白藜芦醇诱导小细胞肺癌H446细胞凋亡机制的研究

doi:10.3877/cma.j.issn.1674-6902.2019.01.003

目的

研究白藜芦醇对小细胞肺癌H446细胞增殖的抑制、凋亡诱导作用及作用特点，并探讨其可能的机制。

方法

采用MTT法观察白藜芦醇对H446细胞的毒性以及细胞抑制率，筛选合适的药物作用浓度。通过不同浓度组及不同作用时间组观察白藜芦醇对H446细胞的毒性及细胞抑制率。采用流式细胞术检测不同浓度白藜芦醇及白藜芦醇不同作用时间对H446细胞的凋亡诱导作用、线粒体膜电位的变化以及细胞内ROS释放量的变化。

结果

白藜芦醇对小细胞肺癌H446细胞增殖有明确的抑制作用，高浓度时可引起细胞死亡，随着药物剂量增加和作用时间的延长，抑制作用更强，具有剂量依赖和时间依赖的特点，差异有统计学意义(P<0.05)。白藜芦醇可诱导H446细胞凋亡、促进细胞内ROS的释放、使线粒体膜电位下降，随着药物剂量增加和作用时间延长，凋亡诱导作用更强、细胞内ROS释放量更多、线粒体膜电位下降速度更快，呈现剂量和时间依赖的特点，差异有统计学意义(P<0.05)。

结论

白藜芦醇对小细胞肺癌H446细胞的增殖具有明确的抑制作用，其诱导小细胞肺癌H446的凋亡可能与线粒体功能障碍有关。

关键词: 白藜芦醇, 小细胞肺癌, 细胞凋亡

Objective

To investigate the inhibition effect of resveratrol on proliferation of H446 cells and the apoptosis effect of resveratrol on H446 cells, further to analyze the characteristic of these effects, and to discuss the possible mechanism of resveratrol inducing apoptosis of H446 cells.

Methods

MTT used to measure the cytotoxicity and the cell inhibitory rate of resveratrol act on H446 cells, to screen the appropriate concentration of drug. The cytotoxicity and the cell inhibitory rate of different concentration and different action time of resveratrol which act on H446 cells were measured. Flow-cytometric analysis used to measure the apoptosis of H446 cells, intracellular ROS and the mitochondrial membrane potential in cells. Two groups were measured, group of different concentration of resveratrol and group of different action time of resveratrol.

Results

Resveratrol has inhibitory effect on proliferation of H446 cells, cells would die when resveratrol is in a higher concentration. Along with the increasing dose of resveratrol and the prolonging action time of resveratrol, the inhibition was stronger, which characterizes concentration dependence and time dependence, the difference has statistically significant(P<0.05). Resveratrol could induce apoptosis, increase the release of ROS, and cause the decrease of mitochondrial membrane potential. With the increase of drug dose and prolonged action time, apoptosis induction was stronger, intracellular ROS release was more, and mitochondrial membrane potential decreased faster, and has the characteristics of concentration dependence and time dependence, the difference has statistically significant(P<0.05).

Conclusion

Resveratrol has obvious inhibitory effect on proliferation of H446 cell. Resveratrol could inducing apoptosis in H446 cells by mediated mitochondrial dysfunction pathway.

Key words: Resveratrol, Small cell lung cancer, Cell apoptosis

图1 白藜芦醇作用于H446细胞后的形态学变化(100×)；注：A：100～120 μg/ml浓度，细胞完全死亡；B：70～90 μg/ml浓度，大部分细胞出现圆缩，失去原有形态，细胞不贴壁；C：40～60 μg/ml浓度，50%以上细胞不透亮，失去活性；D：20～30 μg/ml大部分细胞状态良好，CK组细胞形态展开，透亮，细胞状态良好，活性高；E：DSMO组大部分细胞状态良好

图2 不同浓度白藜芦醇对H446细胞的抑制率

图3 不同浓度白藜芦醇作用时细胞形态变化(100×)

图4 30 μg/ml白藜芦醇作用不同时间后细胞形态变化(100×)

图5 不同浓度白藜芦醇及作用不同时间(30 μg/ml)细胞抑制率

图6 不同浓度白藜芦醇流式检测的细胞凋亡变化

图7 不同浓度白藜芦醇致H446细胞凋亡率

图8 30 μg/ml白藜芦醇作用不同时间流式检测的细胞凋亡变化

图9 30 μg/ml白藜芦醇作用不同时间H446细胞凋亡率

图10 不同浓度白藜芦醇给药时细胞内ROS释放情况

图11 不同浓度白藜芦醇给药时细胞内ROS释放量

图12 30 μg/ml白藜芦醇给药不同时间细胞内ROS释放情况

图13 30 μg/ml白藜芦醇给药不同时间细胞内ROS释放量

图14 不同浓度白藜芦醇给药后线粒体膜电位变化

图15 不同浓度白藜芦醇给药后线粒体膜电位下降率

图16 30 μg/ml白藜芦醇给药不同时间线粒体膜电位变化

图17 30 μg/ml白藜芦醇给药不同时间线粒体膜电位下降率

1	Bishayee A,Sethi G. Bioactive natural products in cancer prevention and therapy: Progress and promise[J]. Semin Cancer Biol, 2016, 40-41: 1-3.
2	姜宁，张峰，郭鲁闽，等. 白藜芦醇对哮喘小鼠肺功能气道炎症的影响[J/CD]. 中华肺部疾病杂志(电子版), 2018, 11(5): 578-582.
3	陈向军，马李杰，侯绍杰，等. 白藜芦醇对脂多糖诱导的急性肺损伤的保护作用[J/CD]. 中华肺部疾病杂志(电子版), 2013, 6(1): 20-24.
4	马李杰，李艳燕，赵诣林，等. 乙酰化白藜芦醇通过调节缝隙连接蛋白43表达减轻海水淹溺型急性肺损伤[J/CD]. 中华肺部疾病杂志(电子版), 2013, 6(1): 15-19.
5	Tome-Carneiro J,Larrosa M,Gonzalez-Sarrias A, et al. Resveratrol and clinical trials: The crossroad from in vitro studies to human evidence[J]. Curr Pharm Des, 2013, 19(34): 6064-6093.
6	Lucas IK,Kolodziej H. Trans-Resveratrol Induces Apoptosis through ROS-Triggered Mitochondria-Dependent Pathways in A549 Human Lung Adenocarcinoma Epithelial Cells[J]. Planta Med, 2015, 81(12-13): 1038-1044.
7	Ko JH,Sethi G,Um JY, et al. The Role of Resveratrol in Cancer Therapy[J]. Int J Mol Sci, 2017, 18(12): 2589.
8	Rauf A,Imran M,Butt MS, et al. Resveratrol as an anti-cancer agent: A review[J]. Crit Rev Food Sci Nutr, 2018, 58(9): 1428-1447.
9	Semenova EA,Nagel R,Berns A, et al. Origins, genetic landscape，and emerging therapies of small cell lung cancer[J]. Genes Dev, 2015, 29(14): 1447-1462.
10	George J,Lim JS,Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer[J]. Nature, 2015, 524(7563): 47-53.
11	Umemura S,Mimaki S,Makinoshima H, et al. Therapeutic priority of the PI3K/AKT/mTOR pathway in small cell lung cancers as revealed by a comprehensive genomic analysis[J]. J Thorac Oncol, 2014, 9(9): 1324-1331.
12	Saunders LR,Bankovich AJ,Anderson WC, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo[J]. Sci Transl Med, 2015, 7(302): 302ra136.
13	Byers LA,Wang J,Nilsson MB, et al. Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1[J]. Cancer Discov, 2012, 2(9): 798-811.
14	Moore AM,Einhorn LH,Estes D, et al. Gefitinib in patients with chemosensitive and chemo refractory relapsed small cell cancers: a Hoosier Oncology Group phase Ⅱ trial[J]. Lung Cancer, 2006, 52(1): 93-97.
15	Helfrich BA,Kim J,Gao DX, et al. Barasertib (AZD1152), a small molecule Aurora B inhibitor, inhibits the growth of SCLC cell lines in vitro and in vivo[J]. Mol Cancer Ther, 2016, 15(10): 2314-2322.
16	Newman DJ,Cragg GM. Natural Products as Sources of New Drugs from 1981 to 2014[J]. J Nat Prod, 2016, 79(3): 629-661.
17	Kundu J．K,Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: Mechanistic perspectives[J]. Cancer Lett, 2008, 269(2): 243-261.
18	Harikumar KB,Kunnumakkara AB,Sethi G, et al. Resveratrol, a multitargeted agent, can enhance antitumor activity of gemcitabine in vitro and in orthotopic mouse model of human pancreatic cancer[J]. Int J Cancer, 2010, 127(2): 257-268.
19	Xu Q,Zong L,Chen X, et al. Resveratrol in the treatment of pancreatic cancer[J]. Ann N Y Acad Sci, 2015, 1348(1): 10-19.
20	Mondal A,Bennett LL. Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7 cells through ROS, cell cycle inhibition, caspase 3 and PARP cleavage[J]. Biomed Pharmacother, 2016, 84: 1906-1914.
21	Ma L,Li W,Wang R, et al. Resveratrol enhanced anticancer effects of cisplatin on non-small cell lung cancer cell lines by inducing mitochondrial dysfunction and cell apoptosis[J]. Int J Oncol, 2015, 47(4): 1460-1468.
22	Lee YJ,Lee GJ,Yi SS, et al. Cisplatin and resveratrol induce apoptosis and autophagy following oxidative stress in malignant mesothelioma cells[J]. Food Chem Toxicol, 2016, 97: 96-107.
23	Tinhofer I,Bernhard D,Senfter M, et al. Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2[J]. FASEB J, 2001, 15(9): 1613-1625.
24	Naik P,Karrim J,Hanahan D. The rise and fall of apoptosis during multistage tumorigenesis: Down-modulation contributes to tumor progression from angiogenic progenitors[J]. Genes Dev, 1996, 10(17): 2105-2116.
25	Ucker DS,Levine JS. Exploitation of Apoptotic Regulation in Cancer[J]. Front Immunol, 2018, 9: 241.
26	Zhou PK,Huang RX. Targeting of the respiratory chain by toxicants：beyond the toxicities to mitochondrial morphology[J]. Toxicol Res (Camb), 2018, 7(6): 1008-1011.
27	Geou YL,Peter S. Reactive oxygen species in cancer[J]. Free Radic Res, 2010, 44(5): 1-5.
28	Tochhawng L,Deng S,Pervaiz S, et al. Redox regulation of cancer cell migration and invasion[J]. Mitochondrion, 2013, 13(3): 246-253.
29	郑弘毅，吴雄健，杨建芬，等. 线粒体形态与功能改变在肿瘤中的研究进展[J]. 中国当代医药，2018, 25(16): 36-40.
30	汤浩，林春龙. 线粒体与细胞调控的研究进展[J]. 医学综述，2015, 21(21): 3876-3879.
31	Luo H,Yang A,Schulte BA, et al. Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage[J]. PLoS One, 2013, 8(3): e60065.
32	Lucas IK,Kolodziej H. Trans-Resveratrol Induces Apoptosis through ROS-Triggered Mitochondria-Dependent Pathways in A549 Human Lung Adenocarcinoma Epithelial Cells[J]. Planta Med, 2015, 81(12-13): 1038-1144.

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Mechanism of resveratoral inducing apoptosis in human small cell lung cancer H446 cells

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Mechanism of resveratoral inducing apoptosis in human small cell lung cancer H446 cells