Expression And Roles Of Chloride Channels In Cell Proliferation In Human Ovarian Cancer Cells

[Background]Ovarian cancer,one of the three common gynecological malignant genital tumors,is the leading cause of gynecological cancer deaths.Due to the lack of effective earlier diagnosis of ovarian cancer,most of patients are diagnosed with advanced-stage,with a resultant poor prognosis.Ovarian cancer cell is characterized by its high potential for unlimited proliferation and metastasis.The factors correlated with the malignant biological behavior of ovarian cancer,however,are poorly understood.There is an imperative need to explore new therapeutic targets and a better understanding of the mechanisms involved in the malignant biological behavior of ovarian cancer.Chloride(Cl^-) channels are ubiquitous transmembrane proteins which have been implicated in cell volume regulation,salt and fluid movements across epithelia, stabilization of membrane potential,intracellular organelle acidification,cell proliferation and differentiation.Chloride channels are regulated by various factors. According to their gating mechanisms,there are,from the functional point of view, five classes of chloride channel,including voltage-gated chloride channels(CLC), volume/swell-regulated/sensitive anion/chloride channels(VRAC),cystic fibrosis transmembrane conductance regulator(CFTR),calcium-activated chloride channels (CLCA) and ligand-activated chloride channels which mainly forms synaptic channels.Chloride channels have been found in control of cell proliferation.Cl^- channel blockers inhibit cell proliferation in many types of cells,including liver cells, pulmonary artery endothelial cells,lymphoctes,vascular smooth muscle cells.Most attractively,recent studies indicate Cl^-channels express in a variety of tumor cells and support the essential role of plasma membrane chloride channels in the proliferation, invasion and migration of tumor cells.For example,CLC has been found to be specifically up-regulated in human glioma membranes,which in turn facilitate rapid changes in cell size and shapes and contribute to glioma cell division or invasion into narrow extracellular brain spaces.VRAC is one of the important factors that modttlate the passage of nasopharyngeal carcinoma cells through the G₁ restriction point and endow nasopharyngeal carcinoma cells with an enhanced proliferation ability.In addition,it is reported that VRAC displays cell cycle-dependent expression in tumor cells.CLC-3,an important member of the CLC superfamily,plays a crucial role in a variety of cellular processes.Moreover,CLC-3 has been considered the most likely molecular candidate of the VRAC.It is implicated that the antiapoptotic protein could up-regulate CLC-3 protein in a dose-dependent manner in the human prostatic carcinoma cells,which thereby protects cells against apoptotic stimuli and increases cell viability.Additionally,the present study demonstrates that inhibition of CLC-3 protein expression down-regulates invasion and migration ability of tumor cell.Up to date,there are no data available as to the expression of Cl^-channels and whether Cl^-channels are related to the cell proliferation and cell cycle progression in ovarian cancer cell.In this study,we aimed to observe the representative Cl^-channels expression,investigate the role of Cl^-channels in proliferation and cell cycle of ovarian cancer cells and study the effect of CLC-3 on human ovarian cancer cell proliferation. Objective:To investigate the expression and the roles of chloride channels in cell proliferation and cell cycle in human epithelial ovarian cancer cell line A2780.Methods:The expression of several representative chloride channels was detected by employing RT-PCR and immunocytochemical methods.The effects of chloride channels on proliferation and cell cycle of human ovarian cancer cell line A2780 were observed by means of MTT assay and flow cytometry.A variety of chloride channel blockers were used in order to observe the effects of chloride channels on human ovarian cancer cell line A2780.Results:CLC-3 and CFTR mRNA was detected by RT-PCR assay,and CLC-3 and CFTR proteins were found to express in A2780 cell plasma membrane by using immunocytochemical assay.The hCLCA mRNA,however,could not be detected in A2780 cells by using RT-PCR.Chloride channel blockers(NPPB,NFA and TAM) significantly inhibited the proliferation of A2780 cells as measured by MTT assay. Treatments with 25μmol/L,50μmol/L,100μmol/L and 200μmol/L NPPB caused inhibitory effects on A2780 cell proliferation potential,and the inhibitory rate was 23.4%,59.2%,90.8%,93.5%,respectively(vs control,P＜0.05).After NFA-treatment, A2780 cell proliferation was greatly inhibited in the presence of 25μmol/L,50μmol/L, 100μmol/L and 200μmol/L NFA,and the inhibitory rate was 18.3%,40.9%,68.6% and 75.1%,respectively(vs control,P＜0.05).TAM,a relatively specific blocker of volume-regulated chloride channels,also produced inhibitory effects on A2780 cell proliferation ability,and the inhibitory rate was 65.8%,73.4%,77.5%after treatment of A2780 cells with 10μmol/L,20μmol/L,30μmol/L TAM,respectively(vs control, P＜0.05).In contrast,Glibenclamide,a relatively specific CFTR blocker,had no inhibitory effects on A2780 cell proliferation.The inhibitory rate was 2.1%,28%, 3.7%and 3.9%after treatment of A2780 cells with 25μmol/L,50μmol/L,100μmol/L and 200μmol/L Glibenclamide,respectively(vs control,P＞0.05).In addition,chloride channel blockers(NPPB,NFA,TAM) also obviously affected the A2780 cell cycle,which increased the percentage of cells in G₀/G₁ phase,and reduced the percentage of cells in S phase.Glibenclamide,however,had no effect on the A2780 cell cycle.After treatment with 50μmol/L,100μmol/L and 200μmol/L NPPB,the proportion of G₀/G₁ phase of A2780 cells increased from 55.4±2.1%to 73.1±3.9%,84.2±2.3%and 86.5±2.6%respectively(vs control,P＜0.35); Surprisingly,the proportion of S phase reduced from 34.0±2.5%to 22.3±4.1%, 12.1±2.3%and 10.7±1.6%,respectively(vs control,P＜0.05).Effects of NFA on A2780 cell cycle progression were similar to those of NPPB.The proportion of G₀/G₁ phase increased from 55.4±2.1%to 69.3±3.5%,74.6±2.1%and 79.5±3.,5%, when 50μmol/L,100μmol/L and 200μmol/L NFA were used,respectively(vs control, P＜0.05).Interestingly,the proportion of S phase reduced from 34.0±2.5%to 24 1±1.5%,18.7±1.9%and 13.4±2.7%,respectively(vs control,P＜0.05).10μmol/L, 20μmol/L and 30μmol/L TAM also affected A2780 cell cycle progression,and the proportion of G₀/G₁ phase went up significantly from 55.4±2.1%to 68.7±2.4%, 73.8±3.3%and 79.4±3.1%,respectively(vs control,P＜0.05).Whereas the proportion of S phase decreased from 34.0±2.5%to 24.6±1.9%,20.3±2.2%and 15.3±1.6%,respectively(vs control,P＜0.05).After treatment with 50μmol/L, 100μmol/L and 200μmol/L Glibenclamide,the proportion of G₀/G₁ phase cells changed from 55.4±2.1%to 55.8±2.9%,53.1±3.2%and 54.2±3.5%,respectively (vs control,P＞0.05);and the proportion of S phase cells changed from 34.0±2.5% to 33.7±2.6%,36.9±1.6%and 35.6±2.8%,respectively(vs control,P＞0.05).Conclusions:Our results suggest that CLC-3 and CFTR are markedly expressed in human epithelial ovarian cancer cells.Chloride channels,CLC and VRAC especially, could play an important role in the proliferation and cell cycle of ovarian cancer cell. Objective:The aim of the present study was to observe the effect of CLC-3 antisense oligonucleotide on proliferation of human ovarian cancer cell line A2780.Methods:CLC-3 antisense oligonucleotides were transfected by lipofectamine into human epithelial ovarian cancer cell line A2780.Western blot was used to detect CLC-3 protein expression.The ability of cell proliferation was determined by using MTT assay,and the cell cycle was measured by employing flow cytometry(FCM). Western blot were performed to detect the cyclinD1 protein expression in A2780 cells.Results:Western blot assay showed that CLC-3 antisense oligonucleotides inhibited the CLC-3 protein expression.Compared with the control group,the inhibitory rates of 50μg/ml,100μg/ml CLC-3 antisense oligonucleotides were 62.5%,78.6%, respectively;however,the CLC-3 sense oligonucleotides had no effects on CLC-3 protein expression of A2780 cells.MTT assay revealed that A2780 cell proliferation could be inhibited by CLC-3 antisense oligonucleotides.The inhibitory rates of 50μg/ml and 100μg/ml CLC-3 antisense oligonucleotides were 48.6%,65.3%, respectively(vs control,P＜0.05).In contrast,the 100μg/ml CLC-3 sense oligonucleotides had no significant effect on cell proliferation(vs control,P＞0.05). Moreover,transfection of A2780 cells with CLC-3 antisense oligonucleotides obviously arrested the progression of cell cycle,which increased the percentage of cells in G₀/G₁ phase,and reduced the percentage of cells in S phase.In 50μg/ml, 100μg/ml CLC-3 antisense-treatment groups,the proportion of G₀/G₁ cells increased from 49.4±3.1%to 70.3±3.4%,75.8±2.6%,respectively(vs control,P＜0.05); while it decreased significantly from 40.3±2.8%to 23.5±2.1%,19.6±1.5%, respectively(vs control,P＜0.05) in the S phase.In contrast,CLC-3 sense oligonucleotides had no significant effects on cell cycle.After transfection with CLC-3 sense oligonucleotides,the cell population changed from 49.4±3.1%to 50.2±3.6%in the G₀/G₁ phase,from 40.3±2.8%to 39.7±2.9%in the S phase, respectively(vs control,P＞0.05).Western blot assays indicated that CLC-3 antisense oligonucleotides inhibited cyclinD1 protein expression in A2780 cell line.However in cells treated with sense oligonucleotides,there exhibited no interference effects on cyclinD1 protein expression in A2780 cells.Conclusions:These results strongly indicate that CLC-3 may get involved in proliferation of human epithelial ovarian cancer cell and thus may be a useful therapeutic target.It is likely that the effects of CLC-3 on A2780 cell proliferation and cell cycle is mediated through downregulation of cyclinD1.