In Conductance Calcium-activated Potassium Channels In The Biological Behavior Of Endometrial Cancer And Control Mechanism

Endometrial cancer(endometrial carcinoma,EC) is common in malignant tumors of female reproductive system,the incidence is at 15-20 per 100000 women per year.Owing to the development of technology and medical treatment,the curability of EC raised,but for the tumors with particular morphological variants, adverse histopathological features and/or advanced stage,the five years survival rate are still very low.Despite extensive etiopathogenisis and pathogenetic studies,the molecular mechanisms of EC remain elusive.The ion channel has been found to play an important role in the proliferation of cancer cells,and our previous study show that intermediate-conductance Ca2+-activated K⁺ channels is highly expressed in endometrial carcinoma tissues and it can promote the proliferation of endometrial carcinoma cells.We will further study the effects of KCa3.1 on endometrial carcinoma cells and to explore the possible molecular mechanisms.Our study contains three parts:(1)The effects of KCa3.1 on the proliferation, cell cycle and apoptosis of endometrial carcinoma cells;(2)The impact of KCa3.1 on the migration and invasion of endometrial carcinoma cells;(3) The effects of EGF on KCa3.1 expression and the possible mechanisms.Sectionâ… Effects of KCa3.1 on proliferation,cell cycle and apoptosis of human endometrial carcinoma cellsObjective The aim of the present study was to explore the effect of KCa3.1 on cell proliferation,cell cycle and apoptosis in endometrial cancer cells.Methods The recombinant plasmid containing small interference RNA of KCa3.1 gene was transfected into endometrial cancer cell lines,HEC-1-A and Ishikawa.Real time-PCR and Western blot were used to examine the gene and protein expression of KCa3.1 channels,while un-transfected cells and transfected negative plasmid cells were served as control groups.The function of KCa3.1 channels in KCa3.1 siRNA transfected endometrial cancer cells was analyzed through MTT,BrdU uptake test,flow cytometry,Real time-RT-PCR and Western blot.In addition, the TRAM-34 was added as inhibitor of KCa3.1.Results KCa3.1 siRNA transfected cells inhibited the mRNA and protein expression of KCa3.1 channels compared with control groups(P＜0.01).TRAM-34 and KCa3.1 siRNA transfectants suppressed the cell proliferation dramatically. Compared with control groups,the ratios of G0-G1 phase cells in experimental groups increased,while the ratios of S phase cells decreased and the apoptotic rate had no significant changes(P＞0.05) among the groups.The protein expressions of Cyclin D1,Cyclin E and survivin had significantly decreased in the experiment groups by Western blot analysis.Conclusion The blockage of the function or expression of KCa3.1 channels could inhibit cell proliferation and cell cycle progression,while may not involved in apoptosis in endometrial cancer cells.Sectionâ…¡The impact of KCa3.1 on the migration and invasion of human endometrial carcinoma cellsObjective To explore the impacts of intermediate-conductance Ca²⁺-activated K⁺ channel on migration and invasion of endometrial carcinoma cells.Methods With TRAM-34,the specific blocker of KCa3.1,we detected the changes of migration and invasion of endometrial carcinoma cells through scratch experiment, migration experiment and Transwell chamber invasion assay.At the same time,we studied the changes of MMP-2 expression before and after treatment with TRAM-34, using RT-PCR and Western blot.Results(1) TRAM-34 can significantly inhibit the ability to migrate and invade in HEC-1-A and Ishikawa cells,compared with the control group(P＜0.01);(2) TRAM-34 could cause the decline of MMP-2 expression and the difference was significant(P＜0.01).Conclusion The inhibition of KCa3.1 channel activity can reduce the ability to migrate and invade in endometrial carcinoma cells.These phenomenon may be related to reduced MMP-2 expression.In endometfial carcinoma cells,KCa3.1 probably can prompted the migration and invasion,and its expression and/or dysfunction maybe plays an important role in the metastasis of endometrial carcinoma.Sectionâ…¢Effect of EGF on KCa3.1 expression and the possible mechanisms Objective To study the effects of EGF on the expression of KCa3.1 and the possible mechanisms.Methods Cultured HEC-1-A and Ishikawa were treated by EGF with different concentration for different time.The cells were then harvested and the mRNA or proteins of KCa3.1 were extracted for RT-PCR or Western blot analysis,and also the expression of p-ERK1/2 and p-AKT with 10ng/ml EGF after different time(0,5, 15,30,60,120min).Moreover,to investigate the molecular mechanism of EGF,the specific inhibitor of MAPK pathway PD98059 or/and the specific inhibitor of PI3K/AKT pathway LY294002 or/and the specific inhibitor of KCa3.1 TRAM-34 were also used to inhibit the phosphorylation of ERK1/2 or AKT or the KCa3.1 and to study the relations in all these molecules.Results(1) With the increase in EGF concentration,HEC-1-A cells or Ishikawa cells KCa3.1 protein expression was significantly increased,compared with the control group;when the dose arrived at 10ng/ml,KCa3.1 expression arrived at the peak with significant difference(P＜0.01);with time extension,KCa3.1 protein expression level gradually increased,peaked at 48 hours,after which the expression level of KCa3.1 was downward trend in HEC-1-A or Ishikawa cells.(2) 10ng/ml EGF can cause significant enhancement of p-ERK1/2 and p-AKT expression,which reached the peak after 60min,while the ERK1/2 and AKT expression had no significant changes,the specific inhibitor of MAPK pathway PD98059 or/and the specific inhibitor of PI3K/AKT pathway LY294002,respectively,inhibited EGF-induced ERK1/2 and AKT phosphorylation,thereby causing the expression of KCa3.1 decline compared with the control group statistically(P＜0.01).(3)The relative specific blocker of KCa3.1 channel TRAM-34 can block the majority of EGF-induced ERK1/2 phosphorylation,but not the AKT phosphorylation or the expression of ERK1/2 or AKT.Conclusion EGF can promote the expression of KCa3.1 through the MAPK and PI3K/AKT signaling pathways;KCa3.1 plays a role in EGF-activated MAPK signaling pathway.Overall,KCa3.1 can promote the proliferation,cell cycle,migration and invasion in endometrial cancer cell lines HEC-1-A and Ishikawa.EGF can up-regulate the expression of KCa3.1 through MAPK and PI3K/AKT signaling pathways,meanwhile,KCa3.1 activity changes plays a regulatory role in the activation of MAPK signaling pathway.Our study provide a new sight for study of pathogenesis and a new target for drug therapy of endometrial cancer in the future.