二烯丙基三硫化物对人胃癌细胞株BGC823和AGS叶酸受体α表达的影响及相关调控机制

Effect of diallyl trisulfide on the expression of folate receptorαin human gastric cancer cell lines BGC823 and AGS and the related regulatory mechanism

目的探讨二烯丙基三硫化物(DATS)对人胃癌细胞叶酸受体α(FRα)表达的影响及相关调控机制。方法选择人胃癌细胞株BGC823、AGS,用10、20、40、80μmol/L DATS处理BGC823细胞48 h,5、10、20、40μmol/L DATS处理AGS细胞48 h,以6μmol/L组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)处理细胞作为表观遗传研究的阳性对照,以未经DATS处理的细胞作为阴性对照。采用流式细胞术检测DATS诱导胃癌细胞凋亡情况。在DATS处理BGC823、AGS细胞48 h后,换无DATS细胞培养液培养不同时间,检测FRα蛋白表达变化。采用6μmol/L TSA或40μmol/L DATS处理BGC823细胞和AGS细胞,蛋白质印迹法检测FRα、HDAC1和HDAC2以及组蛋白H3赖氨酸9位点乙酰化修饰(H3K9ac)和组蛋白H4赖氨酸5位点乙酰化修饰(H4K5ac)蛋白表达水平。应用BGC823细胞接种BALB/c裸鼠,建立移植瘤模型,DATS组裸鼠腹腔注射10 mg/kg DATS 16 d后,提取荷瘤组织蛋白,进行靶蛋白表达的检测;对照组注射等量0.9%NaCl溶液。结果BGC823和AGS细胞经梯度浓度DATS处理后细胞FRα蛋白表达均呈剂量依赖性上调(F=65.68,P<0.01;F=26.65,P<0.01)。撤除DATS后,BGC823和AGS细胞FRα蛋白表达逐渐降低并恢复至初始水平(F=74.57,P<0.01;F=30.92,P<0.01)。随着DATS处理浓度的增加,BGC823、AGS细胞凋亡率均增加(F=32.95,P<0.01;F=38.97,P<0.01)。BGC823、AGS细胞经TSA处理后FRα蛋白表达分别上调约4.5倍(t=-12.62,P<0.01)和3.6倍(t=-10.00,P<0.01)。分别经40、20μmol/L DATS作用后,BGC823和AGS细胞FRα蛋白表达水平上调(均P<0.01),HDAC1、HDAC2的表达受抑制(均P<0.01),H3K9ac和H4K5ac的乙酰化修饰水平升高(均P<0.01)。裸鼠荷瘤实验显示,给药后第16天,DATS组移植瘤体积小于对照组[(214±39)mm3比(577±98)mm3],差异有统计学意义(t=4.86,P<0.01)。与对照组相比,DATS组移植瘤组织FRα蛋白表达上调约2倍(t=-5.29,P<0.01),HDAC1和HDAC2蛋白表达水平均下调(t=9.36,P<0.01;t=9.88,P<0.01)。结论DATS以剂量依赖和可逆性方式上调人胃癌BGC823、AGS细胞FRα蛋白表达,其机制可能与DATS影响肿瘤细胞组蛋白乙酰化修饰有关。

Objective To investigate the effect of diallyl trisulfide(DATS)on the expression of folate receptorα(FRα)in human gastric cancer cells and the related regulatory mechanism.Methods Human gastric cancer cell lines BGC823 and AGS were selected,BGC823 cells were treated with 10,20,40,80μmol/L DATS for 48 hours,and AGS cells were treated with 5,10,20,40μmol/L DATS for 48 hours.Cells treated with 6μmol/L histone deacetylase(HDAC)inhibitor trichostatin A(TSA)were used as positive control for epigenetic study,and cells untreated with DATS were used as negative control.The apoptosis of gastric cancer cells induced by DATS was detected by flow cytometry.After BGC823 and AGS cells were treated by DATS for 48 hours,they were replaced with DATS-free cell culture medium and cultured for different time to detect the changes in FRαprotein expression.BGC823 and AGS cells were treated with 6μmol/L TSA or 40μmol/L DATS.The protein expression levels of FRα,HDAC1 and HDAC2,and histone H3 lysine 9 acetylation(H3K9ac)and histone H4 lysine 5 acetylation(H4K5ac)were detected by Western blot.BGC823 cells were inoculated into BALB/c nude mice to establish tumor bearing model.The nude mice in DATS group were injected intraperitoneally with 10 mg/kg DATS for 16 days,and then the protein in tumor-bearing tissues was extracted to detect the expression of target protein,while the control group was injected with equal dose of 0.9%NaCl solution.Results The expressions of FRαprotein in BGC823 and AGS cells were up-regulated in a dose-dependent way after gradient concentrations of DATS treatment(F=65.68,P<0.01;F=26.65,P<0.01).After changing the cell culture medium without DATS,the expressions of FRαprotein in BGC823 and AGS cells gradually decreased and returned to the initial levels(F=74.57,P<0.01;F=30.92,P<0.01).With the increase of DATS concentration,the apoptosis rates of BGC823 and AGS cells increased(F=32.95,P<0.01;F=38.97,P<0.01).After TSA treatment,FRαprotein expressions in BGC823 and AGS cells were up-regulated by 4.5 times(t=-12.62,P<0.01)and 3.6 times(t=-10.00,P<0.01).After 40μmol/L and 20μmol/L DATS treatment,the expression level of FRαprotein in BGC823 and AGS cells was up-regulated(both P<0.01),the expressions of HDAC1 and HDAC2 were inhibited(all P<0.01),and the levels of H3K9ac and H4K5ac acetylation modification increased(all P<0.01).The results of tumor-bearing nude mice experiment showed that the volume of transplanted tumor in DATS group was smaller than that in the control group[(214±39)mm3 vs.(577±98)mm3],and the difference was statistically significant(t=4.86,P<0.01).Compared with the control group,FRαprotein expression in the transplanted tumor tissues of DATS group was up-regulated about 2 times(t=-5.29,P<0.01),and the expression levels of HDAC1 and HDAC2 proteins were down-regulated(t=9.36,P<0.01;t=9.88,P<0.01).Conclusions DATS up-regulates the expression of FRαprotein in human gastric cancer BGC823 and AGS cells in a dose-dependent and reversible manner.The mechanism may be related to the effect of DATS on histone acetylation modification in tumor cells.