Chloride Channel Protein Is A Stemness Gene Regulatory, And Regulating The Cycle Of Prostate Cancer Cells With Sox2

Chloride channel(ClC)is an important anion channel,which is closely related to the development of tumors.Malignant tumors are tumor stem cell and its stem gene sxpress above the average 。 SRY-related-HMG-box-2(SOX2)is a pivotal transcription factor,its abnormal expression may promote the occurrence and process of many malignant tumors.Here,we used a chloride channel blocker,4,4’-Diisothiocyanatostilbene-2,2’-disulfonic acid(DIDS)and RNA interference(RNAi)to blockade Cl C.It showed that Cl C blockade could inhibit human prostate cancer cell DU145 proliferation,as shown by cell cycle arrest in G0/G1 phase via down-regulation of cyclin D1 and up-regulation of p27.But the specific targets of Cl C regulating cell cycle had not been reported.Then gene chip showed SOX2 changed the most obviously in Cl C blockaded DU145,and this indicated SOX2 might be the key to Cl C-mediated cell cycle.We demonstrated that SOX2 regulated DU145 cell cycle via cyclin D1 and p27,which is conform to the change of cell cycle when interdicted Cl C.The relationship between Cl C and SOX2 had not been reported either.Last we found down-regulating Cl C was accompanied with SOX2 down-regulation,and down-regulating SOX2 was accompanied with Cl C down-regulation as well by CONFOCAL.We further confirmed this closely relation between SOX2 and Cl C by Co-Immunoprecipitation(IP).The cytology results were verified by the experiment in animals.We transplanted DU145 in nude mice and treated with DIDS via intraperitoneal injection.It showed that the tumor size of treatment groups was obviously less than control group.And the expression of Cl C,SOX2,ki67 and cyclin D1 in treatment groups was less than in control group.In sum,we demonstrated Cl C blocking inhibited the proliferation of human prostate cancer cell DU145 via regulating cyclin D1 and p27 to arrest in G0/G1 phase.SOX2 participated in the regulation pathway and it had direct interaction with Cl C protein.Methods and results1.Chloride Channel Blocker DIDS Down-regulates Proliferation and Cell Cycle of the Prostate Cancer Cells(1)Chloride channel blocker DIDS inhibits proliferation of the prostate cancer cells in varying degrees.We cultured human prostate cancer cell line DU145,PC3 and human normal fibroblasts HFF-1 were separately.MTT was used to detect the cell survival rate after being treated by DIDS.It was showed that DIDS had the ability to inhibit the proliferation of DU145 and PC3 of prostate cancer cell line.With a concentration and time dependent manner,DIDS showed an obvious inhibiting effect in DU145 cells,and had no significant effect in human normal fibroblasts.(2)Chloride channel blocker DIDS blocks cell cycle of prostate cancer cells DU145.To determine the cycle of DU145 cells,PI staining was adopted at 0,24,and 48 h after addition of DIDS.Assayed by flow cytometry technique,most DU145 cells were in G1 phase at 0h after addition of DIDS.At 24 h and 48 h,the percentage of G1 phase cells increased while the cell number of G2+S phases decreased.Statistical studies showed that the cells treated by DIDS were arrested in G0/G1 phase.2.Chloride channel proteins are abnormally expressed in prostate cancer cells,CLIC3 and CLIC2 is involved in regulation of cell cycle.(1)Chlorine channel proteins are abnormally expressed in prostate cancer cells.The expression level of chloride channel family proteins from human prostate cancer cell line DU145 and human normal fibroblasts was detected by Quantitative real-time PCR.Chlorine channel proteins CLIC3 and CLIC2 were abnormally expressed in prostate cancer DU145 cells,which indicates that the abnormal expression of chlorine channels might be playing a key role to their growth.In other words,down-regulating the overexpression of chlorine channels is of great significance.(2)Chlorine channel proteins CLIC3 and CLIC2 regulate the cell cycle.To determine whether chlorine channel proteins take part in the regulation of cell cycle,we blocked the expression of CLIC2 and CLIC3 separately and then examined the variation of the cycle through flow cytometry.Compared to control group,the down-regulation of CLIC2 and CLIC3 increased the percentage of G1 phase cells while decreased cells in S phase,which supported the results of cell cycle treated by chloride channel blocker DIDS.The data ascertains that chlorine channel proteins CLIC3 and CLIC2 participate in the regulation of DU145 cell cycle.3.Stemness Gene Sox2 involved in the regulation of cell cycle.(1)Gene chip technology is used to screens the gene of DU145 cells treated by DIDS.To investigate genome variation,we used high-throughput gene microarray to screen the gene of DU145 cells treated by DIDS,and figured out the most significant change of the relevant genome.The variation was most obvious in Stemness gene of all the changed genome.It was shown that Sox2 gene changed notably among others,therefore we speculated that Sox2 gene might be in the key position of cell cycle.(2)Stemness gene Sox2 is related to the block of chlorine channel.To investigate the relationship between Sox2 gene and chloride channel,DU145 cells were cultured in two six-plate wells.At 12,24 and 48 h after being treated by DIDS(100μM),proteins were collected for detecting Sox2 protein through western blot.Compared to control group,Sox2 protein presented little change after 12 h from being treated by DIDS.As time increased,the decrease of Sox2 in 24 h and 48 h were more significant.These statistics corroborated the results of gene microarray.4.The Interaction of chloride channel proteins CLIC2 and CLIC3 with Stemness gene Sox2.(1)Immune Coprecipitation is used to investigate the interaction of chloride channel proteins CLIC2 and CLIC3 with Stemness gene Sox2 in vivo.We used Immune Coprecipitation to investigate the interaction of chloride channel proteins CLIC2 and CLIC3with Stemness gene Sox2.As the result showed,we precipitated Sox2,there was protein precipitation in input positive group,and CLIC2,CLIC3 were precipitated in IP group.On the contrary,we precipitated CLIC2,there was protein precipitation in input positive group,and Sox2 was precipitated in IP group,additionally,negative control Ig G group didn’t have protein precipitation.CLIC3 was precipitated,there was protein precipitation in input positive group,and Sox2 was precipitated in IP group,additionally,negative control Ig G group didn’t have protein precipitation.And the above experimental results proved that Sox2 interacts closely with channel proteins CLIC2 and CLIC3.(2)To test the interaction of channel proteins CLIC2 and CLIC3 with Sox2 in vitro by confocal laser scanning microscope.Prostate cancer cells were seeded in carry sheet glass,and divided into four groups,contains control group,Sox2 si RNA transfection group,CLIC2 si RNA transfection group,CLIC3 si RNA transfection group.Cells were then incubated with the appropriate secondary antibody and the immunofluorescence detected using confocal laser scanning microscope(Olympus).In CLIC2 si RNA transfection group,fluorescence degree of CLIC2 decreased compared with control group,and fluorescence degree of Sox2 increased.In Sox2 si RNA transfection group,fluorescence degree of Sox2 decreased compared with control group,and fluorescence degree of CLIC2 and CLIC3 decreased either.In CLIC3 si RNA transfection group,fluorescence degree of CLIC3 decreased compared with control group,and fluorescence degree of Sox2 decreased either.From the above,Sox2 is positively correlated with channel proteins CLIC2 and CLIC3.5.The Mechanism of Chloride Channel Blocker DIDS and Stemness gene Sox2 participated in cell cycle and the regression of androgen independent prostate cancer.(1)The cell cycle protein of G1 phase variation of DU145 cells were treated by Chloride Channel Blocker DIDS.For the western-blotting analysis of cell cycle protein p27,p16,p53 and cyclinD1,DU145 cells were then treated with chloride channel blocker DIDS(100μmol/L)for 24 h and 48 h.As for the results,the expression of Cyclin D1 decreased over time.P27 increased in 24 h and48h compared with control group,and p53,p16 had no significant changes.Cyclin D1 is closely related G1 phase,which can promote the transition of G1 phase to S phase.P27 belongs to the Cip/Kip family of cyclin dependent kinase(Cdk)inhibitor proteins,which inhibited formation of G1 phase.Together these results,both reduced expression of Cyclin D1 and increase expression of P27 can inhibite the transition of G1 phase.(2)Sox2si RNA transfection changes the expression of cell cycle protein in DU145 cells.DU145 cells were lipofection transfected with ds RNA,after transfection,cells were lysed in lysis buffer to detect cell cycle protein p27 and cyclin D1 by western-blotting analysis.In Sox2 si RNA transfection group,the expression of Sox2 was inhibited,the expression of Cyclin D1 was significant decreased,otherwise,which of p27 was increased.Data are expressed as mean ± SD.(p<0.05 is considered significant)The results are consistent with cells were treated by chloride channel blocker DIDS.(3)Chloride Channel Blocker DIDS Down-regulates severity of DU145 cells.For the western-blotting analysis of expression of androgen receptor AR and Prostate specific antigen,DU145 cells were treated with DIDS(100μmol/L).After 24 h and 48 h,the cells were lysed in lysis buer.There was no significant difference of expression of AR protein between 24 h,48h treated groups and control group,but Prostate specific antigen had significant decrease in 48h-treated group.This suggests that chloride channel was inhibited down-regulates severity of DU145 cells.6.To verify down-regulated Chloride Channel inhibited severity of tumor,and the interaction of chloride channel with Stemness gene Sox2 in vivo(1)Chloride Channel Blocker DIDS Down-regulates volume of tumorThe 3–4 weeks old BALB/c-nude mice(male,10g)were injected with 100 ul DU145 cell suspension(about 1*107 cells)by subcutaneous injection using 1ml injection syringe.One week later,mice’ right hind had hump,and record the length and width of tumor every three days,calculate volume of tumor(volume=length*width* height/2).Continuous dose 1 week,mice’behavioristics and weight were observation,there was no side-effect in mice.We still observation two weeks after off the pill,and then mice were euthanasia.Stripping tumor and calculate the volume of tumor.After injection of DU145 cell suspension one week,each nude mice had Hard hump.Continuous dose 1 week,the volume of tumor in higher DIDS group was significant difference in control group.The volume of tumor in higher DIDS group was about 0.15(cm3),and in control and lower DIDS group,the volume of tumor was 0.45(cm3)and 0.40(cm3)respectively.The results showed that injected with DIDS intraperitoneally inhibited tumor growth.(2)Histopathology examination of Tumor to evaluate the effect of Chloride Channel Blocker DIDS inhibited cell cycle,and verify the interaction of chloride channel with Stemness gene Sox2 in vivoTumor tissue were fixed in 4% paraformaldehyde overnight,dehydrated,embedded in paraffin and then sliced into 5-mm thick sections.After deparaffinization,slices were stained by hematoxylin and eosin(H&E).Pathological changes(contain P27,cyclin D1,Ki67,Sox2)of the tumor tissue were observed in inverted microscope.As for the tumor H&E staining result,P27(brown),cyclinD1(brown),Ki67(brown),Sox2(brown),and nuclei(blue).The expression of Ki-67,Cyclin D1,Sox2 protein in higher DIDS group was decreased,and expression of p27 increased compared with control group.The results showed that injected with DIDS intraperitoneally inhibited tumor growth.Statistics results showed that expression of Ki-67,Cyclin D1,Sox2 protein in higher DIDS group was significant decreased using Gray scale scanning software.Conclusion1)It was showed that DIDS had the ability to inhibit the proliferation of DU145 and PC3 of prostate cancer cell line,with a concentration and time dependent manner,DIDS showed an obvious inhibiting effect in DU145 cells,and had no significant effect in human normal fibroblasts,and cells treated by DIDS were arrested in G0/G1 phase.2)Chloride channel proteins are abnormally expressed in prostate cancer cells,CLIC3 and CLIC2 is involved in regulation of cell cycle.3)Stemness Gene Sox2 involved in the regulation of cell cycle.4)Sox2 interacts closely with channel proteins CLIC2 and CLIC3.5)Chloride Channel Blocker DIDS Down-regulates expression of cyclin D1,up-regulation expression of p27,which regulated cell cycle,and Sox2 involved in it.There was no significant difference of expression of AR protein in all groups,but Prostate specific antigen had significant decrease in DIDS-treated group.This suggests that chloride channel was inhibited down-regulates severity of tumor.6)Animal experiment investigated that inhibited chloride channel regulated expression of cyclin D1 and p27,resulted in inhibiting the transition of G1 phase,expression of stemness gene Sox2 decreased.This proves chloride channel proteins maybe a promising target to regulate expression of stemness gene.