茶多酚对卵巢癌细胞黏附能力影响及其机制探讨

Impact of tea polyphenols on the adhesion ability in ovarian cancer cells and its mechanism

目的探讨茶多酚对人卵巢癌SKOV3和3AO细胞黏附能力及对上皮型钙黏蛋白(epithelial cadherin,E-cadherin)和神经型钙黏蛋白(neural cadherin,N-cadherin)表达的影响。方法体外培养人卵巢癌SKOV3和3AO细胞,采用MTT法检测0、5、10、20、40、80和160μg/mL不同浓度茶多酚处理24h后,SKOV3和3AO细胞体外增殖情况,选取后续实验茶多酚的给药浓度。分别选取5、10及20μg/mL茶多酚处理SKOV3细胞24h,10、20及40μg/mL茶多酚处理3AO细胞24h,各自对照组为不含茶多酚的培养液培养24h,采用细胞分离实验观察细胞-细胞间黏附能力的变化;采用细胞黏附实验观察细胞-基质间黏附能力的变化;利用Real-time PCR和蛋白质印迹法检测E-cadherin和N-cadherin在mRNA及蛋白水平的表达变化。结果 MTT实验结果显示,SKOV3(χ2=18.96,P=0.004)和3AO(χ2=16.98,P=0.009)卵巢癌细胞组内增殖抑制率随浓度增高而增强。细胞分离实验结果显示,随着茶多酚给药浓度的增加,SKOV3和3AO细胞在10和20min的分离率逐渐降低,差异均有统计学意义,P值均<0.05。细胞黏附率的比较,SKOV3细胞对照组、5、10和20μg/mL组的黏附率分别为100.00%、93.89%、82.50%和67.32%,各组间差异有统计学意义,χ2=9.583,P=0.022;3AO细胞对照组、10、20和40μg/mL组黏附率分别为100.00%、92.65%、86.90%和76.71%,差异有统计学意义,χ2=10.532,P=0.015。Real-time PCR结果显示,SKOV3细胞各实验组的E-cadherin mRNA表达水平增加,χ2=10.532,P=0.015,N-cadherin mRNA表达水平减少,χ2=9.583,P=0.022;3AO细胞中各组N-cadherin mRNA表达水平明显减少,χ2=10.532,P=0.015,但E-cadherin mRNA表达水平无明显变化,χ2=4.499,P=0.212。蛋白质印迹法结果显示,SKOV3细胞中各实验组的E-cadherin蛋白表达水平增加,χ2=10.116,P=0.018,N-cadherin蛋白表达水平减少,χ2=9.804,P=0.02;3AO细胞中实验组与对照比较N-cadherin蛋白表达水平明显减少,χ2=8.868,P=0.031,但E-cadherin蛋白表达水平差异无统计学意义,χ2=1.17,P=0.76。结论茶多酚能够提高卵巢癌SKOV3和3AO细胞-细胞间黏附能力,降低细胞-基质间黏附能力,其机制可能与茶多酚上调E-cadherin及下调N-cadherin表达有关。

OBJECTIVE To explore the effect of tea polyphenols （TP） on adhesion in human ovarian cancer SKOV3 and 3AO cells,and investigate the impact of TP on the expression of E-cadhern and N-cadherin. METHODS SKOV3 and 3AO cells were treated with different doses of TP at 0,5,10,20,40,80 and 160 /μg/mL for 24 h in vitro, the inhibitory rate of cell proliferation was assessed by MTT assay. SKOV3 cells were treated with TP at doses of 5,10 and 20 μg/mL and 3AO cells were treated with TP at doses of 10,20 and 40 μg/mL for 24 hs respectively. The control cells were treated with culture medium for 24 hours only. The ability of cell-cell adhesion was detected by cell detachment experiment, and the cell-matrix adhesion was tested by cell adhesion assay. Cellular expression levels of E-cadherin and N-cadherin were examined with real-time fluorescence quantitative PCR and western blot. RESULTS With the increasing concentration of TP, the inhibitory rate of SKOV3 （ 2218.96,P=0.004） and3AO（2=16.98,P=0.009） cells were increased. The re- sults of cell detachment experiment showed that with the increasing concentration of TP,the detachment rates of SKOV3 and 3AO cells were decreased significantly at 10 and 20 minutes among groups,all P values d0.05. The cell adhesion as- say was showed as follows： the adhesion rates of SKOV3 cells in control group,TP 5,10 and 20 /lg/mL group were 100.00% ,93.89%, 82. 50% and 67. 32%, respectively, the differences were statistically significant, x2 = 9. 583, P = 0. 022. The adhesion rates of 3AO cell in control group,TP 10,20 and 40 μg/mL group were 100.00% ,92.65% ,86.90% and 76.71%, the differences were statistically significant, X2 = 10. 532, P = 0. 015. The results of real-time PCR showed that the expression of E-cadherin mRNA increased, X2 = 10. 532, P = 0. 015, and that of N-cadherin mRNA decreased in SKOV3 cells after treatment with TP, X2 = 9. 583, P = 0. 022. The expression of N-cadherin mRNA decreased, X2 = 10.532, P = 0.015, E-cadherin mRNA was not significant in 3 AO cells, X2 = 4.499, P = 0.212. The results of western blot showed that the expression of protein levels of E-cadherin increased, X2 = 10. 116, P= 0. 018, and that of N-cadherin de- creased in SKOV3 after treatment with TP,x2 = 9. 804, P= 0. 02. The expression of protein levels of N-cadherin de- creased, X2 = 8. 868, P = 0.031, and that of E-cadherin was not significant in 3AO cells, x2 = 1.17, P = 0.76. CONCLU- SIONS Tea polyphenols could significantly inhibit the adhesion ability of SKOV3 and 3AO ovarian cancer cells. The un- derlying mechanisms may be correlated with the upregulation of E-cadherin and downregulation of N-cadherin by treat- ment with TP.