葫芦素I对肝癌细胞生长的抑制作用及其与STAT3通路 相关抗凋亡因子的关系
| 作者: |
1王成志,
2彭元亮,
1史夏青,
3周晓庆,
1杨满意,
1赵劲风,
1廖明媚
1 中南大学湘雅医院卫生部纳米生物技术重点实验室，湖南 长沙 410008 2 中南大学生命科学学院，湖南 长沙 410078 3 中南大学湘雅医院神经内科，湖南 长沙 410008 |
| 通讯: |
廖明媚
Email: rosnow@163.com |
| DOI: | 10.3978/.2018.01.010 |
| 基金: | 中南大学中央高校基本科研业务费专项资金资助项目（2017zzts391）；国家自然科学基金资助项目（81302075；81272193；81402001）。 |
摘要
目的：探讨葫芦素I对肝癌细胞生长的抑制作用及机制。
方法：采用CCK-8法检测葫芦素I对不同肝癌细胞（HepG2、QGY-7703、SMMC-7721）增殖活力的影响；葫芦素I处理HepG2细胞后，分别用细胞平板克隆形成实验、流式细胞术、Hochest 33342染色观察细胞克隆形成、细胞周期、凋亡情况，并用Western blot和qRT-PCR检测抗凋亡因子及其相关信号分子蛋白和mRNA表达变化。
结果： 葫芦素I处理后，不同肝癌细胞的增殖均明显抑制，并呈时间与浓度依赖性（均P<0.05）；葫芦素I对HepG2、QGY-7703、SMMC-7721细胞48 h的IC50值分别为0.19、4.16、1.13 μmol/L。HepG2细胞经葫芦素I处理24 h后，克隆形成几乎被完全抑制，出现典型的细胞凋亡形态变化和明显的G2期阻滞；抗凋亡因子Mcl-1、survivin以及转录因子STAT3蛋白与mRNA的表达均明显下调，mRNA半定量分析显示差异均有统计学意义（均P<0.05）。
结论：葫芦素I抑制肝癌细胞的生长，其作用机制可能与STAT3通路下调抗凋亡相关蛋白表达，从而增加细胞凋亡有关。
关键词：
癌，肝细胞；葫芦素类；细胞凋亡；STAT3转录因子；凋亡调节蛋白质类
方法：采用CCK-8法检测葫芦素I对不同肝癌细胞（HepG2、QGY-7703、SMMC-7721）增殖活力的影响；葫芦素I处理HepG2细胞后，分别用细胞平板克隆形成实验、流式细胞术、Hochest 33342染色观察细胞克隆形成、细胞周期、凋亡情况，并用Western blot和qRT-PCR检测抗凋亡因子及其相关信号分子蛋白和mRNA表达变化。
结果： 葫芦素I处理后，不同肝癌细胞的增殖均明显抑制，并呈时间与浓度依赖性（均P<0.05）；葫芦素I对HepG2、QGY-7703、SMMC-7721细胞48 h的IC50值分别为0.19、4.16、1.13 μmol/L。HepG2细胞经葫芦素I处理24 h后，克隆形成几乎被完全抑制，出现典型的细胞凋亡形态变化和明显的G2期阻滞；抗凋亡因子Mcl-1、survivin以及转录因子STAT3蛋白与mRNA的表达均明显下调，mRNA半定量分析显示差异均有统计学意义（均P<0.05）。
结论：葫芦素I抑制肝癌细胞的生长，其作用机制可能与STAT3通路下调抗凋亡相关蛋白表达，从而增加细胞凋亡有关。
Inhibitory effect of cucurbitacin I on hepatocellular carcinoma cells and its relation with anti-apoptotic factors related to STAT3 signaling pathway
CorrespondingAuthor:LIAO Mingmei Email: rosnow@163.com
Abstract
Objective: To investigate the influence of cucurbitacin I on growth of hepatocellular carcinoma (HCC) cells and the mechanism.
Methods: The influences of cucurbitacin I on proliferative activities of different types of HCC cells (HepG2, QGY-7703 and SMMC-7721) were measured by CCK-8 assay. In HepG2 cells after treatment with cucurbitacin I, the colony formation, cell cycle profile and apoptosis were examined by plate colony-forming assay, flow cytometry and Hochest 33342 staining, and the protein and mRNA expressions of anti-apoptotic factors and their related signaling molecules were determined by Western blot analysis and qRT-PCR method, respectively.
Results: The proliferations of all the selected HCC cells were significantly inhibited by cucurbitacin I treatment, with a time- and concentration-dependent manner (all P<0.05), and the 48-h IC50 value of cucurbitacin I for HepG2, QGY-7703 and SMMC-7721 was 0.19, 4.16 and 1.13 μmol/L, respectively. In HepG2 cells after cucurbitacin I treatment for 24 h, the clone formation was almost completely suppressed, typical apoptotic morphological changes and evident G2-phase arrest were presented, and both protein and mRNA expressions of the anti-apoptotic factors Mcl-1 and survivin as well as the transcription factor STAT3 were markedly down-regulated, and the semi-quantitative analysis of mRNA expressions showed all differences had statistical significance (all P<0.05).
Conclusion: Cucurbitacin I can suppress the growth of HCC cells, and the mechanism may be associated with its down-regulating the expressions of anti-apoptotic factors through STAT3 signaling pathway, and thereby inducing cell apoptosis.
Keywords：
Carcinoma
Hepatocellular; Cucurbitacins; Apoptosis; STAT3 Transcription Factor; Apoptosis Regulatory Proteins
Methods: The influences of cucurbitacin I on proliferative activities of different types of HCC cells (HepG2, QGY-7703 and SMMC-7721) were measured by CCK-8 assay. In HepG2 cells after treatment with cucurbitacin I, the colony formation, cell cycle profile and apoptosis were examined by plate colony-forming assay, flow cytometry and Hochest 33342 staining, and the protein and mRNA expressions of anti-apoptotic factors and their related signaling molecules were determined by Western blot analysis and qRT-PCR method, respectively.
Results: The proliferations of all the selected HCC cells were significantly inhibited by cucurbitacin I treatment, with a time- and concentration-dependent manner (all P<0.05), and the 48-h IC50 value of cucurbitacin I for HepG2, QGY-7703 and SMMC-7721 was 0.19, 4.16 and 1.13 μmol/L, respectively. In HepG2 cells after cucurbitacin I treatment for 24 h, the clone formation was almost completely suppressed, typical apoptotic morphological changes and evident G2-phase arrest were presented, and both protein and mRNA expressions of the anti-apoptotic factors Mcl-1 and survivin as well as the transcription factor STAT3 were markedly down-regulated, and the semi-quantitative analysis of mRNA expressions showed all differences had statistical significance (all P<0.05).
Conclusion: Cucurbitacin I can suppress the growth of HCC cells, and the mechanism may be associated with its down-regulating the expressions of anti-apoptotic factors through STAT3 signaling pathway, and thereby inducing cell apoptosis.