Effect Of Capsaicin On The Growth Of Endometrial Cancer ISHIKAWA Cells By TRPV1 Pathway

Background: At present,the incidence of endometrial cancer(EC)in China ranked second in gynecological malignancies,a serious threat to the general health of women.Although the incidence of perimenopausal and postmenopausal women was still the main group,but the incidence of young women increased year by year in recent years.The latest edition of NCCN Guidelines on Clinical Practice of Uterine Tumors pointed out the indications and methods of preserving fertility in endometrioid adenocarcinoma and warned clinicians to pay special attention to the treatment of EC patients who have reproductive needs.Although the treatment of EC is continuously updated and perfected,it is still necessary to explore its pathogenesis and seek more effective non-surgical treatment.Capsaicin(CAP)has the chemical name: trans-8-methyl-N-vanillyl-6-nonenamide,which is the main spicy ingredient in Capsicum.Previously,a large number of studies have shown that it has clinical application prospect in many disciplines,such as pain,respiratory system,cardiovascular system,urinary system,gastrointestinal tract,tumor,obesity and so on.In recent years,its research on anti-tumor has attracted much attention in academic circles.Transient receptor potential vanilloid 1(TRPV1)is a capsaicin-specific receptor.In a variety of tumor cells,the apoptosis-promoting effect of capsaicin is TRPV1-dependent.Our previous study showed that the occurrence and development of type I endometrial cancer may have the involvement of TRPV1,the occurrence of type II endometrial cancer may be related to the loss of TRPV1,and the representative cell line ISHIKAWA of type I endometrial cancer is TRPV1 positive expression.Objective: To investigate the effect of capsaicin on the proliferation,apoptosis and cell cycle of human endometrial carcinoma ISHIKAWA cellsthrough the TRPV1 pathway and the related molecular mechanisms,in order to seek new methods for non-surgical treatment of type I endometrial carcinoma represented by ISHIKAWA cells.Methods:1.Cell culture Human endometrial cancer ISHIKAWA cell line positively expressed estrogen receptor(ER)were cultured in DMEM/F12 medium containing 10% fetal bovine serum,then placed in a 37?,5%CO2saturated humidity incubator.2.Experimental group ISHIKAWA cells were treated with different concentrations of capsaicin(0,50,100,200,400?mol/L)for 24 hours,and an additional group was set up to pretreat ISHIKAWA cells with 10?M capsazepine(CPZ)for 30 min and then to treat ISHIKAWA cells with 200?M CAP for 24 hours.3.Cell proliferation assay ISHIKAWA cells were processed by experimental grouping and MTT assay was used to detect their proliferation in vitro.4.Observation of cell morphology The morphological changes of different groups of cells were observed by bright field photographs and Hoechst 33342.5.Flow cytometric analysis Flow cytometry was used to detect apoptosis and cell cycle distribution in different groups of cells.6.Western-blot The expression of tumor suppressor gene p53,apoptotic protein caspase-3 and cyclin-associated protein CDK2 were detected by Western-blot.7.Statistical analysis Data using SPSS19.0 software for statistical analysis.Measured data are expressed as mean±standard deviation(x±s),count data are expressed in n%(N/N),and test level ? = 0.05.The cell proliferation rate data was in normal distribution,but the variance was not uniform,and multiple independent samples nonparametric tests were performed.Flow cytometry data were in accordance with the normal distribution and homogeneity of variance test,using one-way analysis ofvariance.Results:1.The effect of capsaicin on proliferation activity of human endometrial cancer ISHIKAWA cells: With the increase of capsaicin concentration,the cell proliferation rate of ISHIKAWA cells decreased in a dose-dependent manner,and the effect of TRPV1 receptor antagonist CPZ attenuated this effect.2.Morphological effects of capsaicin on human endometrial cancer ISHIKAWA cells: The bright field photographs show that compared with the control group,ISHIKAWA cells show a decrease in the number of cells and an increase in cell shrinkage as the concentration of the drug increases,cell adhesion worse,and even a large number of broken Split cell.After Hoechst33342 staining,ISHIKAWA cells showed that with the increase of drug concentration,the nuclear staining was deepened and reduced,the chromatin condensed and agglutinated,and the severed cell nuclei fragmented,showing dense granular fluorescence with different sizes.Cell morphological changes induced by CAP can be partially reversed by CPZ.3.The effect of capsaicin on the apoptosis rate of human endometrial cancer ISHIKAWA cells: With the increase of drug concentration,the apoptosis rate of ISHIKAWA cells showed a concentration-dependent increase,and this trend of ISHIKAWA cells can be reversed by CPZ.4.Effects of capsaicin on the cell cycle of human endometrial cancer ISHIKAWA cells: As the drug concentration increases,the percentage of S-phase cells increases in a concentration-dependent manner,and this trend can be reversed by CPZ.5.The expression of related proteins of ISHIKAWA cells after capsaicin treatment: Compared with the control group,the expression of cleaved caspase-3 protein and p53 protein were significantly up-regulated and the expression of CDK2 protein was down-regulated in ISHIKAWA cells after CAP treatment,while CAP+10?M CPZ group reversed the regulation of CAP-induced protein expression.Conclusion:1.In vitro experiments showed that capsaicin can induce proliferation-inhibitory,apoptosis-promoting and S-phase arrest effects in ISHIKAWA cells.2.The growth inhibitory effect of capsaicin on ISHIKAWA cells was TRPV1-dependent,and may be related to the up-regulation of tumor suppressor gene p53,apoptotic protein caspase-3,and down-regulation of the cycle-related protein CDK2.