珍珠梅黄铜纳米粒通过调节MAPK通路诱导人肝癌HepG2细胞凋亡和保护性自噬的实验研究
摘要
目的探讨中药珍珠梅黄酮纳米粒(5,2’,4’-trihydroxy-6,7,5’-trimethoxy flavone nanoparticle,TTF1-NP)处理HepG2细胞后是否发生凋亡和自噬、自噬和凋亡关系及其可能的作用机制。方法不同浓度(0,40,80,120μmol·L~(-1)) TTF1-NP处理HepG2细胞24 h后,MTT法检测细胞的增殖能力;通过Hoechst染色观察TTF1-NP处理HepG2细胞后的形态学变化;利用流式细胞术检测细胞凋亡比例; HepG2细胞转染GFP-LC3质粒后,荧光显微镜观察自噬情况;Western blot检测自噬标志蛋白LC3B I/II,凋亡相关蛋白Cleaved caspase3,以及MAPK通路相关蛋白p38MAPK,ERK1/2,JNK活性变化;采用自噬抑制3-甲基腺苷(3-methyladenine,3-MA)处理细胞后,进一步检测细胞凋亡及MAPK通路相关蛋白的表达。结果 TTF1-NP能够抑制细胞增殖,诱导细胞凋亡及自噬; Western blot结果显示,TTF1-NP增加LC3B-II蛋白表达,并上调MAPK通路相关分子JNK,下调ERK的活性;加入自噬抑制剂3-MA后,细胞增殖进一步下降,细胞凋亡进一步增加。结论 TTF1-NP可以诱导人肝癌HepG2细胞发生凋亡及保护性自噬,其作用机制可能与调节MAPK通路有关。
引文
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[2] Li Y, Bian L, Cui FD, et al. TTF1-induced apoptosis of HepG-2 cells through a mitochondrial pathway [J]. Oncol Rep, 2011,26(3): 651.
[3] Xiao B, Liu C, Liu BT, et al. TTF1-NPs induce ERS-mediated apoptosis and inhibit human hepatoma cell growth in vitro and in vivo [J]. Oncol Res, 2016,23(6):311.
[4] Yuan HX, Russell RC and Guan KL: Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy[J]. Autophagy, 2013,9(12): 1983.
[5] Kim KY, Park KI, Kim SH, et al.Inhibition of autophagy promotes salinomycin-induced apoptosis via reactive oxygen species-mediated PI3K/AKT/mTOR and ERK/p38 MAPK-dependent signaling in human prostate cancer cells[J]. Int J Mol Sci, 2017,18(5): pii:E1088.
[6] Ko A, Kanehisa A, Martins I, et al.Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signaling[J]. Cell Death Differ, 2014, 21(1):92.
[7] Ferlay J ,Soerjomataram I ,Dikshit R,et a.Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[J]. Int J Cancer, 2015, 136 (5): 359.
[8] 郑荣寿, 左婷婷, 曾红梅, 等. 中国肝癌死亡状况与生存分析[J]. 中华肿瘤杂志, 2015, 37(9): 697.
[9] Liu S, Fei W, Shi Q, et al.CHAC2, downregulated in gastric and colorectal cancers, acted as a tumor suppressor inducing apoptosis and autophagy through unfolded protein response[J]. Cell Death Dis, 2016,8(8): e3009.
[10] Wang J, Tan X, Yang Q, et al.Inhibition of autophagy promotes apoptosis and enhances anticancer efficacy of adriamycin via augmented ROS generation in prostate cancer cells[J]. Int J Biochem Cell Biol, 2016, 77: 80.
[11] Cryns V, Yuan J. Proteases to die for[J]. Genes Dev,1998, 12(11): 1551.
[12] Shaid S, Brandts CH, Serve H,et al. Ubiquitination and selective autophagy[J]. Cell Death Differ, 2013, 20(1): 21.
[13] Liu J, Chang F, Li F, et al.Palmitate promotes autophagy and apoptosis through ROS-dependent JNK and p38 MAPK[J]. Biochem Biophys Res Commun, 2015, 463(3):262.