桦木酸通过调节PI3K/Akt/mTOR信号通路诱导人结肠癌细胞SW620凋亡及自噬

Betulinic Acid Induces Autophagy and Apoptosis of Human Colorectal Cancer SW620 Cells by Regulating PI3K/Akt/mTOR Signaling Pathway

目的:研究桦木酸对人结肠癌细胞SW620凋亡和自噬的影响及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在其中的调控作用。方法:采用噻唑蓝(MTT)比色法检测细胞活性,确定最佳给药时间及给药浓度,用于后续实验;设置空白组、桦木酸低、中、高浓度组。采用苏木素-伊红(HE)染色观察桦木酸对SW620细胞形态的影响。采用Annexin-V/碘化丙啶(PI)双染法检测SW620细胞凋亡率。分别采用Hoechst33258染色及细胞自噬染色(MDC)观察细胞凋亡和自噬体发生情况。采用蛋白免疫印迹法(Western blot)检测细胞中B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、胱天蛋白酶-9(Caspase-9)、活化的胱天蛋白酶-3(cleaved Caspase-3),微管相关蛋白Ⅰ轻链3Ⅱ(LC3Ⅱ)、自噬关键分子酵母Atg6同系物-1(Beclin-1)、p62、磷酸化(p)-PI3K、p-Akt、p-mTOR蛋白表达。结果:桦木酸以浓度、时间依赖性的方式抑制SW620、HT29、HCT116细胞的活性;与桦木酸给药24 h比较,W620、HT29、HCT116细胞给药48 h细胞活性明显降低(P<0.05,P<0.01),给药48 h的半数抑制浓度(IC50)显著降低(P<0.01),其中SW620细胞给药48 h的IC;最小。HE染色、Hoechst33258染色显示,桦木酸组可见浓度依赖性细胞凋亡;MDC染色可见细胞自噬体数量增加。与空白组比较,桦木酸低、中、高浓度组细胞凋亡率显著升高(P<0.01)。与空白组比较,桦木酸低、中、高浓度组Bax、Caspase-9、cleaved Caspase-3、LC3Ⅱ蛋白表达水平明显升高(P<0.05,P<0.01),桦木酸中、高浓度组Beclin-1蛋白表达显著升高(P<0.01);桦木酸低、中、高浓度组p62、p-Akt、p-PI3K、p-mTOR蛋白表达显著降低(P<0.01)。结论:桦木酸对SW620细胞活性有明显的抑制作用,其作用机制可能与抑制PI3K/Akt/mTOR信号通路从而诱导人结肠癌细胞SW620的凋亡及自噬有关。

Objective: To investigate the effect of betulinic acid(BA)on apoptosis and autophagy of human colorectal cancer SW620 cells and the regulatory role of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway. Method: Cell viability was detected by methyl thiazolyl tetrazolium(MTT)colorimetry to determine the optimal administration time and dosage for subsequent experiments. Four groups were designed,including blank group and low-,medium-,and high-dose BA groups. Hematoxylin-eosin(HE)staining was conducted for the observation of SW620 cell morphology,and annexin-V/propidium iodide double staining for the determination of apoptosis rate in SW620cells. Hoechst33258 staining and MDC staining were used for the observation of apoptosis and autophagy,respectively. Western blotting was employed to determine the protein levels of B-cell lymphoma/leukemia-2(Bcl-2)-associated X protein(Bax), aspartate proteolytic enzyme-9(Caspase-9), activated aspartate proteolytic enzyme-3(cleaved Caspase-3),microtubule-associated protein 1 light chain 3(LC3),the mammalian homolog of yeast Atg6(Beclin-1), p62, phosphorylated PI3K(p-PI3K), phosphorylated Akt(p-Akt), and phosphorylated mTOR(p-mTOR)in SW620 cells. Result: BA inhibited the activity of SW620,HT29,and HCT116 cells in a concentration-and time-dependent manner. The cells treated with BA for 48 h had lower viability than those treated for 24 h(P<0.05,P<0.01). The half maximal inhibitory concentration(IC50)value of BA at the time point of 48 h was also lower than that at the time point of 24 h(P<0.01),and that for SW620cells was the minimum. BA induced the apoptosis in a concentration-dependent manner and increased the autophagosomes. Compared with the blank group,BA increased the apoptosis rate(P<0.01),up-regulated the protein levels of Bax,Caspase-9,cleaved Caspase-3,and LC3 Ⅱ(P<0.05,P<0.01),and down-regulated the protein levels of p62,p-Akt,p-PI3K,and p-mTOR(P<0.01). Additionally,medium-and high-dose BA upregulated the protein level of beclin-1(P<0.01). Conclusion: BA may inhibit the activity of SW620 cells by hindering the PI3K/Akt/mTOR signaling pathway to induce cell apoptosis and autophagy.