为探讨维药异常黑胆质成熟剂(ASM)对人肝癌细胞(HepG2)增殖、侵袭转移的影响及Rho/ROCK 信号传导通路相关蛋白表达影响,采用四甲基偶氮唑蓝(MMT)法检测不同浓度ASM(10、20、25、50 mg/mL)和10 μmol/L Y-27632 作用24、48、72 h后,对HepG2 细胞增殖的影响;采用扫描电镜技术和细胞侵袭实验测定ASM 不同剂量组和10 μmol/L Y-27632 作用24 h 癌细胞侵袭运动能力,Western Blot 检测ASM 不同剂量组和10 μmol/L Y-27632 作用24 h 癌细胞RhoA、ROCK1、ROCK2 的表达。结果显示,ASM 对肝癌细胞增殖有明显抑制作用,且表现为有明显的剂量效应关系:在10、20 mg/mL ASM 剂量组,ASM 药物作用24、48、72 h 后,HepG2 细胞增殖抑制作用随时间的延长而抑制增加,而25、50 mg/mL 剂量组,ASM 抑制细胞增殖作用不明显;ASM 抑制瘤细胞侵袭运动能力,扫描电镜结果显示ASM 抑制肿瘤细胞伪足的生长,ASM 中高剂量组ROCK1、ROCK2 的表达明显降低,RhoA 表达无明显变化。由此推论,ASM 对人肝癌细胞生长增殖和侵袭运动能力有抑制作用,其机制可能与ROCK酶表达降低有关。
This paper studies the effect of Uyghur medicine abnormal savda munzip (ASM) on the proliferation, invasion and the expression of Rho/ROCK signal transduction pathway related proteins in human hepatocellular carcinoma (HepG2) cells. After being treated with ASM (10, 20, 25, 50 mg/mL) and Y-27632(10 μmol/L) for 24, 48 and 72 h, the proliferation of HepG2 cells was detected using the four methyl thiazolyl tetrazolium (MTT) method. The alteration of the cells invasive ability treated with ASM and Y-27632 for 24h was observed by scanning electron microscope (SEM) and cell invasion assay. Western Blot was used to determine the effect of these drugs on the expression of RhoA, ROCK1 and ROCK2. The results revealed that ASM suppressed the proliferation of HepG2 significantly with a dose-effect relationship: for groups of ASM with 10 and 20 mg/mL, inhibition increased with the time of ASM action; for groups of ASM with 25 and 50 mg/mL, no significant increase of inhibition was found. ASM suppressed the invasive ability of HepG2. SEM results showed that the formation and growth of hepatoma cellular pseudopodia were inhibited by ASM. ASM with doses 25 and 50 mg/mL decreased the expressions of ROCK1 and ROCK2 significantly, but there was no significant effect on RhoA expression. The study suggests that ASM suppresses the proliferation and invasive ability of human hepatoma HepG2, the mechanisms of which could be associated with less expression of ROCK.
[1] 哈木拉提·吾甫尔. 维吾尔医异常黑胆质新论[M]. 乌鲁木齐: 新疆人民出版社, 2009: 1-9. Halmurat Upur. New conception on the theory of abnormal Savda in traditional Uyghur medicine[M]. Xinjiang: Xinjiang Pepole's Press, 2009: 1-9.
[2] 玛依努尔·艾力, 哈木拉提·吾甫尔. 维吾尔药异常黑胆质成熟剂总黄酮逆转肝癌细胞耐药性的作用[J]. 中国中医药信息杂志, 2007, 14 (9): 31-33. Mayinur Ali, Halmurat Upur. The reverse effect of uygur medicine ASM on the drug resistance cells of liver cancer[J]. Chinese Journal of Information on TCM, 2007, 14(9): 31-33.
[3] 胡汉华, 盛磊, 哈木拉提·吾甫尔, 等. 维药异常黑胆质成熟剂的抗肿瘤作用及其对细胞迁移的影响[J]. 科技导报,2011, 29(3): 62-65. Hu Hanhua, Sheng Lei, Halmurat Upur, et al. Anti-cancer effects of uighur medicine abnormal savda munzip and its influence on cell migration[J]. Science & Technology Review, 2011, 29(3): 62-65.
[4] 阿不都热依木·玉苏甫, 哈木拉提·吾甫尔, 吐尔洪·卡迪尔, 等. 异常黑胆质成熟剂乙酸乙酯萃取物对HepG2细胞生长和凋亡及相关基因调控的研究[J]. 中国药科大学学报, 2006,37(2): 161-164. Abdirymu Yusup, Halmurat Upur, Turghun Kadir, et al. Effect of abnormal savda Munziq ethyl acetate extract on the proliferation, apoptosis and correlative gene expression in human hepatoma (HepG2) cells[J]. Journal of China Pharmaceutical University, 2006, 37(2): 161-164.
[5] 王国宁, 李华, 陈珂瑶, 等. 异常黑胆质成熟剂总酚与顺铂、多西他赛联用对宫颈癌HeLa细胞增殖和凋亡的影响[J]. 山东医药, 2011, 51 (22): 22-24. Wang Guoning, Li Hua, Chen Keyao, et al. Effects of total phenolics from abnormal savda munziq combined with cisplation, ocetaxdon HeLa cells proliferation and apoptosis[J]. Shandong Medical Journal, 2011, 51 (22): 22-24.
[6] 张阿丽, 马丁. 细胞迁移调控因子Rho与肿瘤转移[J]. 国外医学生理病理科学与临床分册, 2003, 23(4): 365-367. Zhang Ali, Ma Ding. Cell migration control factor Rho and tumor metastasis[J]. Foreign Medical Sciences·Section of Pathophysiology and Clinical Medicine, 2003, 23(4): 365-367.
[7] 邵静, 王红兵, 杨力, 等. Rho蛋白对肿瘤细胞骨架活动及生长调节的影响[J]. 生物医学工程学杂志, 2008, 25(6): 1462-1465. Shao Jing, Wang Hongbing, Yang Li, et al. Effects of Rho proteins on the cytoskeleton activity and the growth regulation in tumor Cells[J]. Journal of Biomedical Engineering, 2008, 25(6): 1462-1465.
[8] 王德盛, 窦科峰, 李开宗, 等. Rho/Rho激酶对肝癌细胞体外侵袭转移作用的研究[J]. 中华普通外科杂志, 2005, 20(4): 245-247. Wang Desheng, Dou Kefeng, Li Kaizong, et al. Studies on hepatic carcinoma cell invasion in vitro through Rho/Rho kinase pathway[J]. Chinese Journal of General Surgery, 2005, 20(4): 245-247.
[9] Kamai T, Tsujii T, Arai K, et al. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer[J]. Clinical Cancer Research, 2003, 9(7): 2632-2641.
[10] Aikemu A, Yusup A, Umar A, et al. The impact of the Uighur medicine abnormal savda munziq on antitumor and antioxidant activity in a S180 and Ehrlich ascites carcinoma mouse tumor model[J]. Pharmacogn Magazine, 2012, 8(30): 141-148.
[11] 杨靖亚,王锡昌,刘梅芳,等. 表没食子儿茶素没食子酸酯通过下调 GTP结合蛋白RhoA的表达抑制癌细胞的浸润和转移[J]. 中国临床药理学与治疗学, 2006; 11(7): 729-734. Yang Jingya, Wang Xichang, Liu Meifang, et al. (-)-Epigallocatechin-3-gallate suppresses migration and invasion of cancer cell by decreasing expression of small GTP-binding protein RhoA[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2006, 11(7): 729-734.
[12] Liu N,Bi F,Pan Y, et al. Reversal of the malignant phenotype of gastric cancer cells by inhibition of RhoA expression and activity[J]. Clinical Cancer Research, 2004, 10(18 Pt 1): 6239-6247.
[13] 罗元. 细胞迁移运动相关因子与肿瘤浸润转移的关系[J]. 中国医学文摘(肿瘤学), 2004, 18(4): 58-59. Luo Yuan. The relation of the factors related cellular migration and tumor invasion[J]. Journal of Chinese Medical Abstracts (Oncology), 2004, 18(4): 58-59.
[14] 王雪, 王维莉, 任雨, 等. RhoA介导的细胞骨架在肿瘤发生发展中的作用[J]. 中国细胞生物学学报, 2014, 36(2): 267-273. Wang Xue, Wang Weili, Ren Yu, et al. The function of cytoskeleton mediated by RhoA during tumorigenesis and development[J]. Chinese Journal of Cell Biology, 2014, 36(2): 267-273.
[15] 方雷, 吴海滨, 陈晓岗. RhoA基因沉默对胃癌MGC-803细胞增殖和迁移能力的影响[J]. 肿瘤学杂志, 2012, 18(2): 28-32. Fang Lei, Wu Haibing. Effect of RhoA gene silencing on proliferation and migration of gastric cancer cell line MGC-803[J]. Journal of Oncology, 2012, 18(2): 28-32.
