药学学报, 2019, 54(8): 1431-1438
引用本文:
齐世美, 姜琦, 李强, 冯遵永, 张雨晴, 董顺利, 戚之琳, 章尧. 二氢杨梅素调控MAPKs信号通路抑制SMMC-7721细胞凋亡和迁移的分子机制研究[J]. 药学学报, 2019, 54(8): 1431-1438.
QI Shi-mei, JIANG Qi, LI Qiang, FENG Zun-yong, ZHANG Yu-qing, DONG Shun-li, QI Zhi-lin, ZHANG Yao. Ampelopsin inhibited apoptosis and migration of SMMC-7721 cells through MAPKs signaling pathway[J]. Acta Pharmaceutica Sinica, 2019, 54(8): 1431-1438.

二氢杨梅素调控MAPKs信号通路抑制SMMC-7721细胞凋亡和迁移的分子机制研究
齐世美1,4, 姜琦1, 李强2, 冯遵永3, 张雨晴1, 董顺利1, 戚之琳1,4, 章尧1,4
1. 皖南医学院活性生物大分子重点实验室, 安徽 芜湖 241002;
2. 皖南医学院解剖学教研室, 安徽 芜湖 241002;
3. 皖南医学院法医学实验中心, 安徽 芜湖 241002;
4. 皖南医学院生物化学与分子生物学教研室, 安徽 芜湖 241002
摘要:
研究二氢杨梅素(ampelopsin,AMP)在体外诱导人肝癌SMMC-7721细胞凋亡和抑制细胞迁移的作用,并初步探讨其分子机制。用不同浓度的AMP处理SMMC-7721细胞,采用CCK-8法和集落形成实验检测细胞增殖情况;倒置显微镜下观察细胞形态改变;DAPI染色检测细胞核形态变化;Annexin V-FITC/PI流式细胞术检测细胞凋亡率;Transwell和细胞划痕愈合实验检测细胞的迁移和转移能力;运用Western blot法检测相关蛋白。结果表明,AMP对肝癌SMMC-7721细胞具有显著的增殖抑制及凋亡诱导作用,24 h的半数有效抑制浓度(IC50)为38.98μg·mL-1;SMMC-7721细胞发生典型的凋亡形态改变,细胞贴壁性差,细胞皱缩,变圆,细胞核出现高亮的蓝染区;50μg·mL-1AMP处理SMMC-7721细胞24 h后,细胞凋亡率显著增加,从15%增加到55.1%;对凋亡相关蛋白PARP(poly ADPribose polymerase)分析发现,AMP处理SMMC-7721细胞后,PARP出现切割条带,且随着AMP剂量的增加,切割条带显著增强;细胞划痕愈合实验发现AMP能抑制细胞的迁移能力;Transwell实验证实AMP能够显著抑制穿膜细胞数,降低细胞的侵袭能力;检测基底膜降解关键蛋白酶发现,基质金属蛋白酶(matrix metalloproteinase,MMP)-2和9的表达量均减少;进一步分析细胞EMT相关蛋白发现,50μg·mL-1 AMP作用24 h后,E-钙黏蛋白(E-cadherin)表达上调,N-钙黏蛋白(N-cadherin)表达下调;MAPKs家族ERK在AMP处理1 h后达到峰值,JNK的最大活化时间在12 h,而P38在4 h内被AMP激活,2 h磷酸化水平最高,4 h后AMP反而抑制了P38的磷酸化,抑制作用一直持续到24 h,并且AMP对MAPKs家族蛋白的激活具有浓度依赖性。可见,AMP不仅有效抑制SMMC-7721细胞增殖和诱导凋亡,还能显著降低其迁移和侵袭能力,并提示其作用机制可能与激活MAPKs信号通路与逆转肿瘤细胞EMT有关,这为AMP的抗肝癌治疗作用和解析其具体分子机制提供新的思路和理论依据。
关键词:    二氢杨梅素      细胞凋亡      细胞迁移      SMMC-7721细胞      MAPKs信号通路     
Ampelopsin inhibited apoptosis and migration of SMMC-7721 cells through MAPKs signaling pathway
QI Shi-mei1,4, JIANG Qi1, LI Qiang2, FENG Zun-yong3, ZHANG Yu-qing1, DONG Shun-li1, QI Zhi-lin1,4, ZHANG Yao1,4
1. Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu 241002, China;
2. Department of Anatomy, Wannan Medical College, Wuhu 241002, China;
3. Department of Forensic Medicine, Wannan Medical College, Wuhu 241002, China;
4. Department of Biochemistry and Molecular Biology, Wannan Medical College, Wuhu 241002, China
Abstract:
This research is aimed to investigate the effect of ampelopsin on apoptosis and migration of human hepatoma SMMC-7721 cells and explore the molecular mechanism. SMMC-7721 cells were pretreated with different doses of ampelopsin and cells proliferation was detected by CCK8 kit. Cell morphology was observed under an inverted microscope. Nuclear morphology was detected by DAPI staining. Apoptotic rate was detected by Annexin V-FITC/PI flow cytometry. Migration and invasion were detected by Transwell and scratch healing test. Western blotting was used to detect cleavage of poly ADP-ribose polymerase (PARP), expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), E-cadherin, and N-cadherin, and phosphorylation of ERK, P38 and JNK in MAPKs pathway. Our results showed that ampelopsin significantly inhibited proliferation and induced apoptosis of SMMC-7721 cells, with half inhibition dose (IC50) for 24 h was 38.98 μg·mL-1. With 50 μg·mL-1 ampelopsin treatment, typical apoptotic morphological changes occurred, such as cell detachment, shrinkage and nuclear condensation. Apoptotic rate increased from 15% to 55.1%, with PARP cleavage significantly increased. In addition, treatment of ampelopsin reduced scratch healing of cells and transmembrane cells number. The expression levels of MMP-2 and MMP-9 were decreased. Further analysis of EMT-related proteins showed that after ampelopsin treatment, E-cadherin was up-regulated and N-cadherin was down-regulated. During ampelopsin treatment, ERK reached its peak of activation after 1 h, while the maximum activation time of JNK was 12 h. Meanwhile, P38 was activated within 4 h, with the highest point at 2 h. But after 4 h, ampelopsin inhibited phosphorylation of P38. These results indicated that ampelopsin induced apoptosis and reduced migration through activating MAPKs pathway and reversing EMT process in SMMC-7721 cells. This work provides a mechanistic basis for utilizing ampelopsin for anti-hepatocarcinoma treatment.
Key words:    ampelopsin    apoptosis    cell migration    SMMC-7721 cell    MAP kinase signaling pathway   
收稿日期: 2018-12-25
DOI: 10.16438/j.0513-4870.2018-1147
基金项目: 国家自然科学基金资助项目(31301171,81601380,81800082);安徽省2016大学生创新创业训练计划(201610368061);安徽省2017大学生创新创业训练计划(201710368132);活性生物大分子研究安徽省重点实验室项目(1306C083008);安徽高校自然科学研究项目重大项目(KJ2018ZD027,KJ2016SD59);安徽高校自然科学研究项目重点项目(KJ2016A718,KJ2016A734);安徽省优秀青年人才支持计划重点项目(gxyqZD2016173);2018年度皖南医学院重点科研项目培育基金(WK2018Z09).
通讯作者: 章尧,Tel:86-553-3932462,E-mail:zhangyao@ahedu.gov.cn
Email: zhangyao@ahedu.gov.cn
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