Effect Of Capsaicin On Human Endometrial Carcinoma ISHIKAWA Cells

Background: Endometrial carcinoma(EC)is a malignant neoplasm of the epithelial portion of the endometrium.In recent years,the incidence of endometrial carcinoma increases year by year,second only to cervical cancer.EC,a serious threat to women’s health,is the second most commom cancer In malignant tumor of the female reproductive system and has not yet found an effective target for prevention and treatment.Capsaicin,a homovanillic acid derivative,is the main active ingredient of chili peppers,which are widely used as vegetables and spices worldwide.chili pepper has been used as a traditional medicine for thousands of years.Capsaicin topical plaster has been used for the clinical treatment of herpetic neuralgia,diabetic neuropathy pain and rheumatoid arthritis pain.In recent years,a large number of studies have shown that capsaicin plays an anti-cancer effect in the development of a variety of tumors,through its receptor TRPV1 dependent or independent pathway.However,the role of capsaicin in human endometrial cancer has not been reported.Objective: To observe the expression of capsaicin receptor TRPV1 in different human endometrial carcinoma cell lines and to explore the effect of capsaicin on the proliferation,apoptosis and cell cycle of human endometrial carcinoma cell line with positive TRPV1 expression,and then to provide new ideas and new methods for clinical treatment of endometrial cancer.Methods:1 Cell culture.The human endometrial carcinoma cell lines ISHIKAWA,HEC-1A,HEC-1B and KLE were inoculated in DMEM/F12 and DMEM containing 10% fetal bovine serum,then placed 37℃,5%CO2 in a saturated humidity incubator.2 Western-blot detected the expression of TRPV1 in four humanendometrial carcinoma cell lines.Human endometrial cancer cells of the logarithmic growth phase will be extract protein.Western blot was used to detect the expression of TRPV1.3 Cell proliferation assay MTT assay was used to detect the cell proliferation of capsaicin-stimulated.The human endometrial carcinoma cell lines with positive TRPV1 expression were selected and stimulated with different concentrations of capsaicin for different time.MTT assay was used to detect the proliferation of human endometrial carcinoma cells.4 Flow cytometric analysis.Flow cytometry was used to detect the apoptotic rate and cycle distribution after capsaicin stimulation.Detection of apoptosis: After incubation of cells to 70% fusion,different concentrations of capsaicin were added and cells were cultured 24 hours and then the cells in each group were harvested.The cells were washed twice with precooled PBS and stained with propidium iodide(PI)and Annexin V-FITC at room temperature away from light and then detected by flow cytometry.Cell cycle detection: After incubation of cells to 70% fusion,different concentrations of capsaicin were added and cells were cultured 24 hours and then the cells in each group were harvested.70% ethanol was used to fix the cells at 4?C overnight,the cells were washed twice and incubated with PI and Rnase at room temperature for 30 min and then detected by flow cytometry.5 Statistical analysis: all experiments were repeated three times,the data using SPSS21.0 statistical software for processing.Using GraphPad Prism 5.0software for chart production,all data were expressed as mean± standard deviation,using One-way ANOVA analysis,P<0.05 was statistically significant.Results:1 The expression of TRPV1 protein in human endometrial carcinoma cell line: TRPV1 protein was expressed in ISHIKAWA cell line,while was not detected in HEC-1A,HEC-1B and KLE cell lines.2 Effect of Capsaicin on the Proliferation of ISHIKAWA Cells: The proliferation activity of ISHIKAWA cells decreased gradually with theincrease of capsaicin concentration and the prolongation of the time.Capsaicin had obvious inhibitory effect on ISHIKAWA cells in vitro and had a concentration and time-dependent manner.3 Effect of capsaicin on apoptosis of ISHIKAWA cells: Apoptosis rates of ISHIKAWA cells treated with 0,200 and 400 μmol/L capsaicin for 24 h were16.08±7.96%,60.93±2.99%,75.18±3.18%.The early apoptotic rates were8.15±3.80%,54.02±6.46% and 50.39±15.08%,respectively.The late apoptotic rates were 7.93±7.60%,6.91±3.62% and 24.79±13.36% respectively.The overall apoptosis rate in the drug-treated group was significantly higher than that of the control group with concentration-dependent manner,and the difference was statistically significant(F=98.861,P=0.000).Further analysis showed that the apoptotic rate of ISHIKAWA cells was mainly in the early stage,after treatment by 200 and 400μmol/L capsaicin,the apoptotic rate of ISHIKAWA cells was significantly higher than that of the control group(F=20.642,P=0.002),while the late apoptotic was not significantly higher than that of the control group(F=2.209,P=0.191).In general,capsaicin can significantly induce ISHIKAWA cell apoptosis,with the increase in apoptosis rate,the proportion of living cells is also significantly reduced.4 Effect of capsaicin on cycle distribution of ISHIKAWA cells: After treated with 0,200 and 400μmol/L capsaicin for 24 h,The cell cycle distribution of ISHIKAWA cells was as follows: the proportion of cells in G0/G1 phase was 53.84±3.04%,59.43±2.69% and 70.95±0.79%,respectively;S phase cells were 26.38±8.32%,14.81±3.23% and 20.48 ±1.84% respectively;The cells in the G2/M phase were 19.78±7.53%,15.76±3.23% and 8.57±1.28%,respectively.The proportion of G0/G1 phase cells in the drug treatment group was significantly higher than that in the control group(F=7.950,P=0.021).Conclusion:1 TRPV1 protein was expressed in ISHIKAWA endometrial cancer cell lines,while was not detected in HEC-1A,HEC-1B and KLE human endometrial carcinoma cell lines.2 Capsaicin can inhibit the proliferation of ISHIKAWA cells in vitro.3 Capsaicin can induce ISHIKAWA cell apoptosis in vitro.4 Capsaicin can induce G0/G1 phase arrest of ISHIKAWA cells in vitro.