白桦脂酸对HeLa细胞的抑制作用及其机制研究

Inhibitory effect and mechanism of betulin acid on human cervical cancer HeLa cell lines

背景与目的:白桦脂酸(betulinic acid)是一类白桦树皮中提取的天然五环三萜类化合物,具有广泛的药理活性,有研究表明白桦脂酸能诱导多种肿瘤细胞凋亡,但其对人宫颈癌细胞的作用尚缺乏文献资料。本研究旨在探讨白桦脂酸体外对人宫颈癌HeLa细胞的增殖抑制作用及其机制。方法:采用四甲基偶氮唑盐(MTT)法测定白桦脂酸对HeLa细胞生长的抑制作用;Hoechst33258荧光染色检测白桦脂酸处理HeLa细胞后发生凋亡的形态变化;采用流式细胞术(flow cetemotry,FCM)检测白桦脂酸处理HeLa细胞后诱导凋亡作用及凋亡率;采用蛋白质印迹法(Western blot)检测caspase-3、细胞色素C(Cyto C)等凋亡相关蛋白的表达。结果:MTT分析表明,白桦脂酸抑制HeLa细胞增殖,并呈时间和剂量依赖性(P<0.01),其24、48和72 h的半数抑制浓度(50%inhibiting concentration,IC50)分别为20.36、15.45和12.02μg/mL;白桦脂酸处理HeLa细胞后,Hoechst33258染色出现典型的凋亡特征,且流式细胞术检测Annexin-Ⅴ/PI双染出现早期凋亡细胞;Westernblot检测结果显示经白桦脂酸处理后HeLa细胞的caspase-3、Cyto C蛋白表达均上调。结论:白桦脂酸可显著抑制人宫颈癌HeLa细胞的增殖,并可能通过上调caspase-3、Cyto C蛋白的表达诱导人宫颈癌HeLa细胞凋亡。

Background and purpose： Betulinic acid, a pentacyclic triterpenoid isolated from birch bark, has been reported that it could inhibit multiple cancer cells proliferation, but there is no report concerning cervical cancer. Therefore, we investigated the effect of betulinic acid on the proliferation of human cervical cancer HeLa cells in vitro and its mechanisms. Methods： MTT assay was used to determine the inhibitory rate ofbetulinic acid on the proliferation of HeLa cells. Apoptosis induced by betulinic acid was detected by Hoechst33258 staining. Apoptotic rate was quantified by flow cytometry （FCM） using Annexin-V/PI dual staining method. Western blot was used to evaluate the changes of caspase-3 and Cyto C proteins in HeLa cells. Results： Betulinic acid induced HeLa cells apoptosis in a time- and dose-dependent manner. The 50% inhibiting concentration （IC50） at 24, 48 and 72 h of HeLa cells were 20.36, 15.45 and 12.02 μg/mL, respectively. Typical morphological changes of apoptosis were observed in HeLa cells with Hoechst33258 staining after induced by betulinic acid. FCM showed the early apoptotic cells by Annexin-V/ PI staining. The protein expressions of caspase-3 and Cyto C in HeLa cells were both up-regulated after treatment with betulinic acid. Conclusion： Betulinic acid could significantly inhibit the proliferation of HeLa cells and induce apoptosis through caspase-3 pathway bv up-regulating caspase-3 and Cyto C proteins.