自噬在双联苄类化合物诱导肿瘤细胞凋亡中的作用机制  

Bisbibenzyl-induced autophagy delays cell death in PC-3 cells

作　　者：祝芳华[1] 刘永青[2] 娄红祥[1] 

机构地区：[1]山东大学药学院天然药物化学研究所,济南250012 [2]山东大学医学院生化与分子生物学研究所,济南250012

出　　处：《山东大学学报（医学版）》2013年第4期11-17,共7页Journal of Shandong University:Health Sciences

摘　　要：目的探讨自噬在天然萜类、双联苄类化合物诱导肿瘤细胞凋亡中的作用。方法以前期筛选得到的具有抑制肿瘤细胞增殖活性的三萜化合物ST52和乙酰基-11-酮基-β-乳香酸(AKBA)、双联苄化合物H50、F41、H48和Riccardin D(RD)处理稳定表达GFP-LC3的人胶质瘤细胞株U87,荧光显微镜观察GFP-LC3的聚集斑点,确定化合物的自噬诱导作用。Western blot检测人激素非依赖前列腺癌细胞株PC-3细胞中的自噬相关蛋白LC3、Atg5、Beclin1和p62的表达以及凋亡标志蛋白多聚(ADP-核糖)聚合酶(PARP)的剪切情况。自噬抑制剂或siRNA阻断Atg5表达后进行化合物处理,流式细胞技术检测细胞凋亡,MTT法检测细胞的增殖活力,确定自噬对化合物诱导凋亡的影响。结果筛选结果显示,双联苄类化合物均有不同程度的自噬诱导作用,其中RD的作用较强。RD促进PC-3细胞中LC3-I向LC3-II的转化,中等强度诱导Atg5和Beclin1的表达,并导致自噬底物p62的降解。抑制自噬后,能增加RD诱导的细胞凋亡与PARP剪切,但凋亡抑制剂Z-VAD-FMK并不影响RD对LC3-II的诱导程度。结论 RD诱导PC-3细胞产生具有保护性作用的自噬现象,抑制自噬可提高RD的抗肿瘤作用。Objective To study the role of autophagy in the natural terpenoids and bisbibenzyls induced apoptosis in cancer cells. Methods Human astrocytoma U87 cells stably expressing GFP-LC3 were treated with terpenoids ST52 and acetyl-11-keto-f3-boswellic acid （AKBA）, and bisbibenzyls including H50, F41, H48 and Riccardin D（RD）, which were able to inhibite the proliferation of cancer cells, GFP-LC3 punctate dots were observed with a fluorescence microscope. Western blot was used to analyze the expression levels of LC3, Atg5, Beclinl, p62 and cleaved-poly （ADP-ribose） polymerase（PARP） in human androgen-independent prostate cancer PC-3 cells exposed to chemicals. Autophagy inhibitors or Atg5-targeting siRNA was employed to examine the effect of chemicals on autophagy induction. Cellular apoptosis was analyzed by Flow Cytometry with PI staining, and cell viability was detected by MTT assays. Results Compared with other compounds, bisbibenzyls such as RD induced more punctate dots in U87 cells. RD sig- nificantly induced the processing of LC3-I to LC3-II in PC-3 cells, and moderately up-regulated Atg5 and Beclinl ex- pressions, which in aLrn leading to reduction of p62. Blockage of autophagy resulted in enhancement of RD-induced ap- optosis as evidenced by increased cleavage of PARP and reduced cell viability. However, the LC3-II protein expression induced by RD was not altered in the presence of an apoptosis inhibitor Z-VAD-FMK. Conclusion These findings suggest that protetive autophagy is induced in PC-3 cells treated with RD and inhibition of autophagy could be an adjunctive strategy for enhancing antitumor activity.

关 键 词：双联苄类化合物 片叶苔素D 自噬 凋亡 U87细胞 PC-3细胞 

分 类 号：R915[医药卫生—微生物与生化药学]