic acid and 2-methylsulfanyldodecanoic acid were synthesized from methyl decanoate and methyl dodecanoate, respectively, through the reaction of lithium diisopropylamide and dimethyldisulfide in THF followed by saponification with potassium hydroxide in ethanol. Both α-methylsulfanylated FA were cytotoxic to the human chronic myelogenous leukemia K-562 and the human histiocytic lymphoma U-937 cell lines with EC50 values in the 200-300 μM range, which makes them more cytotoxic to these cell lines than either decanoic acid or dodecanoic acid. The cytotoxicity of the studied FA towards K-562 followed the order: 2-SCH3-12:0 > 2-SCH3-10:0 > 10:0 > 12:0 > 2-OCH3-12:0, while towards U-937 the cytotoxicity was found to be: 2-SCH3-10:0 > 2-SCH3-12:0 > 12:0 > 10:0 > 2-OCH3-12:0. These results indicate that the α-methylsulfanyl substitution increases the cytotoxicity of the C10 and C12 fatty acids towards the studied leukemia cell lines. PMID:16382579

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells.

PubMed

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine Kl; Berman, Jason N; Rupasinghe, Hp Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo . We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells

PubMed Central

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine KL; Berman, Jason N; Rupasinghe, HP Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo. We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway. PMID:29312799

Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT

PubMed Central

Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT. PMID:29590128

Osthole inhibits gastric cancer cell proliferation through regulation of PI3K/AKT.

PubMed

Xu, Xiaojun; Liu, Xiaoyuan; Zhang, Yan

2018-01-01

Osthole is an active compound isolated from Chinese herb Cnidium monnieri (L.) Cusson, and had been reported to possess antitumor effect. However, the effect of osthole on the gastric cancer cells has not been investigated. In this study, the effects of osthole on the proliferation of human gastric cancer cells were tested. The data showed that osthole treatment significantly inhibited the proliferation of gastric cancer cells and resulted in the cell cycle arrest at G2/M phase in a dose-dependent manner. Western-blot study showed that the expression of cyclin B1 and cdc2 was markedly reduced by osthole. Moreover, expression of PI3K and pAKT was also significantly suppressed, and the results indicated that the inhibition of pAKT, cyclin B1, and cdc2 levels by osthole was notably enhanced by a PI3K inhibitor. These results demonstrate that osthole could inhibit gastric cancer cells proliferation via induction of cell cycle arrest at G2/M phase by the reduction of PI3K/AKT.

Ester of Quinoxaline-7-carboxylate 1,4-di-N-oxide as Apoptosis Inductors in K-562 Cell Line: An in vitro, QSAR and DFT Study.

PubMed

Rivera, Gildardo; Andrade-Ochoa, Sergio; Romero, Manolo S Ortega; Palos, Isidro; Monge, Antonio; Sanchez-Torres, Luvia Enid

2017-01-01

Quinoxalines have shown a wide variety of biological activities including as antitumor agents. The aims of this study were to evaluate the activity of quinoxaline 1,4-di-N-oxide derivatives on K562 cells, the establishment of the mechanism of induced cell death, and the construction of predictive QSAR models. Sixteen esters of quinoxaline-7-carboxylate 1,4-di-N-oxide were evaluated for antitumor activity on K562 chronic myelogenous leukemia cells and their IC50 values were determined. The mechanism of induced cell death by the most active molecule was assessed by flow cytometry and an in silico study was conducted to optimize and calculate theoretical descriptors of all quinoxaline 1,4-di-N-oxide derivatives. QSAR and QPAR models were created using genetic algorithms. Our results show that compounds C5, C7, C10, C12 and C15 had the lowest IC50 of the series. C15 was the most active compound (IC50= 3.02 μg/mL), inducing caspase-dependent apoptotic cell death via the intrinsic pathway. QSAR and QPAR studies are discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Evidence of K+ channel function in epithelial cell migration, proliferation, and repair

PubMed Central

Girault, Alban

2013-01-01

Efficient repair of epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely expressed family of K+ channels are involved in regulation of cell migration and proliferation, key processes of epithelial repair. First, we briefly summarize the complex mechanisms, including cell migration, proliferation, and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. We also describe the mechanisms whereby K+ channels may control epithelial repair processes. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular Ca2+, and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, we discuss the challenges to efficient, specific, and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo. PMID:24196531

Angiotensin II upregulates K(Ca)3.1 channels and stimulates cell proliferation in rat cardiac fibroblasts.

PubMed

Wang, Li-Ping; Wang, Yan; Zhao, Li-Mei; Li, Gui-Rong; Deng, Xiu-Ling

2013-05-15

The proliferation of cardiac fibroblasts is implicated in the pathogenesis of myocardial remodeling and fibrosis. Intermediate-conductance calcium-activated K⁺ channels (K(Ca)3.1 channels) have important roles in cell proliferation. However, it is unknown whether angiotensin II (Ang II), a potent profibrotic molecule, would regulate K(Ca)3.1 channels in cardiac fibroblasts and participate in cell proliferation. In the present study, we investigated whether K(Ca)3.1 channels were regulated by Ang II, and how the channel activity mediated cell proliferation in cultured adult rat cardiac fibroblasts using electrophysiology and biochemical approaches. It was found that mRNA, protein, and current density of K(Ca)3.1 channels were greatly enhanced in cultured cardiac fibroblasts treated with 1 μM Ang II, and the effects were countered by the angiotensin type 1 receptor (AT₁R) blocker losartan, the p38-MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the PI3K/Akt inhibitor LY294002. Ang II stimulated cell proliferation and the effect was antagonized by the K(Ca)3.1 blocker TRAM-34 and siRNA targeting K(Ca)3.1. In addition, Ang II-induced increase of K(Ca)3.1 expression was attenuated by transfection of activator protein-1 (AP-1) decoy oligodeoxynucleotides. These results demonstrate for the first time that Ang II stimulates cell proliferation mediated by upregulating K(Ca)3.1 channels via interacting with the AT₁R and activating AP-1 complex through ERK1/2, p38-MAPK and PI3K/Akt signaling pathways in cultured adult rat cardiac fibroblasts. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

PubMed

Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

2015-07-01

Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

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

Evaluation of Synergetic Anticancer Activity of Berberine and Curcumin on Different Models of A549, Hep-G2, MCF-7, Jurkat, and K562 Cell Lines.

PubMed

Balakrishna, Acharya; Kumar, M Hemanth

2015-01-01

Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562). All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 10(4) cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1), Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL). The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells), and blank (only medium). The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models.

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.

PubMed

Xiao, Lin; Chen, Can; Li, Zhendong; Zhu, Sumin; Tay, Johan Ck; Zhang, Xi; Zha, Shijun; Zeng, Jieming; Tan, Wee Kiat; Liu, Xin; Chng, Wee Joo; Wang, Shu

2018-03-01

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Suppression of bcr-abl synthesis by siRNAs or tyrosine kinase activity by Glivec alters different oncogenes, apoptotic/antiapoptotic genes and cell proliferation factors (microarray study).

PubMed

Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki; Ewis, Ashraf; Ishikawa, Mitsuru; Shinohara, Yasuo; Baba, Yoshinobu

2004-07-16

Short 21-mer double-stranded/small-interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia. The ds/siRNAs were transfected into bcr-abl-positive K-562 (derived from blast crisis chronic myelogenous leukemia), using lipofectamine. Penetrating of ds/siRNAs into the cells was detected by fluorescent confocal microscopy, using fluorescein-labeled ds/siRNAs. The cells were treated with mix of three siRNA sequences (3 x 60 nM) during 6 days with three repetitive transfections. The siRNA-treatment was accompanied with significant reduction of bcr-abl mRNA, p210, protein tyrosine kinase activity and cell proliferation index. Treatment of cells with Glivec (during 8 days with four repetitive doses, 180 nM single dose) resulted in analogous reduction of cell proliferation activity, stronger suppression of protein tyrosine kinase activity, and very low reduction of p210. siRNA-mix and Glivec did not affect significantly the viability of normal lymphocytes. Microarray analysis of siRNA- and Glivec-treated K-562 cells demonstrated that both pathways of bcr-abl suppression were accompanied with overexpression and suppression of many different oncogenes, apoptotic/antiapoptotic and cell proliferation factors. The following genes of interest were found to decrease in relatively equal degree in both siRNA- and Glivec-treated cells: Bcd orf1 and orf2 proto-oncogene, chromatin-specific transcription elongation factor FACT 140-kDa subunit mRNA, gene encoding splicing factor SF1, and mRNA for Tec protein tyrosine kinase. siRNA-mix and Glivec provoked overexpression of the following common genes: c-jun proto-oncogene, protein kinase C-alpha, pvt-1 oncogene homologue (myc activator), interleukin-6, 1-8D gene from interferon-inducible gene family, tumor necrosis factor receptor superfamily (10b), and STAT-induced STAT inhibitor.

Electric stimulation at 448 kHz promotes proliferation of human mesenchymal stem cells.

PubMed

Hernández-Bule, María Luisa; Paíno, Carlos Luis; Trillo, María Ángeles; Úbeda, Alejandro

2014-01-01

Capacitive-resistive electric transfer (CRET) is a non invasive electrothermal therapy that applies electric currents within the 400 kHz - 450 kHz frequency range to the treatment of musculoskeletal lesions. Evidence exists that electric currents and electric or magnetic fields can influence proliferative and/or differentiating processes involved in tissue regeneration. This work investigates proliferative responses potentially underlying CRET effects on tissue repair. XTT assay, flow cytometry, immunofluorescence and Western Blot analyses were conducted to asses viability, proliferation and differentiation of adipose-derived stem cells (ADSC) from healthy donors, after short, repeated (5 m On/4 h Off) in vitro stimulation with a 448-kHz electric signal currently used in CRET therapy, applied at a subthermal dose of 50 μA/mm(2) RESULTS: The treatment induced PCNA and ERK1/2 upregulation, together with significant increases in the fractions of ADSC undergoing cycle phases S, G2 and M, and enhanced cell proliferation rate. This proliferative effect did not compromise the multipotential ability of ADSC for subsequent adipogenic, chondrogenic or osteogenic differentiation. These data identify cellular and molecular phenomena potentially underlying the response to CRET and indicate that CRET-induced lesion repair could be mediated by stimulation of the proliferation of stem cells present in the injured tissues. © 2014 S. Karger AG, Basel.

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation.

PubMed

Borowiec, Anne-Sophie; Hague, Frédéric; Harir, Noria; Guénin, Stéphanie; Guerineau, François; Gouilleux, Fabrice; Roudbaraki, Morad; Lassoued, Kaiss; Ouadid-Ahidouch, Halima

2007-09-01

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

PubMed

Neri, L M; Bortul, R; Zweyer, M; Tabellini, G; Borgatti, P; Marchisio, M; Bareggi, R; Capitani, S; Martelli, A M

1999-06-01

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA, combined with soluble IL-21.

PubMed

Jiang, Bo; Wu, Xuan; Li, Xi-Ning; Yang, Xi; Zhou, Yulai; Yan, Haowei; Wei, An-Hui; Yan, Weiqun

2014-07-01

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

A nanocomplex of Cu(II) with theophylline drug; synthesis, characterization, and anticancer activity against K562 cell line

NASA Astrophysics Data System (ADS)

Sahlabadi, Maryam; Daryanavard, Marzieh; Hadadzadeh, Hassan; Amirghofran, Zahra

2018-03-01

A new mononuclear of copper (II), [Cu(theophylline)2(H2O)3]·2H2O, has been synthesized by reaction of theophylline (1,3-dimethyl-7H-purine-2,6-dione) with copper (II) nitrate in water. Further, its nanocomplex has been prepared through the three different methods including sonication, grinding, and a combination thereof, sonication-grinding. The prepared nanocomplex was characterized using different techniques including FT-IR, UV-Vis, X-ray diffraction (XRD) analysis, and field-emission scanning electron microscopy (FE-SEM). Moreover, the anticancer activity of the precursor complex, nanocomplex, free theophylline ligand, and the starting copper salt (Cu(NO3)2·3H2O) was investigated against the K562 cell line. The results show that the nanocomplex is an effective nano metal-based anticancer agent with IC50 = 11.7 μM.

Class IA PI3K inhibition inhibits cell growth and proliferation in mantle cell lymphoma.

PubMed

Tabe, Yoko; Jin, Linhua; Konopleva, Marina; Shikami, Masato; Kimura, Shinya; Andreeff, Michael; Raffeld, Mark; Miida, Takashi

2014-01-01

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells. The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis. Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells. These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL. Copyright © 2013 S. Karger AG, Basel.

Diterpenes from Xylopia langsdorffiana inhibit cell growth and induce differentiation in human leukemia cells.

PubMed

Castello Branco, Marianna V S; Anazetti, Maristella C; Silva, Marcelo S; Tavares, Josean F; Diniz, Margareth F F Melo; Frungillo, Lucas; Haun, Marcela; Melo, Patrícia S

2009-01-01

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.

EFFECT OF THAI SARAPHI FLOWER EXTRACTS ON WT1 AND BCR/ABL PROTEIN EXPRESSION IN LEUKEMIC CELL LINES.

PubMed

Sangkaruk, Rungkarn; Rungrojsakul, Methee; Tima, Singkome; Anuchapreeda, Songyot

2017-01-01

Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC 50 values of 2.6 and 77.6 μg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WTlprotein expressions by 75 and 49.5%, respectively when compared to vehicle control. The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.

Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells

PubMed Central

Wang, Jueqiong; Lu, Liu; Kok, Chung H.; Saunders, Verity A.; Goyne, Jarrad M.; Dang, Phuong; Leclercq, Tamara M.; Hughes, Timothy P.; White, Deborah L.

2017-01-01

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in

[Profiles of cell proliferation and apoptosis in the mouse epithelial regeneration model K6b-E6/E7].

PubMed

Bonilla-Delgado, José; Rodríguez-Uribe, Genaro; Cortés-Malagón, Enoc Mariano; Sierra Martínez, Mónica; Acosta-Altamirano, Gustavo; Gariglio-Vidal, Patricio

2012-01-01

Mammals have limited epithelial regeneration capacity. The K6b-E6/E7 mice model has been described as useful for the study of epithelial regeneration. The objective of this study is to compare the expression of E6/E7 oncogenes with those of cell proliferation and apoptosis during epithelization. The hypothesis of this study is that alterations in cell proliferation and apoptosis in K6b-E6/E7 mice will only occur during epithelization. Deep 2 mm punches were performed in the middle of transgenic and control mice's ears. A biopsy was collected from the epithelization zone 72 hours and 2 weeks post-injury. Assays for cell proliferation and apoptosis were carried out by immunohistochemistry and TUNEL techniques, respectively. RT-PCR in situ was performed to compare E6/E7 expressions in the areas studied. Transgenic strain K6b-E6/E7 presented more proliferative cells and less apoptotic cells in epithelizated zones. This effect was limited to suprabasal stratum only, and correlates with E6/E7 oncogenes expression. Two weeks post-injury, cell proliferation and apoptosis were similar in both samples as the E6/E7 expression went down. K6b-E6/E7 mouse model is useful for epithelial regeneration. Its mechanisms should be considered for the treatment of deep wounds.

IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

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

Pan, Deshun; Department of Pharmaceutical science, Guangdong Pharmaceutical University, Guangzhou, Guangdong; Liu, Bing

2012-06-15

Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The resultsmore » showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.« less

Characterization of Compounds with Tumor-Cell Proliferation Inhibition Activity from Mushroom (Phellinus baumii) Mycelia Produced by Solid-State Fermentation.

PubMed

Zhang, Henan; Shao, Qian; Wang, Wenhan; Zhang, Jingsong; Zhang, Zhong; Liu, Yanfang; Yang, Yan

2017-04-27

The inhibition of tumor-cell proliferationbyan organicsolvent extract from the solid-state fermentation of Phellinus baumii mycelia inoculated in rice medium was investigated in vitro. The active compounds inhibiting tumor-cell proliferation were characterized. Results revealed that all (petroleum ether, chloroform, ethyl acetate, and butanol) fractions inhibited tumor-cell proliferation in a dose-dependent fashion. The ethyl acetate extract had the highest inhibitory effecton tumor-cell proliferation, and the butanol fraction had the lowest. Six compounds were isolated and purified from the ethyl acetate extract of P. baumii mycelia by the tandem application of silica-gel column chromatography (SGCC), high-speed countercurrent chromatography (HSCCC), and preparative HPLC. These compounds were identified by NMR and electrospray ionization-mass spectrometry (ESI-MS) spectroscopic methods as ergosterol (RF1), ergosta-7,22-dien-3β-yl pentadecanoate (RF3), 3,4-dihydroxy benzaldehyde(RF6), inoscavinA (RF7), baicalein(RF10), and 24-ethylcholesta-5,22-dien-3β-ol (RF13). To further clarify the activity of these compounds, the cell-proliferation-inhibition tests of these compounds on various tumor cells were carried out and evaluatedin vitro. Results suggested that compounds RF6, RF7, and RF10 had potent inhibition effects on the proliferation of a series of tumor cell lines, including K562, L1210, SW620, HepG2, LNCaP, and MCF-7cells. These findings indicated that P. baumii mycelia produced by solid-state fermentation in rice canbe used to obtain active compounds with the ability to inhibittumor-cell proliferation.

Tramadol regulates proliferation, migration and invasion via PTEN/PI3K/AKT signaling in lung adenocarcinoma cells.

PubMed

Xia, M; Tong, J-H; Ji, N-N; Duan, M-L; Tan, Y-H; Xu, J-G

2016-06-01

Tramadol is used mainly for the treatment of moderate to severe chronic cancer pain. However, the effect of tramadol on lung cancer remains unclear. Therefore, it is important to explore the mechanism accounting for the function of tramadol on lung cancer. We investigated the effects of tramadol on the proliferation, migration and invasion in human lung adenocarcinoma cells in vitro by CCK-8 assay, wound healing assay and Transwell assay, respectively. We also explored the potential mechanism of tramadol on lung cancer cells by Western blotting. A549 and PC-9 cells were incubated with 2 µM tramadol for different time (0, 7, 14 and 28 d). The in vitro experiments showed that tramadol treatment significantly inhibited cell proliferation, migration and invasion in a time-dependent manner. Moreover, administration of tramadol suppressed tumor growth in vivo. The data also revealed that tramadol could up-regulate the protein expression level of PTEN and consistently inhibit the phosphorylation level of PI3K and Akt, whereas the total level of PI3K and Akt remain unchanged. These findings indicated that tramadol inhibited proliferation, migration and invasion of human lung adenocarcinoma cells through elevation of PTEN and inactivation of PI3K/Akt signaling.

PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

PubMed Central

Que, Jianwen; Lian, Qizhou; El Oakley, Reida M; Lim, Bing; Lim, Sai-Kiang

2007-01-01

Background We have previously derived highly similar lineage-restricted stem cell lines, RoSH and E-RoSH cell lines from mouse embryos and CD9hi SSEA-1- differentiated mouse embryonic stem cells, respectively. These cell lines are not pluripotent and differentiate readily into endothelial cells in vitro and in vivo. Results We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines. Conclusion This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells. PMID:17892597

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

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

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

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

Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

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

Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp; Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Wada, Eiji, E-mail: gacchu1@yahoo.co.jp

2015-05-01

Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc onmore » differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.« less

miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein

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

Li, Yajuan; Wang, Haixia; Tao, Kun

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3′-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3′UTR and this activity is not observed in cells transfected with mutated ABL1-3′UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colonymore » formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1. - Highlights: ► miR-29b expression was downregulated in CML patients. ► ABL1 was identified as a direct target gene of miR-29b. ► Enforced expression of miR-29b inhibits cell proliferation and induces apoptosis. ► miR-29b might be a therapeutic target to CML.« less

The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.

PubMed

Tian, Yuwei; Wang, Hui; Li, Bing; Ke, Mengyun; Wang, Jing; Dou, Jie; Zhou, Changlin

2013-11-01

The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-fluorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate.

Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

PubMed

Jia, Wei; Feng, Jie; Zhang, Jing-Song; Lin, Chi-Chung; Wang, Wen-Han; Chen, Hong-Ge

2017-01-01

FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

Insulin-mediated upregulation of K(Ca)3.1 channels promotes cell migration and proliferation in rat vascular smooth muscle.

PubMed

Su, Xing-Li; Wang, Yan; Zhang, Wei; Zhao, Li-Mei; Li, Gui-Rong; Deng, Xiu-Ling

2011-07-01

The detailed molecular mechanisms underlying pathogenesis of various vascular diseases such as atherosclerosis are not fully understood in type-2 diabetes. The present study was designed to investigate whether insulin regulates K(Ca)3.1 channels and participates in vasculopathy in type-2 diabetes. A rat model with experimental insulin-resistant type-2 diabetes was used for detecting pathological changes in the aorta wall, and cultured vascular smooth muscle cells (VSMCs) were employed to investigate the regulation of K(Ca)3.1 channels by insulin and roles of K(Ca)3.1 channels in cell migration and proliferation using molecular biology and electrophysiology. Early pathological changes were observed and expression of K(Ca)3.1 channels increased in the aorta wall of the type 2 diabetic rats. K(Ca)3.1 channel mRNA, protein levels and current density were greatly enhanced in cultured VSMCs treated with insulin, and the effects were countered in the cells treated with the ERK1/2 inhibitor PD98059, but not the p38-MAPK inhibitor SB203580. In addition, insulin stimulated cell migration and proliferation in cultured VSMCs, and the effects were fully reversed in the cells treated with the K(Ca)3.1 blocker TRAM-34 or PD98059, but not SB203580. These results demonstrate the novel information that insulin increases expression of K(Ca)3.1 channels by stimulating ERK1/2 phosphorylation thereby promoting migration and proliferation of VSMCs, which likely play at least a partial role in the development of vasculopathy in type-2 diabetes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

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

Inhibition of the K+ channel K(Ca)3.1 reduces TGF-β1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

PubMed

Fu, Rong-Guo; Zhang, Tao; Wang, Li; Du, Yan; Jia, Li-Ning; Hou, Jing-Jing; Yao, Gang-Lian; Liu, Xiao-Dan; Zhang, Lei; Chen, Ling; Gui, Bao-Song; Xue, Rong-Liang

2014-01-01

K(Ca)3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca)3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca)3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca)3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca)3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01). Targeted disruption of K(Ca)3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells

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

Li, Xiting; Shu, Rong, E-mail: shurong123@hotmail.com; Liu, Dali

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF),more » gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 {mu}g/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.« less

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

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

Wu, Weijie; Liu, Qingqing; Liu, Yuxi

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

ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.

PubMed

Mchaourab, Zenab F; Perreault, Andrea A; Venters, Bryan J

2018-03-06

The human K562 chronic myeloid leukemia cell line has long served as an experimental paradigm for functional genomic studies. To systematically and functionally annotate the human genome, the ENCODE consortium generated hundreds of functional genomic data sets, such as chromatin immunoprecipitation coupled to sequencing (ChIP-seq). While ChIP-seq analyses have provided tremendous insights into gene regulation, spatiotemporal insights were limited by a resolution of several hundred base pairs. ChIP-exonuclease (ChIP-exo) is a refined version of ChIP-seq that overcomes this limitation by providing higher precision mapping of protein-DNA interactions. To study the interplay of transcription initiation and chromatin, we profiled the genome-wide locations for RNA polymerase II (Pol II), the histone variant H2A.Z, and the histone modification H3K4me3 using ChIP-seq and ChIP-exo. In this Data Descriptor, we present detailed information on parallel experimental design, data generation, quality control analysis, and data validation. We discuss how these data lay the foundation for future analysis to understand the relationship between the occupancy of Pol II and nucleosome positions at near base pair resolution.

Selected terpenes from leaves of Ocimum basilicum L. induce hemoglobin accumulation in human K562 cells.

PubMed

Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Torricelli, Piera; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

2018-06-01

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for β-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in β-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on β-thalassemia and sickle cells anemia. Copyright © 2018 Elsevier B.V. All rights reserved.

miR-181a promotes G1/S transition and cell proliferation in pediatric acute myeloid leukemia by targeting ATM.

PubMed

Liu, Xiaodan; Liao, Wang; Peng, Hongxia; Luo, Xuequn; Luo, Ziyan; Jiang, Hua; Xu, Ling

2016-01-01

Abnormal expression of miRNAs is intimately related to a variety of human cancers. The purpose of this study is to confirm the expression of miR-181a and elucidate its physiological function and mechanism in pediatric acute myeloid leukemia (AML). Pediatric AML patients and healthy controls were enrolled, and the expression of miR-181a and ataxia telangiectasia mutated (ATM) in tissues were examined using quantitative PCR. Moreover, cell proliferation and cell cycle were evaluated in several cell lines (HL60, NB4 and K562) by using flow cytometry after transfected with miR-181a mimics and inhibitors, or ATM siRNA and control siRNA. Finally, ATM as the potential target protein of miR-181a was examined. We found that miR-181a was significantly increased in pediatric AML, which showed an inverse association with ATM expression. Overexpressed miR-181a in cell lines significantly enhanced cell proliferation, as well as increased the ratio of S-phase cells by miR-181a mimics transfection in vitro. Luciferase activity of the reporter construct identified ATM as the direct molecular target of miR-181a. ATM siRNA transfection significantly enhanced cell proliferation and increased the ratio of S-phase cells in vitro. The results revealed novel mechanism through which miR-181a regulates G1/S transition and cell proliferation in pediatric AML by regulating the tumor suppressor ATM, providing insights into the molecular mechanism in pediatric AML.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

SASH1 inhibits proliferation and invasion of thyroid cancer cells through PI3K/Akt signaling pathway

PubMed Central

Sun, Dawei; Zhou, Rui; Liu, Huamin; Sun, Wenhai; Dong, Anbing; Zhang, Hongmei

2015-01-01

The SASH1 (SAM- and SH3-domain containing 1) gene, a member of the SLY-family of signal adapter proteins, has an important regulatory role in tumorigenesis, but its implication in thyroid carcinoma has not been yet investigated. In this study, we investigated the role of SASH1 in proliferation and invasion of thyroid cancer cells and the underlying mechanism. Our results demonstrated that SASH1 is down-regulated in thyroid cancer cells. Overexpression of SASH1 inhibits thyroid cancer cell proliferation, migration and invasion with decreased epithelial-mesenchymal transition (EMT). Mechanistically, overexpression of SASH1 inhibits thyroid cancer cell proliferation and invasion through down-regulation of PI3K and Akt phosphorylation. Taken together, the present study showed that the loss or inhibition of SASH1 expression may play an important role in thyroid cancer development, invasion, and metastasis and that SASH1 may be a potential therapeutic target for the treatment of thyroid cancer. PMID:26722413

SASH1 inhibits proliferation and invasion of thyroid cancer cells through PI3K/Akt signaling pathway.

PubMed

Sun, Dawei; Zhou, Rui; Liu, Huamin; Sun, Wenhai; Dong, Anbing; Zhang, Hongmei

2015-01-01

The SASH1 (SAM- and SH3-domain containing 1) gene, a member of the SLY-family of signal adapter proteins, has an important regulatory role in tumorigenesis, but its implication in thyroid carcinoma has not been yet investigated. In this study, we investigated the role of SASH1 in proliferation and invasion of thyroid cancer cells and the underlying mechanism. Our results demonstrated that SASH1 is down-regulated in thyroid cancer cells. Overexpression of SASH1 inhibits thyroid cancer cell proliferation, migration and invasion with decreased epithelial-mesenchymal transition (EMT). Mechanistically, overexpression of SASH1 inhibits thyroid cancer cell proliferation and invasion through down-regulation of PI3K and Akt phosphorylation. Taken together, the present study showed that the loss or inhibition of SASH1 expression may play an important role in thyroid cancer development, invasion, and metastasis and that SASH1 may be a potential therapeutic target for the treatment of thyroid cancer.

Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells

PubMed Central

Sheng, Lingling; Mao, Xiyuan; Yu, Qingxiong; Yu, Dong

2017-01-01

Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor. PMID:28123468

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

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

Mizutani, Naoki; College of Life and Health Sciences, Chubu University, Kasugai; Omori, Yukari

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increasedmore » by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.« less

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

Effects of FR235222, a novel HDAC inhibitor, in proliferation and apoptosis of human leukaemia cell lines: role of annexin A1.

PubMed

Petrella, Antonello; D'Acunto, Cosimo Walter; Rodriquez, Manuela; Festa, Michela; Tosco, Alessandra; Bruno, Ines; Terracciano, Stefania; Taddei, Maurizio; Paloma, Luigi Gomez; Parente, Luca

2008-03-01

FR235222, a novel histone deacetylase inhibitor (HDACi), at 50nM caused accumulation of acetylated histone H4, inhibition of cell proliferation and G1 cycle arrest accompanied by increase of p21 and down-regulation of cyclin E in human promyelocytic leukaemia U937 cells. The compound was also able to increase the protein and mRNA levels of annexin A1 (ANXA1) without effects on apoptosis. Similar effects were observed in human chronic myelogenous leukaemia K562 cells and human T cell leukaemia Jurkat cells. Cycle arrest and ANXA1 expression, without significant effects on apoptosis, were also induced by different HDACi like suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). FR235222 at 0.5 microM stimulated apoptosis of all leukaemia cell lines associated to an increased expression of the full-length (37kDa) protein and the appearance of a 33kDa N-terminal cleavage product in both cytosol and membrane. These results suggest that ANXA1 expression may mediate cycle arrest induced by low doses FR235222, whereas apoptosis induced by high doses FR235222 is associated to ANXA1 processing.

TET1-GPER-PI3K/AKT pathway is involved in insulin-driven endometrial cancer cell proliferation.

PubMed

Xie, Bing-Ying; Lv, Qiao-Ying; Ning, Cheng-Cheng; Yang, Bing-Yi; Shan, Wei-Wei; Cheng, Ya-Li; Gu, Chao; Luo, Xue-Zhen; Zhang, Zhen-Bo; Chen, Xiao-Jun; Xi, Xiao-Wei; Feng, You-Ji

2017-01-22

Large amount of clinical evidence has demonstrated that insulin resistance is closely related to oncogenesis of endometrial cancer (EC). Despite recent studies showed the up-regulatory role of insulin in G protein-coupled estrogen receptor (GPER/GPR30) expression, GPER expression was not decreased compared to control when insulin receptor was blocked even in insulin treatment. The purpose of this study was to explore the possible mechanism by which insulin up-regulates GPER that drives EC cell proliferation. For this purpose, we first investigated the GPER expression in tissues of endometrial lesions, further explored the effect of GPER on EC cell proliferation in insulin resistance context. Then we analyzed the role of Ten-Eleven Translocation 1 (TET1) in insulin-induced GEPR expression and EC cell proliferation. The results showed that GPER was highly expressed in endometrial atypical hyperplasia and EC tissues. Mechanistically, insulin up-regulated TET1 expression and the latter played an important role in up-regulating GPER expression and activating PI3K/AKT signaling pathway. TET1 mediated GPER up-regulation was another mechanism that insulin promotes EC cell proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

Anticancer activity of Sargassum oligocystum water extract against human cancer cell lines.

PubMed

Zandi, K; Ahmadzadeh, S; Tajbakhsh, S; Rastian, Z; Yousefi, F; Farshadpour, F; Sartavi, K

2010-08-01

Antitumor drug resistance and side effects of antitumor compounds are the most common problems in medicine. Therefore, finding new antitumor agents with low side effects could be interesting. This study was designed to assay antitumor activity of the extract from brown alga Sargassum oligocystum, gathered from Persian Gulf seashore, against K562 and Daudi human cancer cell lines. The research was performed as an in vitro study. The effect of the alga extract on proliferation of cell lines were measured by two methods: MTT assay and trypan blue exclusion test. The most effective antitumor activity has been shown at concentrations 500 microg/ml and 400 microg/ml of the alga extract against Daudi and K562 cell lines, respectively. The results showed that the extracts of brown alga Sargassum oligocystum have remarkable antitumor activity against K562 and Daudi cell lines. It is justified to be suggested for further research such as algal extract fractionation and purification and in vivo studies in order to formulate natural compounds with antitumor activities.

Xylitol induces cell death in lung cancer A549 cells by autophagy.

PubMed

Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

2015-05-01

Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

Newly developed PPAR-alpha agonist (R)-K-13675 inhibits the secretion of inflammatory markers without affecting cell proliferation or tube formation.

PubMed

Kitajima, Ken; Miura, Shin-Ichiro; Mastuo, Yoshino; Uehara, Yoshinari; Saku, Keijiro

2009-03-01

Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a key regulator of lipid and glucose metabolism and has been implicated in inflammation. The vascular effects of activator for PPARs, particularly PPAR-alpha, on vascular cells remain to be fully elucidated. Therefore, we analyzed the hypothesis that newly developed (R)-K-13675 decreases the secretion of inflammatory markers without affecting cell proliferation or tube formation. Human coronary endothelial cells (HCECs) were maintained in different doses of (R)-K-13675 under serum starvation. After 20h, the levels of monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T expressed and secreted (RANTES), interleukin-6 (IL-6) and interferon-gamma (INF-gamma) secreted in the medium and nuclear factor kappa B (NFkappaB) in cell lysate were analyzed using enzyme-linked immunosorbent assays (ELISA). Upon treatment with (R)-K-13675 at 0, 10, 20, 50 and 100nM, with the inflammatory markers at 0nM as 100 (arbitrary units), MCP-1 levels were significantly suppressed (94+/-9, 88+/-2, 80+/-5 and 74+/-11, respectively). RANTES, IL-6 and INF-gamma levels were also significantly suppressed (RANTES: 92+/-2, 74+/-9, 64+/-7 and 60+/-2, respectively, IL-6: 97+/-2, 89+/-10, 82+/-1 and 66+/-7, respectively, INF-gamma: 98+/-7, 94+/-3, 76+/-8 and 64+/-8, respectively). NFkappaB levels were also decreased to 91+/-5, 90+/-5, 84+/-7 and 82+/-8, respectively. In addition, (R)-K-13675 did not affect HCEC proliferation or tube formation at up to 100nM. Thus, (R)-K-13675 was associated with the inhibition of inflammatory responses without affecting cell proliferation or angiogenesis, and subsequently may induce an anti-atherosclerotic effect.

[miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

PubMed

Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

2016-12-23

Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

PubMed

Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells

PubMed Central

Liang, Xinyue; Gu, Junlian; Yu, Dehai; Wang, Guanjun; Zhou, Lei; Zhang, Xiaoying; Zhao, Yuguang; Chen, Xiao; Zheng, Shirong; Liu, Qiang; Cai, Lu

2016-01-01

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′ -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy. PMID:26788032

Detecting T-cell reactivity to whole cell vaccines

PubMed Central

Brusic, Ana; Hainz, Ursula; Wadleigh, Martha; Neuberg, Donna; Su, Mei; Canning, Christine M.; DeAngelo, Daniel J.; Stone, Richard M.; Lee, Jeng-Shin; Mulligan, Richard C.; Ritz, Jerome; Dranoff, Glenn; Sasada, Tetsuro; Wu, Catherine J.

2012-01-01

BCR-ABL+ K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8+ T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown. PMID:23170257

Effects of vitamin K3 and K5 on proliferation, cytokine production, and regulatory T cell-frequency in human peripheral-blood mononuclear cells.

PubMed

Hatanaka, Hiroshige; Ishizawa, Hitomi; Nakamura, Yurie; Tadokoro, Hiroko; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

2014-03-18

The effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Anti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. VK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10-100μM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10-100μM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10μM (p<0.001). Our data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of Vitamin K3 and K5 on Daunorubicin-resistant Human T Lymphoblastoid Leukemia Cells.

PubMed

Nakaoka, Eri; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

2015-11-01

Anticancer efficacy of vitamin K derivatives on multidrug-resistant cancer cells has been scarcely investigated. The effects of vitamins K3 and K5 on proliferation of human leukemia MOLT-4 cells and on daunorubicin-resistant MOLT-4/DNR cells were estimated by a WST assay. Apoptotic cells were detected by Annexin V and propidium iodide staining, followed by flow cytometry. Vitamins K3 and K5 significantly inhibited proliferation of leukemic cells at 10 and 100 μM (p<0.05), and these effects were almost equally observed in both MOLT-4 and MOLT/DNR drug-resistant cells. Vitamin K3 induced cell apoptosis at 10 and 100 μM in both MOLT-4 and MOLT-4/DNR cells (p<0.05). Vitamin K5 also increased apoptotic cells, while rather inducing necrotic cell death. Vitamins K3 and K5 suppress MOLT-4 and MOLT-4/DNR cell-proliferation partially through induction of apoptosis, and these vitamin derivatives can overcome drug resistance due to P-glycoprotein expression. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The cell proliferation antigen Ki-67 organises heterochromatin

PubMed Central

Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

2016-01-01

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

PubMed

Goudarzi, Fariba; Asadi, Asadollah; Afsharpour, Maryam; Jamadi, Robab Hassanvand

2018-05-01

The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

Five novel naphthylisoquinoline alkaloids with growth inhibitory activities against human leukemia cells HL-60, K562 and U937 from stems and leaves of Ancistrocladus tectorius.

PubMed

Jiang, Chao; Li, Zhan-Lin; Gong, Ping; Kang, Sheng-Li; Liu, Ming-Sheng; Pei, Yue-Hu; Jing, Yong-Kui; Hua, Hui-Ming

2013-12-01

Two new 7,6'-coupled naphthylisoquinolines, namely ancistrotectorines A (1) and B (2), two new 5,3'-coupled naphthylisoquinolines, namely ancistrotectorines C (3) and D (4), and one new 7,8-coupled naphthylisoquinoline, namely ancistrotectorine E (5), together with 9 known naphthylisoquinoline alkaloids, hamatine (6), ancistrobertsonine B (7), ancistrocladinine (8), hamatinine (9), ancistrotanzanine A (10), ancistrotanzanine B (11), ancistrotectoriline B (12), 7-epi-ancistrobrevine D (13), and ancistrotectorine (14), were isolated from the 70% EtOH extract of Ancistrocladus tectorius. Their structures were elucidated based on the extensive analysis of spectroscopic data (1D, 2D NMR and MS). Compound 5 exhibited inhibitory activities against HL-60, K562 and U937 cell lines with IC50 values of 1.70, 4.18 and 2.56 μM respectively. © 2013.

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

PubMed

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

2017-01-01

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

Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI{sub 3}K and mTOR

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

Wang, Yanling; Sato, Masaaki; Guo, Yuan

2014-10-15

The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI{sub 3}K system and the mTOR complexes were all activated by cAMP, but these activations weremore » not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias. - Highlights: • cAMP can mimick norepinephrine-induced proliferation of brown preadipocytes. • The cAMP-induced proliferation can occur in the absence of serum, of any other growth factors, and of insulin. • Erk1/2, PI{sub 3}K and mTOR are cAMP activated but not involved in induction of proliferation. • A Protein Kinase C member may be in the signalling cascade. • This pathway analysis may also be of importance for certain endocrine hyper- and neoplasias.« less

Extracellular matrix stiffness causes systematic variations in proliferation and chemosensitivity in myeloid leukemias.

PubMed

Shin, Jae-Won; Mooney, David J

2016-10-25

Extracellular matrix stiffness influences biological functions of some tumors. However, it remains unclear how cancer subtypes with different oncogenic mutations respond to matrix stiffness. In addition, the relevance of matrix stiffness to in vivo tumor growth kinetics and drug efficacy remains elusive. Here, we designed 3D hydrogels with physical parameters relevant to hematopoietic tissues and adapted them to a quantitative high-throughput screening format to facilitate mechanistic investigations into the role of matrix stiffness on myeloid leukemias. Matrix stiffness regulates proliferation of some acute myeloid leukemia types, including MLL-AF9 + MOLM-14 cells, in a biphasic manner by autocrine regulation, whereas it decreases that of chronic myeloid leukemia BCR-ABL + K-562 cells. Although Arg-Gly-Asp (RGD) integrin ligand and matrix softening confer resistance to a number of drugs, cells become sensitive to drugs against protein kinase B (PKB or AKT) and rapidly accelerated fibrosarcoma (RAF) proteins regardless of matrix stiffness when MLL-AF9 and BCR-ABL are overexpressed in K-562 and MOLM-14 cells, respectively. By adapting the same hydrogels to a xenograft model of extramedullary leukemias, we confirm the pathological relevance of matrix stiffness in growth kinetics and drug sensitivity against standard chemotherapy in vivo. The results thus demonstrate the importance of incorporating 3D mechanical cues into screening for anticancer drugs.

Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation.

PubMed

Woessner, David W; Lim, Carol S

2013-01-07

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell. Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which block ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use is required in order to treat CML. The primary failure for TKIs is through the development of a resistant population due to mutations in the TKI binding regions. This led us to develop the mutant coiled-coil, CC(mut2), an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation. In this article, we explore additional pathways that are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach, we test the combination of CC(mut2) and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox5 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-1 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy using chloroquine in addition to blocking BCR-ABL signaling with the CC(mut2) was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways.

Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells.

PubMed

Mendes, Anderson Nogueira; Filgueiras, Lívia Alves; Siqueira, Monica Regina Pimentel; Barbosa, Gleyce Moreno; Holandino, Carla; de Lima Moreira, Davyson; Pinto, José Carlos; Nele, Marcio

2017-01-01

This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n -hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia.

Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells

PubMed Central

Mendes, Anderson Nogueira; Filgueiras, Lívia Alves; Siqueira, Monica Regina Pimentel; Barbosa, Gleyce Moreno; Holandino, Carla; de Lima Moreira, Davyson; Pinto, José Carlos; Nele, Marcio

2017-01-01

This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n-hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia. PMID:29200848

Abrogation of Cbl-PI3K interaction increases bone formation and osteoblast proliferation.

PubMed

Brennan, Tracy; Adapala, Naga Suresh; Barbe, Mary F; Yingling, Vanessa; Sanjay, Archana

2011-11-01

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (Cbl(YF/YF), YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745-36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl-PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.

Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

PubMed

Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

2017-06-01

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways

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

Fang, Ling, E-mail: fangling_1984@126.com; Zhan, Shuxiang; Huang, Cheng

2013-11-01

TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl{sub 4}-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increasemore » of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 mRNA and protein in the fibrotic livers from CCl{sub 4}-treated rats. • Increasing expression of TRPM7 mRNA and protein during HSC activation. • Blockade of TRPM7 inhibited the PDGF-BB induced proliferation of HSC-T6 cells. • Blockade of TRPM7 decreased α-SMA and Col1α1 expressions in activated HSC-T6 cells. • TRPM7 up-regulation contributes to the activation of ERK and AKT pathways.« less

Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells

PubMed Central

Kawasaki, Akira; Matsumura, Itaru; Miyagawa, Jun-ichiro; Ezoe, Sachiko; Tanaka, Hirokazu; Terada, Yasuhiko; Tatsuka, Masaaki; Machii, Takashi; Miyazaki, Hiroshi; Furukawa, Yusuke; Kanakura, Yuzuru

2001-01-01

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes. PMID:11266445

Menin determines K-RAS proliferative outputs in endocrine cells

PubMed Central

Chamberlain, Chester E.; Scheel, David W.; McGlynn, Kathleen; Kim, Hail; Miyatsuka, Takeshi; Wang, Juehu; Nguyen, Vinh; Zhao, Shuhong; Mavropoulos, Anastasia; Abraham, Aswin G.; O’Neill, Eric; Ku, Gregory M.; Cobb, Melanie H.; Martin, Gail R.; German, Michael S.

2014-01-01

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors. PMID:25133424

miR-489 inhibits proliferation, cell cycle progression and induces apoptosis of glioma cells via targeting SPIN1-mediated PI3K/AKT pathway.

PubMed

Li, Yan; Ma, Xiaolin; Wang, Yanpeng; Li, Guohua

2017-09-01

microRNA-489 (miR-489), a newly identified tumor-related miRNA, functions as an oncogene or tumor suppressor via regulating growth and metastasis of human cancers. But, the clinical significance, biological function and underlying mechanisms of miR-489 in glioma remain rarely known. Here, we showed that the levels of miR-489 in glioma tissues were notably underexpressed compared to corresponding non-tumor tissues. In accordance, the relative levels of miR-489 were decreased in glioma cell lines compared with NHA cells. Kaplan-Meier plots indicated that miR-489 low expressing glioma patients showed a prominent shorter overall survival. In addition, miR-489 overexpression prohibited proliferation and cell cycle progression, and promoted apoptosis in U251 cells. While, miR-489 knockdown showed opposite effects on these cellular processes of U87 cells. In vivo experiments demonstrated that miR-489 restoration reduced the tumor volume and weight of subcutaneous glioma xenografts in nude mice. Notably, Spindlin 1 (SPIN1) was inversely and directly regulated by miR-489 in glioma cells. A negative correlation between the expression of miR-489 and SPIN1 mRNA was confirmed in glioma tissues. Interestingly, miR-489 inversely modulated activation of PI3K/AKT pathway and expression of downstream targets including p-mTOR, Cyclin D1 and BCL-XL. SPIN1 re-expression abolished the effects of miR-489 on U251 cells with enhanced activation of PI3K/AKT pathway and malignant phenotype. Meanwhile, AKT inhibitor MK-2206 blocked activation of PI3K/AKT pathway and resulted in reduced proliferation, cell cycle arrest and increased apoptosis in miR-489 down-regulating U87 cells. Altogether, our data support that miR-489 loss facilitates malignant phenotype of glioma cells probably via SPIN1-mediated PI3K/AKT pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

PubMed Central

Everett, Peter; Clish, Clary B.; Sukhatme, Vikas P.

2010-01-01

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for

PI3K-mediated proliferation of fibroblasts by Calendula officinalis tincture: implication in wound healing.

PubMed

Dinda, Manikarna; Dasgupta, Uma; Singh, Namrata; Bhattacharyya, Debasish; Karmakar, Parimal

2015-04-01

Calendula officinalis, a member of the Asteraceae family, is a flowering plant and has been used for its antibacterial, antifungal, antiviral, antiinflammatory, anticancer and wound healing activity. The mode of action of C. officinalis tincture on wound healing is poorly understood. Here, we investigated the role of C. officinalis tincture (CDOT) on cell viability and wound closure. C. officinalis tincture stimulated both proliferation and migration of fibroblasts in a statistically significant manner in a PI3K-dependent pathway. The increase in phosphorylation of FAK (Tyr 397) and Akt (Ser 473) was detected after treatment of CDOT. Inhibition of the PI3K pathway by wortmannin and LY294002 decreased both cell proliferation and cell migration. HPLC-ESI MS revealed the presence of flavonol glycosides as the major compounds of CDOT. Altogether, our results showed that CDOT potentiated wound healing by stimulating proliferation and migration of fibroblast in a PI3K-dependent pathway, and the identified compounds are likely to be responsible for wound healing activity. Copyright © 2015 John Wiley & Sons, Ltd.

miR-18a promotes cell proliferation of esophageal squamous cell carcinoma cells by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis

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

Zhang, Weiguo, E-mail: weiguozhangHU@gmail.com; Lei, Caipeng; Fan, Junli

Esophageal squamous cell carcinoma (ESCC) is one of the lethal cancers with a high incidence rate in Asia. Cyclin D1 is overexpressed and plays an important role in the carcinogenesis of ESCC; however the mechanism of the deregulation of Cyclin D1 in ESCC remains to be determined. In the study, we found that miR-18a promotes the expression Cyclin D1 by targeting PTEN in eophageal squamous cell carcinoma TE13 and Eca109 cells. Transfection of miR-18a mimetics increased cyclin D1, while transfection of miR-18a antagomir decreased D1. Moreover, miR-18a-mediated upregulation of cyclin D1 was accompanied with downregulation of PTEN, which is a directmore » target of miR-18a, and increase of the phosphorylation of AKT and S6K1. In addition, pharmacologic inhibition of AKT or mTOR kinases abolished the increase of cyclinD1 by miR-18a, which was accompanied with decreased phosphorylation of Rb−S780 and inhibition of cell proliferation. Our results demonstrated the upregulation of miR-18a promoted cell proliferation by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis, suggesting that small molecule inhibitors of AKT-mTOR signaling are potential agents for the treatment of ESCC patients with upregulation of miR-17-92 cluster. - Highlights: • miR-18a promotes the proliferation of ESCC cells. • miR-18a increase cyclin D1 expression in ESCC cells. • miR-18a directly targets PTEN in ESCC cells. • Inhibition of AKT-mTOR prevents miR-18a-induced cyclin D1 in ESCC cells. • miR-18a antagomir sensitizes ESCC cells to cisplatin.« less

Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

PubMed

Kornasio, Reut; Riederer, Ingo; Butler-Browne, Gillian; Mouly, Vincent; Uni, Zehava; Halevy, Orna

2009-05-01

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.

Inhibition of Siah2 Ubiquitin Ligase by Vitamin K3 Attenuates Chronic Myeloid Leukemia Chemo-Resistance in Hypoxic Microenvironment.

PubMed

Huang, Jixian; Lu, Ziyuan; Xiao, Yajuan; He, Bolin; Pan, Chengyun; Zhou, Xuan; Xu, Na; Liu, Xiaoli

2018-02-05

BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

In vitro vitamin K3 effect on conjunctival fibroblast migration and proliferation.

PubMed

Pinilla, I; Izaguirre, L B; Gonzalvo, F J; Piazuelo, E; Garcia-Gonzalez, M A; Sanchez-Cano, A I; Sopeña, F

2014-01-01

To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [(3)H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Inhibition of brain tumor cell proliferation by alternating electric fields

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

Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr

2014-11-17

This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway.

PubMed

Xiao, Mingbing; Li, Tao; Ji, Yifei; Jiang, Feng; Ni, Wenkai; Zhu, Jing; Bao, Baijun; Lu, Cuihua; Ni, Runzhou

2018-01-01

S100A11, a member of S100 calcium-binding protein family, is associated with the numerous processes of tumorigenesis and metastasis. In the present study, the role of S100A11, and its possible underlying mechanisms in cell proliferation, apoptosis and cell cycle distribution in human pancreatic cancer were explored. Immunohistochemical analyses of S100A11 and phosphorylated (p)-AKT serine/threonine kinase (AKT) were performed in 30 resected specimens from patients with pancreatic cancer. PANC-1 cells were transfected with pcDNA3.1-S100A11 or treated with 50 µmol/l LY294002 for 48 h. Cell proliferation was determined using a cell counting kit-8 assay, whereas apoptosis and cell cycle distribution were determined by flow cytometry analysis. The mRNA and protein levels of S100A11, and AKT were determined using semi quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. Pearson correlation analysis revealed that the expression levels of S100A11 and p-AKT were positively correlated (r, 0.802; P<0.05). Compared with the control group, S100A11 overexpression significantly promoted PANC-1 cell proliferation and reduced the percentage of early apoptotic cells. Flow cytometric analysis indicated that the proportion of PANC-1 cells in the S phase was significantly elevated and cell percentage in the G0/G1 phase declined in response to S100A11 overexpression (all P<0.05). S100A11 overexpression also significantly increased AKT mRNA and p-AKT protein expression levels (both P<0.05). The phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, significantly inhibited PANC-1 cell proliferation, promoted apoptosis and caused G1/S phase arrest in PANC-1 cells (all P<0.05). These findings together suggest that S100A11 promotes the viability and proliferation of human pancreatic cancer PANC-1 cells through the upregulation of the PI3K/AKT signaling pathway. Thus, S100A11 may be considered as a novel drug target for targeted therapy of

Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.

PubMed

Ren, Yan-Ping; Zhang, Ming-Juan; Zhang, Ting; Huang, Ruo-Wen

2014-01-01

To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.

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

PubMed

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

2014-09-01

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

Leaf extracts from Moricandia arvensis promote antiproliferation of human cancer cells, induce apoptosis, and enhance antioxidant activity.

PubMed

Skandrani, Ines; Boubaker, Jihed; Bhouri, Wissem; Limem, Ilef; Kilani, Soumaya; Ben Sghaier, Mohamed; Neffati, Aicha; Bouhlel, Ines; Ghedira, Kamel; Chekir-Ghedira, Leila

2010-01-01

The in vitro antiproliferative, apoptotic, and antioxidant activities from leaf extracts of Moricandia arvensis, which are used in traditional cooking and medicines, were investigated. The MTT assay revealed that only TOF (total oligomer flavonoids), ethyl acetate (EA), chloroform (Chl), and petroleum ether (PE) extracts inhibited the proliferation of K562 cells. Apoptosis plays a very important role in the treatment of cancer by promoting the apoptosis of cancer cells and limiting the concurrent death of normal cells. Thus, the possible effects of M. arvensis extracts on the induction of apoptosis in human leukemic cells (K562 cells) were investigated. The electrophoretic analysis of DNA fragmentation confirms that TOF, Chl, PE, and EA extracts provoke DNA fragmentation. Using the lipid peroxidation inhibitory assay, the antioxidant capacity of M. arvensis extracts was evaluated by the ability of each extract to inhibit malondialdehyde formation. It was revealed that EA and TOF extracts are the most active in scavenging the hydroxyl radicals.

BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways

PubMed Central

Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-01-01

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer. PMID:24805814

BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

PubMed

Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-01-01

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

An essential role for the Id1/PI3K/Akt/NFkB/survivin signalling pathway in promoting the proliferation of endothelial progenitor cells in vitro.

PubMed

Li, Wei; Wang, Hang; Kuang, Chun-Yan; Zhu, Jin-Kun; Yu, Yang; Qin, Zhe-Xue; Liu, Jie; Huang, Lan

2012-04-01

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.

Akt SUMOylation regulates cell proliferation and tumorigenesis.

PubMed

Li, Rong; Wei, Jie; Jiang, Cong; Liu, Dongmei; Deng, Lu; Zhang, Kai; Wang, Ping

2013-09-15

Proto-oncogene Akt plays essential roles in cell proliferation and tumorigenesis. Full activation of Akt is regulated by phosphorylation, ubiquitination, and acetylation. Here we report that SUMOylation of Akt is a novel mechanism for its activation. Systematically analyzing the role of lysine residues in Akt activation revealed that K276, which is located in a SUMOylation consensus motif, is essential for Akt activation. Ectopic or endogenous Akt1 could be modified by SUMOylation. RNA interference-mediated silencing of UBC9 reduced Akt SUMOylation, which was promoted by SUMO E3 ligase PIAS1 and reversed by the SUMO-specific protease SENP1. Although multiple sites on Akt could be SUMOylated, K276 was identified as a major SUMO acceptor site. K276R or E278A mutation reduced SUMOylation of Akt but had little effect on its ubiquitination. Strikingly, these mutations also completely abolished Akt kinase activity. In support of these results, we found that expression of PIAS1 and SUMO1 increased Akt activity, whereas expression of SENP1 reduced Akt1 activity. Interestingly, the cancer-derived mutant E17K in Akt1 that occurs in various cancers was more efficiently SUMOylated than wild-type Akt. Moreover, SUMOylation loss dramatically reduced Akt1 E17K-mediated cell proliferation, cell migration, and tumorigenesis. Collectively, our findings establish that Akt SUMOylation provides a novel regulatory mechanism for activating Akt function. ©2013 AACR.

Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi

PubMed Central

Parker, Aimee; Maclaren, Oliver J.; Fletcher, Alexander G.; Muraro, Daniele; Kreuzaler, Peter A.; Byrne, Helen M.; Maini, Philip K.; Watson, Alastair J. M.; Pin, Carmen

2017-01-01

The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.—Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi. PMID:27811059

Forkhead box K2 inhibits the proliferation, migration, and invasion of human glioma cells and predicts a favorable prognosis.

PubMed

Wang, Bo; Zhang, XueBin; Wang, Wei; Zhu, ZhiZhong; Tang, Fan; Wang, Dong; Liu, Xi; Zhuang, Hao; Yan, XiaoLing

2018-01-01

Forkhead box K2 (FOXK2) is a member of the forkhead box family of transcription factors. Recently, researchers discovered that overexpression of FOXK2 inhibits the proliferation and metastasis of breast cancer, non-small cell lung cancer, and colorectal cancer, and is related to the clinical prognosis. However, in hepatocellular carcinoma, FOXK2 results in the opposite phenotypes. Currently, the contribution of FOXK2 to glioma pathogenesis is not clear. We evaluated the expression of FOXK2 in 151 glioma patients using immunohistochemistry assays. The associations among the expression of FOXK2, clinicopathological parameters, and the prognosis of glioma patients were statistically analyzed. We downregulated and upregulated the level of FOXK2 in glioma cells by transfections with small interfering RNA and plasmids. Then, we investigated the effects on tumor cell behavior in vitro by Cell Counting Kit-8 assays, colony-formation assay, transwell assay, and the epithelial-to-mesenchymal transition (EMT) biomarker levels. The clinical data showed that expression of FOXK2 gradually decreased with increasing World Health Organization (WHO) grades and a low level of FOXK2 indicates a poor prognosis. FOXK2 expression is negatively correlated with Ki67 expression and the WHO degree but is not correlated with other clinicopathological parameters, including sex, age, Karnofsky Performance Status, tumor diameter, O -6-methylguanine-DNA methyltransferase, and glutathione S -transferase pi. FOXK2 knockdown enhances glioma cell proliferation, migration, invasion, and EMT process, and, in contrast, FOXK2 overexpression inhibits glioma cell proliferation, migration, invasion, and the EMT process. Expression of FOXK2 gradually decreases with increasing WHO grades. FOXK2 inhibits tumor proliferation, migration, and invasion. FOXK2 is a critical mediator of the EMT process.

Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

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

Liu, Wenrui; Kong, Hui; Zeng, Xiaoning

Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation andmore » migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

PubMed

Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S; Li, Wanda Y; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H Y; Snow, Bryan E; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C; He, Housheng Hansen; Ohashi, Pamela S; Mak, Tak W

2017-01-13

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8 + T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells.

PubMed

Mizutani, Naoki; Omori, Yukari; Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi; Suzuki, Motoshi; Kyogashima, Mamoru; Nakamura, Mitsuhiro; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Effectiveness of imatinib mesylate over etoposide in the treatment of sensitive and resistant chronic myeloid leukaemia cells in vitro.

PubMed

Husaini, Roslina; Ahmad, Munirah; Zakaria, Zubaidah

2017-06-01

Chronic myeloid leukaemia (CML) is a form of leukaemia derived from the myeloid cell lineage. Imatinib mesylate, the breakpoint cluster region-abelson murine leukeamia kinase inhibitor, is a specific reagent used in the clinical treatment of CML. The DNA topoisomerase II inhibitor, etoposide, is also employed as a therapeutic, though it is used to a lesser extent. The present study aims to evaluate the effects of CML-targeted therapy, utilising imatinib mesylate and etoposide in the in vitro treatment of parental sensitive and adriamycin-resistant CML in the K562 and K562/ADM cell lines, respectively. Preliminary work involved the screening of multidrug resistant (MDR) gene expression, including MDR1, MRP1 and B-cell lymphoma 2 (BCL-2) at the mRNA levels. The sensitive and resistant CML cell lines expressed the MRP1 gene, though the sensitive K562 cells expressed low, almost undetectable levels of MDR1 and BCL-2 genes relative to the K562/ADM cells. Following treatment with imatinib mesylate or etoposide, the IC50 for imatinib mesylate did not differ between the sensitive and resistant cell lines (0.492±0.024 and 0.378±0.029, respectively), indicating that imatinib mesylate is effective in the treatment of CML regardless of cell chemosensitivity. However, the IC50 for etoposide in sensitive K562 cells was markedly lower than that of K562/ADM cells (50.6±16.5 and 194±8.46 µM, respectively), suggesting that the higher expression levels of MDR1 and/or BCL-2 mRNA in resistant cells may be partially responsible for this effect. This is supported by terminal deoxynucleotidyl transferase dUTP nick-end labeling data, whereby a higher percentage of apoptotic cells were found in the sensitive and resistant K562 cells treated with imatinib mesylate (29.3±0.2 and 31.9±16.7%, respectively), whereas etoposide caused significant apoptosis of sensitive K562 cells (18.3±8.35%) relative to K562/ADM cells (5.17±3.3%). In addition, the MDR genes in K562/ADM cells were knocked

Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma

PubMed Central

Wang, H; Wu, Q; Liu, Z; Luo, X; Fan, Y; Liu, Y; Zhang, Y; Hua, S; Fu, Q; Zhao, M; Chen, Y; Fang, W; Lv, X

2014-01-01

It is largely recognized that fibroblast activation protein (FAP) is expressed in cancer-associated fibroblasts (CAFs) of many human carcinomas. Furthermore, FAP was recently also reported to be expressed in carcinoma cells of the breast, stomach, pancreatic ductal adenocarcinoma, colorectum, and uterine cervix. The carcinoma cell expression pattern of FAP has been described in several types of cancers, but the role of FAP in oral squamous cell carcinoma (OSCC) is unknown. The role of endogenous FAP in epithelium-derived tumors and molecular mechanisms has also not been reported. In this study, FAP was found to be expressed in carcinoma cells of OSCC and was upregulated in OSCC tissue samples compared with benign tissue samples using immunohistochemistry. In addition, its expression level was closely correlated with overall survival of patients with OSCC. Silencing FAP inhibited the growth and metastasis of OSCC cells in vitro and in vivo. Mechanistically, knockdown of FAP inactivated PTEN/PI3K/AKT and Ras-ERK and its downstream signaling regulating proliferation, migration, and invasion in OSCC cells, as the inhibitory effects of FAP on the proliferation and metastasis could be rescued by PTEN silencing. Our study suggests that FAP acts as an oncogene and may be a potential therapeutic target for patients with OSCC. PMID:24722280

Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma.

PubMed

Wang, H; Wu, Q; Liu, Z; Luo, X; Fan, Y; Liu, Y; Zhang, Y; Hua, S; Fu, Q; Zhao, M; Chen, Y; Fang, W; Lv, X

2014-04-10

It is largely recognized that fibroblast activation protein (FAP) is expressed in cancer-associated fibroblasts (CAFs) of many human carcinomas. Furthermore, FAP was recently also reported to be expressed in carcinoma cells of the breast, stomach, pancreatic ductal adenocarcinoma, colorectum, and uterine cervix. The carcinoma cell expression pattern of FAP has been described in several types of cancers, but the role of FAP in oral squamous cell carcinoma (OSCC) is unknown. The role of endogenous FAP in epithelium-derived tumors and molecular mechanisms has also not been reported. In this study, FAP was found to be expressed in carcinoma cells of OSCC and was upregulated in OSCC tissue samples compared with benign tissue samples using immunohistochemistry. In addition, its expression level was closely correlated with overall survival of patients with OSCC. Silencing FAP inhibited the growth and metastasis of OSCC cells in vitro and in vivo. Mechanistically, knockdown of FAP inactivated PTEN/PI3K/AKT and Ras-ERK and its downstream signaling regulating proliferation, migration, and invasion in OSCC cells, as the inhibitory effects of FAP on the proliferation and metastasis could be rescued by PTEN silencing. Our study suggests that FAP acts as an oncogene and may be a potential therapeutic target for patients with OSCC.

Loss of MAP3K1 enhances proliferation and apoptosis during retinal development

PubMed Central

Mongan, Maureen; Wang, Jingcai; Liu, Hongshan; Fan, Yunxia; Jin, Chang; Kao, Winston Y.-W.; Xia, Ying

2011-01-01

Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1+/ΔKDJnk1–/– and Map3k1+/ΔKDJnk+/–Jnk2+/– mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration. PMID:21862560

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

PubMed

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

2016-04-01

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

Neural control of colonic cell proliferation.

PubMed

Tutton, P J; Barkla, D H

1980-03-15

The mitotic rate in rat colonic crypts and in dimethylhydrazine-induced colonic carcinomas was measured using a stathmokinetic technique. In sympathectomized animals cell proliferation was retarded in the crypts but not in the tumors, whereas in animals treated with Metaraminol, a drug which releases norepinephrine from nerve terminals, crypt cell but not tumor cell proliferation was accelerated. Blockade of alpha-adrenoceptors also inhibited crypt cell proliferation. However, stimulation of beta-adrenoceptors inhibited and blockade of beta-adrenoceptors accelerated tumor cell proliferation without influencing crypt cell proliferation. Injection of either serotonin or histamine stimulated tumor but not crypt cell proliferation and blockade or serotonin receptors or histamine H2-receptors inhibited tumor cell proliferation. It is postulated that cell proliferation in the colonic crypts, like that in the jejunal crypts, is under both endocrine and autonomic neural control whereas colonic tumor cell division is subject to endocrine regulation alone.

MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

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

Ma, Yanfei; Qin, Huadong; Cui, Yunfu, E-mail: yfma77@126.com

Highlights: •MiR-34a is up- and GAS1 is down-regulated in papillary thyroid carcinoma. •GAS1 is a direct target for miR-34a. •MiR-34a promotes PTC cells proliferation and inhibits apoptosis through PI3K/Akt/Bad pathway. -- Abstract: MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors.more » However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.« less

NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway

PubMed Central

Xie, Yufeng; He, Yang; Wang, Zhenxin; Qin, Lei

2017-01-01

The nuclear receptor coactivator 5 (NCOA5) displays both coactivator and corepressor functions. Previous studies showed that alteration of NCOA5 participates in carcinogenesis and progression. However, its roles in colorectal cancer (CRC) remain unknown. Herein, we demonstrated that expression of NCOA5 in human CRC tissues was notably higher than that in adjacent tissues, which significantly correlated with clinicopathological features such as length of tumor, regional lymph node staging and cancer staging. Knockdown of NCOA5 markedly suppressed proliferation, migration and invasion of SW620 high malignant CRC cells. Silencing of NCOA5 also inhibited in vivo growth of SW620 CRC subcutaneously xenografted tumors in athymic BALB/c nude mice. Meanwhile, Overexpression of NCOA5 facilitated these processes in SW480 low malignant CRC cells. Furthermore, knockdown of NCOA5 induced cell cycle G1 phase arrest in SW620 cells, whereas overexpression of NCOA5 promoted G1 to S phase transition in SW480 cells. Mechanistic studies revealed that NCOA5 upregulated phospho-protein kinase B (p-PKB/AKT), Cyclin D1 and matrix metalloproteinase 9 (MMP9) as well as downregulated P27 in CRC cells. Notably, PI3K inhibitor LY294002 obviously attenuated the effects of NCOA5 on p-AKT, Cyclin D1, P27 and MMP9. Moreover, LY294002 and knockdown of Cyclin D1 or MMP9 remarkably blocked the tumor-promoting activity of NCOA5. Collectively, NCOA5 promoted CRC cell proliferation, migration and invasion by upregulating Cyclin D1 and MMP9 while downregulating P27 to a great extent via activating PI3K/AKT signaling pathway. These findings suggested that NCOA5 exhibits an oncogenic effect in human CRC and represents a novel therapeutic target for CRC. PMID:29296214

Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism.

PubMed

Bonilla-Porras, Angelica R; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

2011-06-10

Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia. It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM) or 100:1 (300 μM: 3 μM), respectively. We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia.

Synergistic effect of atorvastatin and cyanidin-3-glucoside against angiotensin II-mediated vascular smooth muscle cell proliferation and migration through MAPK and PI3K/Akt pathways.

PubMed

Pantan, Rungusa; Tocharus, Jiraporn; Phatsara, Manussabhorn; Suksamrarn, Apichart; Tocharus, Chainarong

2016-09-13

This study aimed to investigate the mechanism of cyanidin-3-glucoside (C3G) in synergy with atorvastatin, even when it is used in low concentrations. Human aortic smooth muscle cells (HASMCs) were used to verify the synergistic mechanism of atorvastatin and C3G against angiotensin II-induced proliferation and migration. BrdU incorporation assay was used to evaluate cell proliferation. Wound healing and Boyden chamber assays were used to investigate cell migration. The cell cycle was examined using flow cytometry. The results revealed that atorvastatin and C3G exhibit a synergistic effect in ameliorating HASMC proliferation and migration by enhancing cell cycle arrest. In addition, these effects also decreased mitogen-activated protein kinase (MAPK) activity by attenuating the expression of phospho-p38, phospho-extracellular signaling-regulated kinase 1/2, and phospho-c-Jun N-terminal kinase. Furthermore, the combination of atorvastatin and C3G modulated the PI3K/Akt pathway and upregulated p21 Cip1 , which was associated with decreases in cyclin D 1 and phospho-retinoblastoma expressions. The synergistic effect of atorvastatin and C3G induced anti-proliferation and anti-migration through MAPK and PI3K/Akt pathways mediated by AT 1 R. These results suggest that the synergistic effect of atorvastatin and C3G may be an alternative therapy for atherosclerosis patients.

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

PubMed

Lee, Jin; Lim, Kye-Taek

2011-08-01

Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.

Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

PubMed Central

Peng, Xing-Xiang; Tiwari, Amit K.; Wu, Hsiang-Chun; Chen, Zhe-Sheng

2012-01-01

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance (MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells. PMID:22098951

Branches of the NF-κB signaling pathway regulate proliferation of oval cells in rat liver regeneration.

PubMed

Zhao, W M; Qin, Y L; Niu, Z P; Chang, C F; Yang, J; Li, M H; Zhou, Y; Xu, C S

2016-03-24

The NF-kB (nuclear factor kB) pathway is involved in the proliferation of many cell types. To explore the mechanism of the NF-kB signaling pathway underlying the oval cell proliferation during rat liver regeneration, the Rat Genome 230 2.0 Array was used to detect expression changes of NF-kB signaling pathway-related genes in oval cells. The results revealed that the expression levels of many genes in the NF-kB pathway were significantly changed. This included 48 known genes and 16 homologous genes, as well as 370 genes and 85 homologous genes related to cell proliferation. To further understand the biological significance of these changes, an expression profile function was used to analyze the potential biological processes. The results showed that the NF-kB pathway promoted oval cell proliferation mainly through three signaling branches; the tumor necrosis factor alpha branch (TNF-a pathway), the growth factor branch, and the chemokine branch. An integrated statistics method was used to define the key genes in the NF-kB pathway. Seven genes were identified to play vital roles in the NF-kB pathway. To confirm these results, the protein content, including two key genes (TNF and FGF11) and two non-key genes (CCL2 and TNFRSF12A), were analyzed using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The results were generally consistent with those of the array data. To conclude, three branches and seven key genes were involved in the NF-kB signaling pathway that regulates oval cell proliferation during rat liver regeneration.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

PubMed

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies

Insulin-like growth factor-I stimulates H{sub 4}II rat hepatoma cell proliferation: Dominant role of PI-3'K/Akt signaling

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

Alexia, Catherine; Fourmatgeat, Pascal; Delautier, Daniele

2006-04-15

Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H{sub 4}II rat hepatoma cell proliferation, as estimated by {sup 3}H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H{sub 4}II cells, stimulation being maximal 3 h aftermore » the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H{sub 4}II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H{sub 4}II cells. Altogether, these data support the notion that the stimulation of H{sub 4}II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signal0009i.« less

The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus.

PubMed

Sengupta, P K; Lavelle, D E; DeSimone, J

1994-03-01

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta-globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation*

PubMed Central

Fukushima, Toshiaki; Nakamura, Yusaku; Yamanaka, Daisuke; Shibano, Takashi; Chida, Kazuhiro; Minami, Shiro; Asano, Tomoichiro; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2012-01-01

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G1 phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G1 to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G1 phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr1316-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR−/− fibroblasts expressing exogenous mutant IGF-IR in which Tyr1316 was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation. PMID:22767591

Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells

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

Tang, Chunling; Yang, Liqun; Jiang, Xiaolan

Highlights: • Tigecycline inhibited cell growth and proliferation in human gastric cancer cells. • Tigecycline induced autophagy not apoptosis in human gastric cancer cells. • AMPK/mTOR/p70S6K pathway was activated after tigecycline treatment. • Tigecycline inhibited tumor growth in xenograft model of human gastric cancer cells. - Abstract: Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cellmore » proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer.« less

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

PubMed

Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells: Importance of ERK1/2 and AKT Signaling Pathways.

PubMed

Liang, Xinyue; Gu, Junlian; Yu, Dehai; Wang, Guanjun; Zhou, Lei; Zhang, Xiaoying; Zhao, Yuguang; Chen, Xiao; Zheng, Shirong; Liu, Qiang; Cai, Lu; Cui, Jiuwei; Li, Wei

2016-01-01

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3' -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

microRNA-188 is downregulated in oral squamous cell carcinoma and inhibits proliferation and invasion by targeting SIX1.

PubMed

Wang, Lili; Liu, Hongchen

2016-03-01

microRNA-188 expression is downregulated in several tumors. However, its function and mechanism in human oral squamous cell carcinoma (OSCC) remains obscure. The present study aims to identify the expression pattern, biological roles, and potential mechanism by which miR-188 dysregulation is associated with oral squamous cell carcinoma. Significant downregulation of miR-188 was observed in OSCC tissues compared with paired normal tissues. In vitro, gain-of-function, loss-of-function experiments were performed to examine the impact of miR-188 on cancer cell proliferation, invasion, and cell cycle progression. Transfection of miR-188 mimics suppressed Detroit 562 cell proliferation, cell cycle progression and invasion, with downregulation of cyclin D1, MMP9, and p-ERK. Transfection of miR-188 inhibitor in FaDu cell line with high endogenous expression exhibited the opposite effects. Using fluorescence reporter assays, we confirmed that SIX1 was a direct target of miR-188 in OSCC cells. Transfection of miR-188 mimics downregulated SIX1 expression. SIX1 siRNA treatment abrogated miR-188 inhibitor-induced cyclin D1 and MMP9 upregulation. In addition, we found that SIX1 was overexpressed in 32 of 80 OSCC tissues. In conclusion, this study indicates that miR-188 downregulation might be associated with oral squamous cell carcinoma progression. miR-188 suppresses proliferation and invasion by targeting SIX1 in oral squamous cell carcinoma cells.

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition

PubMed Central

Chang, Yi; Li, Jiun-Yi; Jayakumar, Thanasekaran; Hung, Shou-Huang; Lee, Wei-Cheng; Manubolu, Manjunath; Sheu, Joen-Rong; Hsu, Ming-Jen

2017-01-01

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a (pp2a) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition.

PubMed

Chang, Yi; Li, Jiun-Yi; Jayakumar, Thanasekaran; Hung, Shou-Huang; Lee, Wei-Cheng; Manubolu, Manjunath; Sheu, Joen-Rong; Hsu, Ming-Jen

2017-11-27

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a ( pp2a ) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing

Inhibition of DNA-dependent protein kinase catalytic subunit by small molecule inhibitor NU7026 sensitizes human leukemic K562 cells to benzene metabolite-induced apoptosis.

PubMed

You, Hao; Kong, Meng-meng; Wang, Li-ping; Xiao, Xiao; Liao, Han-lin; Bi, Zhuo-yue; Yan, Hong; Wang, Hong; Wang, Chun-hong; Ma, Qiang; Liu, Yan-qun; Bi, Yong-yi

2013-02-01

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

32 CFR 562.6 - Responsibilities.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Responsibilities. 562.6 Section 562.6 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.6 Responsibilities. (a) The Commanding General, US Army Military Personnel Center, 200...

32 CFR 562.2 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Applicability. 562.2 Section 562.2 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.2 Applicability. This regulation applies to the program given at college level...

Oxyfadichalcone C inhibits melanoma A375 cell proliferation and metastasis via suppressing PI3K/Akt and MAPK/ERK pathways.

PubMed

Peng, Xiaolin; Wang, Zhengming; Liu, Yang; Peng, Xin; Liu, Yao; Zhu, Shan; Zhang, Zhe; Qiu, Yuling; Jin, Meihua; Wang, Ran; Zhang, Qingying; Kong, Dexin

2018-08-01

Melanoma remains to be one of the most incurable cancers. Discovery of novel antitumor agent for melanoma therapy is expected. We recently isolated Oxyfadichalcone C from Oxytropis falcate and investigated the anti-proliferative and anti-metastatic activity on human melanoma A375 cells in vitro. Cell viability was determined using MTT assay and soft agar cloning formation assay. The effect of Oxyfadichalcone C on cell cycle distribution and apoptosis were analyzed by flow cytometry. Cell metastasis was determined by wound healing assay, Transwell assay and Gelatin zymography assay. The effect of Oxyfadichalcone C on signal proteins of PI3K/Akt and MAPK/ERK pathways was examined by western blot analysis. Synergism assay was employed to determine whether combination of Oxyfadichalcone C with Vemurafenib would enhance the anti-proliferative effect. Oxyfadichalcone C potently inhibited proliferation, induced G1 phase arrest and weak apoptosis in A375 cells. Anti-migration and anti-invasion activities were also indicated. Such effects were associated with upregulation of p27, reduction of cyclin D1, p-pRb, p-Integrin β1, as well as the proteolytic activity of metalloproteinase (MMP)-2/9. Meanwhile, key molecules of PI3K/Akt and MAPK/ERK pathways were downregulated, which might be involved in the inhibition against proliferation and metastasis of A375 cells by Oxyfadichalcone C. In addition, combination of Oxyfadichalcone C with Vemurafenib at a ratio of IC50 Oxyfadichalcone C : 5 × IC 50 Vemurafenib exhibited synergistic anti-proliferative effect on A375 cells. Our findings suggest that Oxyfadichalcone C has the potential to be developed as a promising drug candidate for the treatment of melanoma. Copyright © 2018 Elsevier Inc. All rights reserved.

IGF-1 Regulates Cyr61 Induced Breast Cancer Cell Proliferation and Invasion

PubMed Central

Sarkissyan, Suren; Sarkissyan, Marianna; Wu, Yanyuan; Cardenas, Jessica; Koeffler, H. Phillip; Vadgama, Jaydutt V.

2014-01-01

Background Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation. Methods Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation. Results Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells. Conclusion The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion. PMID

31 CFR 562.303 - Entity.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Entity. 562.303 Section 562.303 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS... § 562.303 Entity. The term entity means a partnership, association, trust, joint venture, corporation...

32 CFR 562.1 - Purpose.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Purpose. 562.1 Section 562.1 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.1 Purpose. This regulation gives policies for conducting the Army's Senior Reserve Officers...

32 CFR 562.4 - Objectives.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Objectives. 562.4 Section 562.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.4 Objectives. The objectives of the ROTC program are to: (a) Attract, motivate, and prepare...

Activation of the PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation.

PubMed

Palacios, F; Abreu, C; Prieto, D; Morande, P; Ruiz, S; Fernández-Calero, T; Naya, H; Libisch, G; Robello, C; Landoni, A I; Gabus, R; Dighiero, G; Oppezzo, P

2015-01-01

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.

The silencing of Pokemon attenuates the proliferation of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the PI3K/Akt pathway.

PubMed

Lin, Chan-Chan; Zhou, Jing-Ping; Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner.

The Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Inhibiting the PI3K/Akt Pathway

PubMed Central

Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner. PMID:23300578

Voltage-Gated Ion Channels in Cancer Cell Proliferation

PubMed Central

Rao, Vidhya R.; Perez-Neut, Mathew; Kaja, Simon; Gentile, Saverio

2015-01-01

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation. PMID:26010603

32 CFR 562.3 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Definitions. 562.3 Section 562.3 National Defense... § 562.3 Definitions. The following terms apply to the Army's Senior Reserve Officers' Training Corps... installation. This is part of the advanced course and normally attended between Military Science (MS)-III and...

Novel Combined Ato-C Treatment Synergistically Suppresses Proliferation of Bcr-Abl-Positive Leukemic Cells In Vitro and In Vivo.

PubMed

Wahiduzzaman, Md; Ota, Akinobu; Karnan, Sivasundaram; Hanamura, Ichiro; Mizuno, Shohei; Kanasugi, Jo; Rahman, Md Lutfur; Hyodo, Toshinori; Konishi, Hiroyuki; Tsuzuki, Shinobu; Takami, Akiyoshi; Hosokawa, Yoshitaka

2018-06-23

Chronic myelogenous leukemia (CML) accounts for 15-20% of all leukemias affecting adults. Despite recent advances in the development of specific Bcr-Abl tyrosine kinase inhibitors (TKIs), some CML patients suffer from relapse due to TKI resistance. Here, we assessed the efficacy of a novel combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment (Ato-C) in human Bcr-Abl-positive leukemic cells. Combination index analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in K562, KU-812, MEG-A2, and KCL-22 cells. Notably, Ato-C synergistically enhanced apoptosis and decreased the survival of both acquired TKI-resistant CML cells and the cells expressing mutant Bcr-Abl T315I . In addition, Ato-C dramatically decreased the phosphorylation level of forkhead transcription factor FOXO1/3a and STAT5 as well as c-Myc protein level. Interestingly, results of gene set enrichment analysis showed that Ato-C significantly downregulates the expression of MYC- and/or E2F1-targets genes. Furthermore, Ato-C significantly suppressed the proliferation of MEG-A2-derived tumor when compared with that following monotherapy in vivo. Collectively, these results suggest that combined Ato-C treatment could be a promising alternative to the current therapeutic regime in CML. Copyright © 2018. Published by Elsevier B.V.

PI3K-mediated PDGFRα signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways

PubMed Central

Fantauzzo, Katherine A.; Soriano, Philippe

2014-01-01

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRα signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRα is unable to bind PI3K (PdgfraPI3K/PI3K) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRα signaling, we immunoprecipitated Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates and analyzed the peptides by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Our analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. We demonstrate that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that PdgfraPI3K/PI3K-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. We show that Ybox1 binds both the Trp53 promoter and the p53 protein and that expression of Trp53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, we demonstrate that introduction of a Trp53-null allele attenuates the vertebral defects found in PdgfraPI3K/PI3K neonates. Our findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRα signaling that regulates survival and proliferation during skeletal development in vivo. PMID:24788519

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

PubMed

Li, Qianyin; Huang, Zhenglan; Gao, Miao; Cao, Weixi; Xiao, Qin; Luo, Hongwei; Feng, Wenli

2017-03-02

The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

Irradiated KHYG-1 retains cytotoxicity: potential for adoptive immunotherapy with a natural killer cell line.

PubMed

Suck, G; Branch, D R; Keating, A

2006-05-01

To evaluate gamma-irradiation on KHYG-1, a highly cytotoxic natural killer (NK) cell line and potential candidate for cancer immunotherapy. The NK cell line KHYG-1 was irradiated at 1 gray (Gy) to 50 Gy with gamma-irradiation, and evaluated for cell proliferation, cell survival, and cytotoxicity against tumor targets. We showed that a dose of at least 10 Gy was sufficient to inhibit proliferation of KHYG-1 within the first day but not its cytolytic activity. While 50 Gy had an apoptotic effect in the first hours after irradiation, the killing of K562 and HL60 targets was not different from non-irradiated cells but was reduced for the Ph + myeloid leukemia lines, EM-2 and EM-3. gamma-irradiation (at least 10 Gy) of KHYG-1 inhibits cell proliferation but does not diminish its enhanced cytolytic activity against several tumor targets. This study suggests that KHYG-1 may be a feasible immunotherapeutic agent in the treatment of cancers.

IDO decreases glycolysis and glutaminolysis by activating GCN2K, while it increases fatty acid oxidation by activating AhR, thus preserving CD4+ T‑cell survival and proliferation.

PubMed

Eleftheriadis, Theodoros; Pissas, Georgios; Liakopoulos, Vassilios; Stefanidis, Ioannis

2018-07-01

It is generally hypothesized in the literature that indoleamine 2,3‑dioxygenase (IDO), by degrading L‑tryptophan along the kynurenine pathway, suppresses CD4+ T‑cell function by inducing apoptosis, inhibiting proliferation and promoting differentiation towards a regulatory phenotype. These effects are either accompanied or directly lead to alterations in cell metabolism. The present study evaluated the pathways that govern the effect of IDO on the utilization of the three main energy sources in CD4+ T‑cells. Two‑way mixed lymphocyte reactions were performed with or without oleate and/or the IDO inhibitor 1‑methyl‑DL‑tryptophan. In addition, isolated CD4+ T‑cells cultured in an oleate‑containing medium were activated in the presence or not of the general control nonderepressible 2 kinase (GCN2K) activator tryptophanol. L‑tryptophan, glucose and free fatty acid consumption, cell proliferation, apoptosis and the levels of key proteins involved in IDO‑mediated signal transduction, and glucose, glutamine and free fatty acid utilization were assessed. The results indicate that IDO decreased glycolysis and glutaminolysis by activating GCN2K, resulting in activation of AMP‑activated protein kinase (AMPK). In parallel with AMPK activation, IDO‑induced activation of aryl hydrocarbon receptor increased the expression of all carnitine palmitoyltransferase I isoenzymes, leading ultimately to increased free fatty acid oxidation and preservation of CD4+ T‑cell survival and proliferation. Thus, contrary to what is generally hypothesized, in a normal environment containing fatty acids, the immunosuppressive effect of IDO may not be due to a decrease in CD4+ T‑cell survival and proliferation, since IDO supplies the required energy for cell survival and proliferation by increasing free fatty acid oxidation.

PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways.

PubMed

Wu, Dong-Dong; Gao, Ying-Ran; Li, Tao; Wang, Da-Yong; Lu, Dan; Liu, Shi-Yu; Hong, Ya; Ning, Hui-Bin; Liu, Jun-Ping; Shang, Jia; Shi, Jun-Feng; Wei, Jian-She; Ji, Xin-Ying

2018-05-02

PEST-containing nuclear protein (PCNP), a novel nuclear protein, is involved in cell proliferation and tumorigenesis. However, the precise mechanism of action of PCNP in the process of tumor growth has not yet been fully elucidated. ShRNA knockdown and overexpression of PCNP were performed in human neuroblastoma cells. Tumorigenic and metastatic effects of PCNP were examined by tumor growth, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. PCNP over-expression decreased the proliferation, migration, and invasion of human neuroblastoma cells and down-regulation of PCNP showed reverse effects. PCNP over-expression increased protein expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase, as well as ratios of B-cell lymphoma-2 (Bcl-2)-associated X protein/Bcl-2 and Bcl-2-associated death promoter/B-cell lymphoma-extra large in human neuroblastoma cells, however PCNP knockdown exhibited reverse trends. PCNP over-expression increased phosphorylations of extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, as well as decreased phosphorylations of phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), nevertheless PCNP knockdown exhibited opposite effects. Furthermore, PCNP over-expression significantly reduced the growth of human neuroblastoma xenograft tumors by down-regulating angiogenesis, whereas PCNP knockdown markedly promoted the growth of human neuroblastoma xenograft tumors through up-regulation of angiogenesis. PCNP mediates the proliferation, migration, and invasion of human neuroblastoma cells through mitogen-activated protein kinase and PI3K/AKT/mTOR signaling pathways, implying that PCNP is a therapeutic target for patients with neuroblastoma.

Genistein Promotes Proliferation of Human Cervical Cancer Cells Through Estrogen Receptor-Mediated PI3K/Akt-NF-κB Pathway

PubMed Central

Chen, Hai-Hong; Chen, Shu-Ping; Zheng, Qiu-Ling; Nie, Shao-Ping; Li, Wen-Juan; Hu, Xiao-Juan; Xie, Ming-Yong

2018-01-01

Phytoestrogens are polyphenol compounds which have similar structure to 17β-estradiol (E2), a kind of main estrogen in women. Thus, phytoestrogens may affect the reproductive and endocrine systems, leading to the development of estrogen-related cancers. The effect of genistein (Gen), one of the most studied phytoestrogens, on human cervical cancer cells (HeLa) was investigated in this study. It was found that Gen at concentrations of 0.001, 0.01, 0.1 and 1 µmol·L-1 promoted the proliferation of HeLa cells in a dose-dependent manner. Gen increased the portion of HeLa cells in S phase and decreased the portion of the cells in G1 phase. Besides, apoptosis rate of the cells was significantly lower when treated with Gen compared with the control group. It was also found that the expression of ERα, Akt or nuclear NF-κB p65 protein was activated by Gen. The correlation between these three proteins may be as following: ERα was the upstream, followed by Akt, and then nuclear NF-κB p65 protein. In addition, the downstream genes of activated nuclear NF-κB p65 were found to be associated with cell cycle and apoptosis of cancer cells. Our results suggested that Gen may stimulate cell proliferation partially through the estrogen receptor-mediated PI3K/Akt-NF-κB pathway and the further activation of the downstream genes of nuclear NF-κB p65. PMID:29344275

Erythroleukemia cells acquire an alternative mitophagy capability.

PubMed

Wang, Jian; Fang, Yixuan; Yan, Lili; Yuan, Na; Zhang, Suping; Xu, Li; Nie, Meilan; Zhang, Xiaoying; Wang, Jianrong

2016-04-19

Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

PubMed

Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

2015-01-01

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

Proliferating cell nuclear antigen (PCNA): a new marker to study human colonic cell proliferation.

PubMed Central

Kubben, F J; Peeters-Haesevoets, A; Engels, L G; Baeten, C G; Schutte, B; Arends, J W; Stockbrügger, R W; Blijham, G H

1994-01-01

Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied as an alternative to ex-vivo bromodeoxyuridine (BrdU) immunohistochemistry for assessment of human colonic cell proliferation. From 16 subjects without colonic disease biopsy specimens were collected from five different sites along the colorectum and processed for BrdU and PCNA immunohistochemistry. The mean proliferation index of PCNA was significantly higher at 133% of the value obtained with BrdU. There was, however, a good correlation between the results from both techniques (r = 0.6275; p < 0.05). Decrease in proliferation index along the colorectum was seen with both staining methods but was clearer with PCNA immunohistochemistry (caecum/ascending colon v rectum: 12.0 v 7.2; p < 0.004). The total number of crypt cells also decreased from proximal to distal (134 to 128; p < 0.06) but at no site correlated significantly with the proliferation index. It is concluded that in clinical cell kinetic studies staining for PCNA may serve as an attractive alternative to the BrdU incorporation assay. Images Figure 4 PMID:7909785

The activation of the G protein-coupled estrogen receptor (GPER) inhibits the proliferation of mouse melanoma K1735-M2 cells.

PubMed

Ribeiro, Mariana P C; Santos, Armanda E; Custódio, José B A

2017-11-01

The activation of the G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 inhibits prostate cancer and 17β-estradiol-stimulated breast cancer cell proliferation. Tamoxifen (TAM), which also activates the GPER, decreases melanoma cell proliferation, but its action mechanism remains controversial. Here we investigated the expression and the effects of GPER activation by G-1, TAM and its key metabolite endoxifen (EDX) on melanoma cells. Mouse melanoma K1735-M2 cells expressed GPER and G-1 reduced cell biomass, and the number of viable cells, without increasing cell death. Rather, G-1 decreased cell division by blocking cell cycle progression in G2. Likewise, TAM and EDX exhibited an antiproliferative activity in melanoma cells due to decreased cell division. Both G-1 and the antiestrogens showed a trend to decrease the levels of phosphorylated ERK 1/2 after 1 h treatment, although only EDX, the most potent antiproliferative antiestrogen, induced significant effects. Importantly, the targeting of GPER with siRNA abolished the cytostatic activity of both G-1 and antiestrogens, suggesting that the antitumor actions of antiestrogens in melanoma cells involve GPER activation. Our results unveil a new target for melanoma therapy and identify GPER as a key mediator of antiestrogen antiproliferative effects, which may contribute to select the patients that benefit from an antiestrogen-containing regimen. Copyright © 2017 Elsevier B.V. All rights reserved.

Antiproliferative Effects of Bacillus coagulans Unique IS2 in Colon Cancer Cells.

PubMed

Madempudi, Ratna Sudha; Kalle, Arunasree M

2017-10-01

In the present study, the in vitro anticancer (antiproliferative) effects of Bacillus coagulans Unique IS2 were evaluated on human colon cancer (COLO 205), cervical cancer (HeLa), and chronic myeloid leukemia (K562) cell lines with a human embryonic kidney cell line (HEK 293T) as noncancerous control cells. The Cytotoxicity assay (MTT) clearly demonstrated a 22%, 31.7%, and 19.5% decrease in cell proliferation of COLO 205, HeLa, and K562 cells, respectively, when compared to the noncancerous HEK 293T cells. Normal phase-contrast microscopic images clearly suggested that the mechanism of cell death is by apoptosis. To further confirm the induction of apoptosis by Unique IS2, the sub-G0-G1 peak of the cell cycle was quantified using a flow cytometer and the data indicated 40% of the apoptotic cells in Unique IS2-treated COLO cells when compared with their untreated control cells. The Western blot analysis showed an increase in pro-apoptotic protein BAX, decrease in antiapoptotic protein, Bcl2, decrease in mitochondrial membrane potential, increase in cytochrome c release, increase in Caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. The present study suggests that the heat-killed culture supernatant of B. coagulans can be more effective in inducing apoptosis of colon cancer cells and that can be considered for adjuvant therapy in the treatment of colon carcinoma.

CIP-36, a novel topoisomerase II-targeting agent, induces the apoptosis of multidrug-resistant cancer cells in vitro.

PubMed

Cao, Bo; Chen, Hong; Gao, Ying; Niu, Cong; Zhang, Yuan; Li, Ling

2015-03-01

The need to overcome cancer multidrug resistance (MDR) has fueled considerable interest in the development of novel synthetic antitumor agents with cytotoxicity against cancer cell lines with MDR. In this study, we aimed to investigate CIP-36, a novel podophyllotoxin derivative, for its inhibitory effects on human cancer cells from multiple sources, particularly cells with MDR in vitro. The human leukemia cell line, K562, and the adriamycin-resistant subline, K562/A02, were exposed to CIP-36 or anticancer agents, and various morphological and biochemical properties were assessed by Hoechst 33342 staining under a fluorescence microscope. Subsequently, cytotoxicity, cell growth curves and the cell cycle were analyzed. Finally, the effects of CIP-36 on topoisomerase IIα (Topo IIα) activity were determined. Treatment with CIP-36 significantly inhibited the growth of the K562 and MDR K562/A02 cells. Our data demonstrated that CIP-36 induced apoptosis, inhibited cell cycle progression and inhibited Topo IIα activity. These findings suggest that CIP-36 has the potential to overcome the multidrug resistance of K562/A02 cells by mediating Topo IIα activity.

LSD1 sustains estrogen-driven endometrial carcinoma cell proliferation through the PI3K/AKT pathway via di-demethylating H3K9 of cyclin D1.

PubMed

Chen, Chunqin; Wang, Yanan; Wang, Shiyu; Liu, Yuan; Zhang, Jiawen; Xu, Yuyao; Zhang, Zhenbo; Bao, Wei; Wu, Sufang

2017-03-01

A recent study reported that histone lysine specific demethylase 1 (LSD1, KDM1A) is overexpressed in endometrioid endometrial carcinoma (EEC) and associated with tumor progression as well as poor prognosis. However, the physiological function and mechanism of LSD1 in endometrial cancer (EC) remains largely unknown. In this study, we demonstrate that β-estradiol (E2) treatment increased LSD1 expression via the GPR30/PI3K/AKT pathway in endometrial cancer cells. Both siGPR30 and the PI3K inhibitor LY294002 block this effect. RNAi-mediated silencing of LSD1 abolished estrogen-driven endometrial cancer cell (ECC) proliferation, and induced G1 cell arrest and apoptosis. Mechanistically, we find that LSD1 silencing results in PI3K/AKT signal inactivation, but without the elevation of PTEN expression as expected. This is because the inhibition of LSD1 induces dimethylation of lysine 9 on histone H3 (H3K9m2) accumulation at the promoter region of cyclin D1. Interfering with cyclin D1 leads to PI3K/AKT signal suppression. Re-overexpression of cyclin D1 in LSD1-knockdown ECCs reverses the LSD1 inhibitory action. Our finding connects estrogen signaling with epigenetic regulation in EEC and provides novel experimental support for LSD1 as a potential target for endometrial cancer therapeutics.

Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.

PubMed

Kariya, Ryusho; Matsuda, Kouki; Gotoh, Kumiko; Vaeteewoottacharn, Kulthida; Hattori, Shinichiro; Okada, Seiji

2014-01-01

Nude mice are used in human xenograft research; however, only 25-35% of human tumors have been successfully transplanted into nude mice and their application is limited due to high natural killer (NK) cell activity. More severely immunodeficient mice with loss of NK activity are needed to overcome this limitation. Balb/c nude Rag-2(-/-)Jak3(-/-) (Nude-RJ) mice were established by crossing Rag-2(-/-)Jak3(-/-) mice and nude mice. The K562 cell line was implanted subcutaneously to compare tumorigenicity between Nude-RJ mice and Nude mice. The cholangiocarcinoma mCherry expressing cell line (KKU-M213) was implanted subcutaneously, and fluorescence intensity and tumor weight were measured. Nude R/J mice showed complete loss of lymphocytes and NK cells. Xeno-transplantation of K562 cells showed higher proliferation in Nude R/J mice than nude mice. Subcutaneously-transplanted mCherry-transduced KKU-M213 cells were successfully detected with a fluorescence imager. Nude-R/J mice are valuable tools for in vivo imaging studies in biomedical research. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

PubMed

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

2013-02-01

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

Regulation of Schwann Cell Differentiation and Proliferation by the Pax-3 Transcription Factor

PubMed Central

Moate, Roy M.; Jessen, Kristjan R.; Mirsky, Rhona; Parkinson, David B.

2017-01-01

Pax-3 is a paired domain transcription factor that plays many roles during vertebrate development. In the Schwann cell lineage, Pax-3 is expressed at an early stage in Schwann cells precursors of the embryonic nerve, is maintained in the nonmyelinating cells of the adult nerve, and is upregulated in Schwann cells after peripheral nerve injury. Consistent with this expression pattern, Pax-3 has previously been shown to play a role in repressing the expression of the myelin basic protein gene in Schwann cells. We have studied the role of Pax-3 in Schwann cells and have found that it controls not only the regulation of cell differentiation but also the survival and proliferation of Schwann cells. Pax-3 expression blocks both the induction of Oct-6 and Krox-20 (K20) by cyclic AMP and completely inhibits the ability of K20, the physiological regulator of myelination in the peripheral nervous system, to induce myelin gene expression in Schwann cells. In contrast to other inhibitors of myelination, we find that Pax-3 represses myelin gene expression in a c-Jun-independent manner. In addition to this, we find that Pax-3 expression alone is sufficient to inhibit the induction of apoptosis by TGFβ1 in Schwann cells. Expression of Pax-3 is also sufficient to induce the proliferation of Schwann cells in the absence of added growth factors and to reverse K20-induced exit from the cell cycle. These findings indicate new roles for the Pax-3 transcription factor in controlling the differentiation and proliferation of Schwann cells during development and after peripheral nerve injury. PMID:22532290

32 CFR 562.7 - Program information.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Program information. 562.7 Section 562.7 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.7 Program information. (a) The Senior ROTC is conducted at military colleges...

32 CFR 562.7 - Program information.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 3 2012-07-01 2009-07-01 true Program information. 562.7 Section 562.7 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.7 Program information. (a) The Senior ROTC is conducted at military colleges, civilian...

Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

PubMed Central

Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

2017-01-01

Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

31 CFR 562.309 - United States.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia.

PubMed

Usuludin, Suaidah Binte Mohamed; Cao, Xue; Lim, Mayasari

2012-05-01

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion. Copyright © 2011 Wiley Periodicals, Inc.

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

JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells.

PubMed

Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

2017-03-01

Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels.

JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells

PubMed Central

Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

2017-01-01

Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels. PMID:28454225

Apigenin inhibits cell proliferation, migration, and invasion by targeting Akt in the A549 human lung cancer cell line.

PubMed

Zhou, Zhongping; Tang, Miaomiao; Liu, Yi; Zhang, Zhuyi; Lu, Rongzhu; Lu, Jian

2017-04-01

Apigenin (APG), a widely distributed flavonoid in vegetables and fruits, with low toxicity, and a nonmutagenic characteristic, has been reported to have many targets. Evidence indicates that APG can inhibit the proliferation, migration, invasion, and metastasis of some tumor cells, but the mechanism, specifically in lung cancer, is unclear. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway regulates a diverse set of cellular functions relevant to the growth and progression of lung cancer, including proliferation, survival, migration, and invasion. Our results showed that APG exerted anti-proliferation, anti-migration, and anti-invasion effects in A549 human lung cancer cells by targeting the PI3K/Akt signaling pathway. 3-(4, 5-dimethylthiszol-2-yl)-2, 5-diphenytetrazolium bromide assay and colony formation assay showed that APG suppressed cell proliferation in a dose-dependent and time-dependent manner. Cell motility and invasiveness were assayed using a wound healing and Transwell assay, suggesting that APG inhibited the migration and invasion of A549 cells. Western blot analyses were carried out to examine the Akt signaling pathways. The results confirmed that APG decreased Akt expression and its activation. Then, cells were transfected with Akt-active and Akt-DN plasmids separately. The migration and invasion of A549 cells were significantly changed, constitutively activating Akt or knocking down Akt, indicating that APG can suppress the migration and invasion of lung cancer cells by modulating the PI3K/Akt signaling pathway. Furthermore, the results indicated that APG not only suppressed phosphorylation of Akt, thereby preventing its activation, but also inhibited its downstream gene expression of matrix metalloproteinases-9, glycogen synthase kinase-3β, and HEF1. Together, APG is a new inhibitor of Akt in lung cancer and a potential natural compound for cancer chemoprevention.

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at prices offered through the competitive...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance: Treasury 1 2013-07-01 2013-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance: Treasury 1 2012-07-01 2012-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance: Treasury 1 2014-07-01 2014-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance: Treasury 1 2011-07-01 2011-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway.

PubMed

Zhang, Hui; Zhao, Xingbo; Liu, Shu; Li, Jijun; Wen, Zeqing; Li, Mingjiang

2010-04-12

The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition. Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.

PubMed

Davitt, Katlin; Babcock, Blake D; Fenelus, Maly; Poon, Chi Kong; Sarkar, Abhishek; Trivigno, Vincent; Zolkind, Paul A; Matthew, Sheena M; Grin'kina, Natalia; Orynbayeva, Zulfiya; Shaikh, Mohammad F; Adler, Victor; Michl, Josef; Sarafraz-Yazdi, Ehsan; Pincus, Matthew R; Bowne, Wilbur B

2014-01-01

We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

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

Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko

2013-06-07

Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzedmore » the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.« less

Regulator of G protein signaling 4 inhibits human melanoma cells proliferation and invasion through the PI3K/AKT signaling pathway

PubMed Central

Xue, Xiaotong; Wang, Lihua; Meng, Xianguang; Jiao, Jing; Dang, Ningning

2017-01-01

Melanoma is a tumor produced by skin melanocytes, which has a high metastatic rate and poor prognosis. So far, plenty of work has been done on melanoma, but mechanisms underlying melanoma development have not been fully elucidated. Here we identified regulator of G protein signaling 4(RGS4) as novel therapeutic target for malignant melanoma and its regulating effect on melanoma. We found that endogenous RGS4 expression was much lower in melanoma tissues and cells. In A375 cell line with low endogenous RGS4 expression, the function of RGS4 was detected by up-regulation its expression with pcDNA3.1-RGS4 and knockdown its expression with siRNA. Our results showed that RGS4 could significantly reduce the proliferation, migration and invasion of melanoma cells. RGS4 is an important regulator for the apoptosis of melanocyte, and the apoptosis rate is significantly decreased in low RGS4 enviroment. RGS4 induced non-activation of PI3K/AKT pathway, resulting in decreased expression of E2F1 and Cyclin D1, thus constraining cell proliferation and invasion. These results were further confirmed in M14 cell lines. Collectively, our findings show that RGS4 plays an important role in multiple cellular functions of melanoma development and is valuable to be a therapeutic target. PMID:29108247

Naringin enhances endothelial progenitor cell (EPC) proliferation and tube formation capacity through the CXCL12/CXCR4/PI3K/Akt signaling pathway.

PubMed

Zhao, Zhihu; Ma, Xinlong; Ma, Jianxiong; Sun, Xiaolei; Li, Fengbo; Lv, Jianwei

2018-04-25

Endothelial progenitor cells (EPCs) have been shown to be involved in the process of physiological neovascularization in vivo. Because increasing evidence has indicated that naringin, a major active ingredient in the Chinese herb Drynaria fortunei, can promote angiogenesis and inhibit endothelial cell apoptosis, our study was designed to determine the role of naringin in EPC proliferation and tube formation capacity and examine the potential mechanism for these effects. EPCs were isolated from bone marrow and treated with naringin. An MTT assay was used to investigate EPC proliferation and the tube formation capacity of these EPCs, which were seeded on Matrigel. The protein levels of CXCL12, its receptor (chemokine receptor 4 (CXCR4)) and a downstream signaling molecule (Akt and phosphorylated Akt (pAkt)) were examined using Western blotting. A CXCR4 antagonist (AMD3100) and a phosphatidylinositol 3-kinase (PI3K) antagonist (LY294002) were used to characterize the underlying mechanisms. The results showed that naringin-induced EPC proliferation reached a maximum at day 3 and that the optimal dose of naringin was 500 ng/ml. Treatment with naringin facilitated the EPC tube formation capacity and increased the levels of CXCL12, CXCR4 and pAkt (P < 0.05) relative to those in the control group. Moreover, the naringin-induced EPC tube formation capacity was significantly attenuated by AMD3100 or LY294002. In conclusion, we showed here that the naringin-enhanced EPC proliferation and tube formation were mediated by the activation of the PI3K/Akt signaling pathway via the CXCL12/CXCR4 axis, which suggests that naringin could serve as a new therapeutic medicine and has the potential to be applied for the treatment of ischemic disease. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of spaceflight on natural killer cell activity

NASA Technical Reports Server (NTRS)

Rykova, Marina P.; Sonnenfeld, Gerald; Lesniak, A. T.; Taylor, Gerald R.; Meshkov, Dimitrii O.; Mandel, Adrian D.; Medvedev, Andrei E.; Berry, Wallace D.; Fuchs, Boris B.; Konstantinova, Irina V.

1992-01-01

The effects of spaceflight on immune cell function were determined in rats flown on Cosmos 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for Cr-51-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

PubMed

Kawano, M; Matsushima, K; Oppenheim, J J

1987-08-01

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

HP1α is highly expressed in glioma cells and facilitates cell proliferation and survival.

PubMed

Lai, Xianliang; Deng, Zhifeng; Guo, Hua; Zhu, Xingen; Tu, Wei

2017-08-19

Epigenetic alteration plays critical roles in gliomagenesis by regulating gene expression through modifications of Histones and DNA. Trimethylation of H3K9, an essential repressed transcription mark, and one of its methyltransferase, SUV39H1, are implicated in glioma pathogenesis and progression. We find that the protein level of HP1α, a reader of H3K9me3 is elevated in cultured glioma cell lines and glioma tissues. H3K9me3 is also upregulated. Depletion of HP1α and SUV39H1 weakens glioma cell proliferation capacity and results in apoptosis of cells. Furthermore, we find that HP1α and H3K9me3 are enriched in the FAS and PUMA promoters, which suggests that upregulated HP1α and H3K9me3 contribute to cell survival by suppressing apoptotic activators. These data suggests that up-regulated HP1α and H3K9me3 in glioma cells are functionally associated with glioma pathogenesis and progression and may serve as novel biomarkers for diagnostic and therapeutic targeting of brain tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

Calcium signaling and cell proliferation.

PubMed

Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

2015-11-01

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

32 CFR 562.6 - Responsibilities.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Responsibilities. 562.6 Section 562.6 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS... Stovall Street, Alexandria, VA 22332, is the adminstrator of the Department of the Army for ROTC. (b) The...

40 CFR 721.562 - Substituted alkylamine salt.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted alkylamine salt. 721.562 Section 721.562 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.562 Substituted alkylamine salt...

32 CFR 562.4 - Objectives.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Objectives. 562.4 Section 562.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS... students with potential to serve as commissioned officers in the Regular Army or the US Army Reserve. (b...

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

PAQR3 inhibits the proliferation, migration and invasion in human glioma cells.

PubMed

Tang, Shi-Lei; Gao, Yuan-Lin; Hu, Wen-Zhong

2017-08-01

Progestin and AdipoQ Receptor 3 (PAQR3), a member of the PAQR family, is down-regulated in several types of cancers and has been closely associated with tumor progression and development. However, little is known about the functions of PAQR3 in the tumorigenesis of human glioma. Therefore, in this report, we investigated the role of PAQR3 in human glioma. Our results showed that the expression of PAQR3 was significantly reduced in human glioma tissues and cell lines. PAQR3 overexpression inhibited the proliferation of glioma cells in vitro and attenuated tumor xenograft growth in vivo. In addition, PAQR3 overexpression suppressed the migration and invasion of glioma cells, as well as prevented the EMT process. Mechanistic studies demonstrated that PAQR3 overexpression significantly down-regulated the levels of phosphorylated PI3K and Akt in U251 cells. In conclusion, these results demonstrated that PAQR3 inhibited the proliferation, migration and invasion in glioma cells, at least in part, through the inactivation of PI3K/Akt signaling pathway. Therefore, PAQR3 may be a therapeutic target for the treatment of glioma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Rapamycin attenuates BAFF-extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B-lymphoid cells.

PubMed

Zeng, Qingyu; Qin, Shanshan; Zhang, Hai; Liu, Beibei; Qin, Jiamin; Wang, Xiaoxue; Zhang, Ruijie; Liu, Chunxiao; Dong, Xiaoqing; Zhang, Shuangquan; Huang, Shile; Chen, Long

2018-01-01

B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases. © 2017 Wiley Periodicals, Inc.

Human germinal center CD4+CD57+ T cells act differently on B cells than do classical T-helper cells.

PubMed

Bouzahzah, F; Bosseloir, A; Heinen, E; Simar, L J

1995-01-01

We have isolated two subtypes of helper T cells from human tonsils: CD4+CD57+ cells, mostly located in the germinal center (GC), and CD4+CD57- cells, distributed through the interfollicular areas but also present in the GC. In a functional study, we have compared the capacities of these T-cell subtypes to stimulate B cells in cocultures. In order to block T-cell proliferation while maintaining their activation level, we pretreated isolated T cells with mitomycin C prior to culture in the presence of B cells and added polyclonal activators such as PHA and Con A, combined or not with IL-2. Contrary to CD4+ CD57- cells, CD4+CD57+ cells did not markedly enhance B-cell proliferation. Even when sIgD.B cells typical of germinal center cells were tested, the CD4+CD57+ cells had no significant effect. This is in accordance with the location of these cells: They mainly occupy the light zones of the GC where few B cells divide. Even when added to preactivated, actively proliferating cells, CD4+CD57 cells failed to modulate B-cell multiplication. On the supernatants of B-cell-T-cell cocultures, we examined by the ELISA technique the effect of T cells on Ig synthesis. Contrary to CD57+ T cells, whose effect was strong, CD57- T cells weakly stimulated Ig synthesis. More IgM than IgG was generally found. Because CD57 antigen is a typical marker of natural killer cells, we tested the cytolytic activity of tonsillar CD4+CD57+ cells on K562 target cells. Unlike NK cells, neither CD4+CD57+ nor CD4+CD57- cells exhibit any cytotoxicity. Thus, germinal center CD4+CD57+ cells are not cytolytic and do not strongly stimulate either B-cell proliferation or Ig secretion. CD4+CD57- cells, however, enhance B-cell proliferation and differentiation, thus acting like the classical helper cells of the T-dependent areas.

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

PubMed

Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

2016-06-01

Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells.

PubMed

Nogami, Haruo; Hiraoka, Yoshiki; Aiso, Sadakazu

2016-08-01

Estrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). Effects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. Estradiol and the specific agonist for both estrogen receptor (ER) α and ERβ stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17β-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1μM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. The results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

MiR-374b Promotes Proliferation and Inhibits Apoptosis of Human GIST Cells by Inhibiting PTEN through Activation of the PI3K/Akt Pathway.

PubMed

Long, Zi-Wen; Wu, Jiang-Hong; Hong, Cai-; Wang, Ya-Nong; Zhou, Ye

2018-06-14

Gastrointestinal stromal tumours (GIST) are the most common mesenchymal tumors of the gastrointestinal (GI) tract. In order to investigate a new treatment fot GIST, we hypothesized the effect of miR-374b targeting PTEN gene-mediated PI3K/Akt signal transduction pathway on proliferation and apoptosis of human gastrointestinal stromal tumor (GIST) cells. We obtained GIST tissues and adjacent normal tissues from 143 patients with GIST to measure the levels of miR- 374b, PTEN, PI3K, Akt, caspase9, Bax, MMP2, MMP9, ki67, PCNA, P53 and cyclinD1. Finally, cell viability, cell cycle and apoptosis were detected. According to the KFGG analysis of DEGs, PTEN was involved in a variety of signaling pathways and miRs were associated with cancer development. The results showed that MiR-374b was highly expressed, while PTEN was downregulated in the GIST tissues. The levels of miR-374b, PI3K, AKT and PTEN were related to tumor diameter and pathological stage. Additionally, miR-374b increased the mRNA and protein levels of PI3K, Akt, MMP2, MMP9, P53 and cyclinD1, suggesting that miR-374b activates PI3K/Akt signaling pathway in GIST-T1 cells. Moreover, MiR- 374b promoted cell viability, migration, invasion, and cell cycle entry, and inhibited apoptosis in GIST cells. Taken together, the results indicated that miR-374b promotes viability and inhibits apoptosis of human GIST cells by targeting PTEN gene through the PI3K/Akt signaling pathway. Thus, this study provides a new potential target for GIST treatment.