裂果薯皂苷对人肝癌BEL-7402细胞自噬的影响
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摘要
目的:探讨裂果薯皂苷中化合物Ⅰ(SPHSⅠ)和化合物Ⅱ(SPHSⅡ)对人肝癌BEL-7402细胞自噬的影响,并对其分子机制进行初步探究。方法:采用MTT、Western blotting、Lyso-Tracker Red染色、Magic Red Cathepsin B染色、流式细胞仪分析分别检测SPHSⅠ和SPHSⅡ对人肝癌BEL-7402细胞增殖、自噬和凋亡的影响。结果:SPHSⅠ和SPHSⅡ可抑制BEL-7402细胞增殖并诱导细胞凋亡,同时可抑制细胞自噬,引起自噬标志性蛋白LC3-Ⅰ转变为LC3-Ⅱ,p62蛋白表达无明显变化;加入溶酶体抑制剂氯喹后,SPHSⅠ和SPHSⅡ未能进一步提高用氯喹处理的细胞中的LC3-Ⅱ水平。Lyso-Tracker Red染色、Magic Red Cathepsin B染色及Western blotting检测结果表明,SPHSⅠ和SPHSⅡ可阻断自噬体-溶酶体融合,并通过改变溶酶体酸化和下调溶酶体组织蛋白酶的表达来抑制溶酶体蛋白水解活性。利用Earle′s平衡盐溶液(EBSS)饥饿处理细胞后,SPHSⅠ和SPHSⅡ诱导的细胞凋亡进一步增加。结论:SPHSⅠ和SPHSⅡ可抑制自噬并诱导细胞凋亡,饥饿显著增加人肝癌BEL-7402细胞对SPHSⅠ和SPHSⅡ诱导的细胞凋亡的敏感性。
Objective:To study the effects of compound Ⅰ(SPHSⅠ)and compound Ⅱ(SPHSⅡ)in Schizocapsa plantaginea saponins(SPS)on autophagy in human liver cancer BEL-7402 cells,and to explore its preliminary molecular mechanism.Methods:MTT assay,Western blotting,Lyso-Tracker Red staining,Magic Red Cathepsin B staining and flow cytometric analysis were used to analysis and detect the effect of SPHS Ⅰand SPHS Ⅱ on proliferation,autophagy and apoptosis of human liver cancer BEL-7402 cells,respectively.Results:SPHS Ⅰand SPHS Ⅱ inhibited the proliferation and induced apoptosis of BEL-7402 cells and inhibited autophagy at the same time,induced the transformation of the representative proteins of autophagy LC3-Ⅰinto LC3-Ⅱ,and the protein expression of p62 had no significant change.After joining lysosomal inhibitors chloroquine,SPHS Ⅰ and SPHS Ⅱ failed to further improve the the level of LC3-Ⅱ in the cells treated with chloroquine.The results of Lyso-Tracker Red staining,Magic Red Cathepsin B staining and Western blotting showed that SPHS Ⅰand SPHS Ⅱ could block autophagic-lysosomal fusion and inhibit lysosomalproteolytic activity by changing lysosomal acidification and down-regulating lysosomal tissue protease expression.The cell apoptosis induced by SPHS Ⅰand SPHS Ⅱ was futher increased under the treatment with Earle′s balanced salt solution(EBSS).Conclusion:SPHS Ⅰand SPHS Ⅱ can inhibit autophagy and induce cell apoptosis.Starvation can significantly increase the sensitivity of human liver cancer BEL-7402 cells to apoptosis induced by SPHS Ⅰand SPHS Ⅱ.
Objective:To study the effects of compound Ⅰ(SPHSⅠ)and compound Ⅱ(SPHSⅡ)in Schizocapsa plantaginea saponins(SPS)on autophagy in human liver cancer BEL-7402 cells,and to explore its preliminary molecular mechanism.Methods:MTT assay,Western blotting,Lyso-Tracker Red staining,Magic Red Cathepsin B staining and flow cytometric analysis were used to analysis and detect the effect of SPHS Ⅰand SPHS Ⅱ on proliferation,autophagy and apoptosis of human liver cancer BEL-7402 cells,respectively.Results:SPHS Ⅰand SPHS Ⅱ inhibited the proliferation and induced apoptosis of BEL-7402 cells and inhibited autophagy at the same time,induced the transformation of the representative proteins of autophagy LC3-Ⅰinto LC3-Ⅱ,and the protein expression of p62 had no significant change.After joining lysosomal inhibitors chloroquine,SPHS Ⅰ and SPHS Ⅱ failed to further improve the the level of LC3-Ⅱ in the cells treated with chloroquine.The results of Lyso-Tracker Red staining,Magic Red Cathepsin B staining and Western blotting showed that SPHS Ⅰand SPHS Ⅱ could block autophagic-lysosomal fusion and inhibit lysosomalproteolytic activity by changing lysosomal acidification and down-regulating lysosomal tissue protease expression.The cell apoptosis induced by SPHS Ⅰand SPHS Ⅱ was futher increased under the treatment with Earle′s balanced salt solution(EBSS).Conclusion:SPHS Ⅰand SPHS Ⅱ can inhibit autophagy and induce cell apoptosis.Starvation can significantly increase the sensitivity of human liver cancer BEL-7402 cells to apoptosis induced by SPHS Ⅰand SPHS Ⅱ.
引文
[1] 邱芳龙,周俊,濮全龙.裂果薯的化学成分研究——皂甙甲和乙[J].植物分类与资源学报,1985,7(2):225-231.
[2] 王金妮.利用抗肿瘤药靶——酪氨酸激酶从广西特色中草药中筛选和研究选择性抗肿瘤药[D].南宁:广西医科大学,2012.
[3] 孙悦文.广西特色中草药靶向抗肿瘤筛选及裂果薯皂苷的分离与抗肿瘤作用研究[D].南宁:广西医科大学,2013.
[4] 罗舜仁,邱汉琛,陈燕燕,等.裂果薯皂苷的特征图谱及总皂苷中有效组分的抗肿瘤活性研究[J].天然产物研究与开发,2018,30(2):294-298,245.
[5] Petrizzo A,Mauriello A,Tornesello ML,et al.Cellular prognostic markers in hepatitis-related hepatocellular carcinoma[J].Infectious Agents & Cancer,2018,13(1):10.
[6] Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].Ca A Cancer Journal for Clinicians,2016,66(2):115-132.
[7] Katayama M,Kawaguchi T,Berger MS,et al.DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells[J].Cell Death & Differentiation,2007,14(4):548-558.
[8] Tanida I.Autophagosome formation and molecular mechanism of autophagy.[J].Antioxid Redox Signal,2011,14(11):2201-2214.
[9] Zhou J,Li G,Zheng Y,et al.A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission[J].Autophagy,2015,11(8):1259-1279.
[10] Lao Y,Wan G,Liu Z,et al.The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation[J].Autophagy,2014,10(5):736-749.
[11] Zhou J,Hu SE,Tan SH,et al.Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells[J].Autophagy,2012,8(3):338-349.
[12] Kawai A,Uchiyama H,Takano S,et al.Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells[J].Autophagy,2007,3(2):154-157.
[13] Kaminskyy V,Zhivotovsky B.Proteases in autophagy[J].BBA-Proteins and Proteomics,2012,1824(1):44-50.
[14] Janku F,Mcconkey DJ,Hong DS,et al.Autophagy as a target for anticancer therapy[J].Nature Reviews Clinical Oncology,2011,8(9):528-539.
[15] Mathew R,Karantzawadsworth V,White E.Role of autophagy in cancer[J].Medical Recapitulate,2010,7(12):961-967.
[2] 王金妮.利用抗肿瘤药靶——酪氨酸激酶从广西特色中草药中筛选和研究选择性抗肿瘤药[D].南宁:广西医科大学,2012.
[3] 孙悦文.广西特色中草药靶向抗肿瘤筛选及裂果薯皂苷的分离与抗肿瘤作用研究[D].南宁:广西医科大学,2013.
[4] 罗舜仁,邱汉琛,陈燕燕,等.裂果薯皂苷的特征图谱及总皂苷中有效组分的抗肿瘤活性研究[J].天然产物研究与开发,2018,30(2):294-298,245.
[5] Petrizzo A,Mauriello A,Tornesello ML,et al.Cellular prognostic markers in hepatitis-related hepatocellular carcinoma[J].Infectious Agents & Cancer,2018,13(1):10.
[6] Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].Ca A Cancer Journal for Clinicians,2016,66(2):115-132.
[7] Katayama M,Kawaguchi T,Berger MS,et al.DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells[J].Cell Death & Differentiation,2007,14(4):548-558.
[8] Tanida I.Autophagosome formation and molecular mechanism of autophagy.[J].Antioxid Redox Signal,2011,14(11):2201-2214.
[9] Zhou J,Li G,Zheng Y,et al.A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission[J].Autophagy,2015,11(8):1259-1279.
[10] Lao Y,Wan G,Liu Z,et al.The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation[J].Autophagy,2014,10(5):736-749.
[11] Zhou J,Hu SE,Tan SH,et al.Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells[J].Autophagy,2012,8(3):338-349.
[12] Kawai A,Uchiyama H,Takano S,et al.Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells[J].Autophagy,2007,3(2):154-157.
[13] Kaminskyy V,Zhivotovsky B.Proteases in autophagy[J].BBA-Proteins and Proteomics,2012,1824(1):44-50.
[14] Janku F,Mcconkey DJ,Hong DS,et al.Autophagy as a target for anticancer therapy[J].Nature Reviews Clinical Oncology,2011,8(9):528-539.
[15] Mathew R,Karantzawadsworth V,White E.Role of autophagy in cancer[J].Medical Recapitulate,2010,7(12):961-967.