重楼活性单体pp-10通过抑制PI3K/Akt通路诱导人胃癌细胞BGC-823凋亡和自噬

doi:10.13523/j.cb.20150205

中国生物工程杂志

2015, Vol. 35

Issue (2): 31-37 DOI: 10.13523/j.cb.20150205

研究报告

重楼活性单体pp-10通过抑制PI3K/Akt通路诱导人胃癌细胞BGC-823凋亡和自噬

王娟娟¹, 张鹏¹, 黄志宏², 陈家劲¹, 李强¹, 王国才³, 李药兰³, 蒋建伟¹

1. 暨南大学医学院广州 510632;
2. 暨南大学附属第一医院广州 510632;
3. 暨南大学药学院中药及天然产物研究所广州 510632

Pp-10, a Monomer Purified from Paris polyphylla, Induces Apoptosis and Autophagy of Human Gastric Carcinoma BGC-823 Cells by Inhibiting PI3K/Akt Signaling Pathway

WANG Juan-juan¹, ZHANG Peng¹, HUANG Zhi-hong², CHEN Jia-jin¹, LI Qiang¹, WANG Guo-cai³, LI Yao-lan³, JIANG Jian-wei¹

1. Department of Biochemistry, Medical College, Jinan University, Guangzhou 510630, China;
2. Department of Pulmonary, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China;
3. Institute of Traditional Chinese Medicine and Nature Products, College of Pharmacy, Guangzhou 510630, China

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摘要：

目的: 探讨重楼/七叶一枝花(Paris polyphylla)的醇提物单体pp-10诱导人胃癌BGC-823细胞凋亡和自噬及其分子机制。方法:采用MTT法和克隆形成抑制实验观察不同浓度的重楼单体pp-10对人胃癌BGC-823细胞的增殖抑制作用;Hoechst33342染色法检测pp-10作用于人胃癌BGC-823细胞后细胞核形态的改变;AnnexinV-FITC/PI双染法检测细胞凋亡;Western blotting检测重楼单体pp-10对细胞凋亡和自噬相关蛋白Caspase-3、Caspase-9、(ADP-核糖)聚合酶(PARP)、Bax、Bcl-2、LC3、P62以及PI3K /Akt信号通路相关蛋白(Akt、p-Akt、mTOR、p-mTOR、P70s6k、p-P70s6k)表达的影响。结果:重楼单体pp-10能显著抑制BGC-823细胞的生长,作用呈时间-效应关系及剂量-效应关系;克隆形成抑制实验表明随着pp-10浓度的增加,细胞克隆形成逐渐减少,与对照组相比有显著差异; 在荧光显微镜下观察可见其细胞核固缩、边聚、裂解等细胞凋亡形态学变化;流式细胞术检测显示,随着作用药物浓度的增高,其凋亡率逐渐升高;Western blotting结果表明,随着药物浓度的增加,线粒体相关凋亡信号通路蛋白Caspase9、Caspase3及PARP均出现酶切活化条带,细胞促凋亡蛋白 Bax的表达水平增加,抗凋亡蛋白Bcl-2减少,自噬相关蛋白Ⅱ型LC3增加,P62蛋白减少,p-Akt蛋白的表达水平下降,Akt下游蛋白p-mTor、p-p70S6K表达减少。结论: 重楼单体pp-10通过抑制BGC-823细胞增殖,诱导细胞凋亡和自噬,与下调P13K/Akt信号通路有关。

关键词： 重楼; 胃癌; BGC-823; 细胞; Akt; 凋亡和自噬

Abstract:

Objective: To investigate pp-10, a monomer purified from Paris Polyphylla, induced apoptosis of human gastric carcinoma BGC-823 cells and the possible mechanism behind this effect. Methods: The effects of pp-10 on the proliferation of BGC-823 Cells were evaluated by the MTT assays and colony formation assays.The cellular morphology changes of BGC-823 cells were observed by fluorescence microscopy after Hoechst 33342 staining. Cell apoptosis was detected by Annexin V-FITC/Propidium iodide (PI) double staining using flow cytometry. The expression of apoptotic and autophagy associated proteins as LC3,P62,Caspase-9, Caspase-3, the Caspase substrate poly(ADP-ribose) polymerase (PARP), Bcl-2 proteins family such as Bax, Bcl-2, PI3k/Akt signaling pathway numbers such as Akt, p-Akt, mTOR, p-mTOR, P70s6k and p-P70s6k were detected by Western blotting. Results: Paris Polyphylla monomer pp-10 inhibited the proliferation of BGC-823 cells significantly in dose- and time- dependent manner (P<0.05). Cell clone formation inhibition assay demonstrated that cell clones decreased with the increase of drug concentrations. Apoptotic morphology such as cell shrinkage, nuclear condensation, nuclear fragmentation, chromatin condensation were also observed by staining with Hoechst33342 under fluorescence microscope. Flow cytometry analysis showed an increase of the percentage of apoptotic cells in a dose-dependent manner treated with pp-10 for 24 h. Active bands of mitochondria associated apoptotic signaling pathway proteins such as Caspase-3, Caspase-9 and PARP could be seen with western blotting analysis after BGC-823 cells were incubated with different concentrations of pp-10 for 24 h. In addition, pp-10 upregulated the expression of Bax, a pro-apoptotic protein, down-regulated the expression of Bcl-2 protein, an anti-apoptotic protein, up-regulated the expression of LC3, down-regulated the expression of P62 protein,and also down-regulated PI3K/Akt signal pathway as decreasing the phosphorylation of p-mTor, p-p70S6K, downstream of Akt. Conclusions: pp-10 inhibits cell proliferation and induces the apoptosis and autophagy of human Gastric carcinoma BGC-823 cells by activation of Bax, down-regulation of Bcl-2 and reduction of PI3K/Akt signaling pathway.

Key words: Paris polyphylla Gastric carcinoma BGC-823 cell lines Akt Apoptosis and autophagy

收稿日期: 2014-11-18 出版日期: 2015-02-25

ZTFLH:

R966

基金资助:

广东省科技计划项目(2011B031800012)、广东省医学科学技术研究基金(A2014379)资助项目

通讯作者: 蒋建伟 E-mail: jjw703@163.com

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引用本文:

王娟娟, 张鹏, 黄志宏, 陈家劲, 李强, 王国才, 李药兰, 蒋建伟. 重楼活性单体pp-10通过抑制PI3K/Akt通路诱导人胃癌细胞BGC-823凋亡和自噬[J]. 中国生物工程杂志, 2015, 35(2): 31-37.

WANG Juan-juan, ZHANG Peng, HUANG Zhi-hong, CHEN Jia-jin, LI Qiang, WANG Guo-cai, LI Yao-lan, JIANG Jian-wei. Pp-10, a Monomer Purified from Paris polyphylla, Induces Apoptosis and Autophagy of Human Gastric Carcinoma BGC-823 Cells by Inhibiting PI3K/Akt Signaling Pathway. China Biotechnology, 2015, 35(2): 31-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150205 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I2/31

[1] 段纪俊,陈万青,张思维. 中国恶性肿瘤死亡率的国际比较.中国社会医学杂志,2009,26(6):377-378. Duan J J, Chen W Q, Zhang S W. International comparison of maligant tumor mortality. Chinese Journal of Social Medicine, 2009,26(6):377-378.

[2] 汪济东, 许扬, 章银秀,等. 顺铂、亚叶酸钙及氟脲嘧啶方案治疗晚期胃癌.临床肿瘤学杂志,2004,9(2):190-191. Wang J D, Xu Y,Zhang Y X, et al. Cisplatin, leucovorin and fluorouracil in the treatment of advanced gastric cancer. Chinese Clinical Oncology, 2004,9(2):190-191.

[3] 陈志红龚先玲刘义. 重楼总皂苷对人肺癌细胞 A549 的增殖抑制作用及对细胞周期的影响.实用医学杂志,2010,15(26):2685-2687. Chen Z H, Gong X L, Liu Y.Effect of steroidal saponins from P.polyp hylla on proliferation and cell cycle of A549 cells. The Journal of Practical Medicine,2010,15,(26):2685-2687.

[4] 萧梅芳,戴霞红,贺新春,等. 重楼皂苷Ⅰ对肝癌细胞的增殖及凋亡的影响. 生命科学研究, 2011,15(6):519-523. Xiao M F, Dai X H, He X C, et al. Growth and apoptosis effects of Paris saponinⅠon human hepatocellular carcinoma cells. Life Science Research, 2011,15(6):519-523.

[5] 贾科,吴庆琛,张成. 重楼总皂苷对胃癌细胞株MGC-803生长的抑制作用.中国生化药物杂志,2011,32(4):284-286. Jia K, Wu Q S, Zhang C. Inhibition Paridis saponins on gastric cancer cell line MGC-803 growth. Chin J Biochem Pharm,2011,32(4):284-286.

[6] 胡静,钱晓萍,刘宝瑞,等. 重楼醇提物体外抑制血管生成作用研究.现代肿瘤医学,2008,16(8):1273-1278. Hu J, Qian X P, Liu B R, et al. Inhibitory effect of parid is on angiogenes is in vitro. Modern Oncology, 2008,16(8):1273-1278.

[7] 赵鹏军,冯建国,马胜林. 重楼皂苷Ⅰ对肺腺癌A549 细胞系放射增敏作用研究. 肿瘤学杂志,2012,18(2):108-110. Zhao P J, Feng J G, Ma S L. In vitro study of radiosensitization of saponinⅠon lung adenocarcinoma A549 cell line. Journal of Chinese Oncology, 2012,18(2):108-110.

[8] WU X, Wang L, Wang G C, et al. Triterpenoid saponins from rhizomes of Paris polyphylla var.yunnanensis.Carbohydr Res,2013,368:1-7.

[9] Wu X, Wang L, Wang H,et al. Steroidal saponins from Paris polyphylla var.yunnanensis.Phytochemistry,2012,81:133-143.

[10] Kang M H and Reynolds C P. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res,2009,15(4):1126-1132.

[11] Zhou F F, Yang Y, Xing D. Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. FEBS J,2011,278(3): 403-413.

[12] Fan J, Li R, Zhang R, et al. Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells. World J Gastroenterology, 2007, 13(45): 6053-6059.

[13] Adams J M, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene,2007, 26(9):1324-1337.

[14] 吴秀勤,杨炼红,钟健强,等.米诺环素通过调控PI3K /Akt通路抑制硝普钠诱导的PC12 细胞凋亡.中国病理生理杂志,2013,9(4):660-663. Wu X Q,Yang L H, Zhong J Q, et al. Minocycline suppress essodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling. Chinese Journal of Pathophysiology,2013,9(4):660-663.

[15] Du C X,Wang Y.Expression of p-Akt,NF-κB and their correlation with human papillomavirus infection in cervical carcinoma. Eur J Gynaecol Oncol,2012, 33(3):274-277.

[16] Khor T O, Gul Y A, Ithnin H, et al. Positive correlation between overexpression of phospho-BAD with phosphorylated Akt at serine 473 but not threonine 308 in colorectal carcinoma. Cancer Lett. 2004, 210 (2):139-150.

[17] 唐莉,刘小鹰,王林曦,等.氧化应激诱导内皮细胞凋亡与PI3k/Akt-Bcl-2通路的关系.细胞与分子免疫学杂志,2011,27(8):859-861. Tang L, Liu X Y, Wang L X, et al. The relationship between oxidative stress induced endothelial cell apoptosis and the PI3k/Akt - the Bcl - 2 pathway. Chin J Cell Mol Immunol,2011,27(8):859-861.

[18] Xin M G, Deng X M. Nicotine inactivation of the proapoptotic function of bax through phosphorylation. J Biol Chem, 2005,280 (11):10781-10789.

[19] Song G, Ouyang G L, Bao S D. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med, 2005, 9(1):59-71.

[20] 陈松峰,邵增务.自噬相关信号转导通路研究进展.国际骨科学杂志,2013,4(34):268-280. Chen S F, Shao Z W. Autophagy-related signal transduction pathway.Int J Orthop, 2013,4(34):268-280.

[21] 于春艳,李洪岩,钟加滕,等. 维生素K3通过下调mTOR信号途径而诱导HeLa细胞自噬.中国药理学通报,2009,25(9):1173-1176. Yu C Y, Li H Y, Zhong J T, et al. Vitamin K3 induced HeLa cell autophagy by down-regulating mTOR signaling pathway. Chinese Pharmacological Bulletin, 2009,25(9):1173-1176.

[22] Glick D, Barth S, Macleod K F. Autophagy: cellular and molecular mechanisms.J Pathol, 2010,221(1):3-12.

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