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目的:研究白花地胆草(Elephantopus mollis H.B.K)的乙醇提取物EM-3抗肿瘤作用分子机制。方法:MTT、克隆形成抑制和细胞划痕实验检测EM-3对鼻咽癌细胞增殖、迁移能力的影响;Annexin V-FITC/PI双染法检测细胞凋亡;PI单染检测细胞周期;超速流式分选细胞仪检测鼻咽癌CNE2-S18肿瘤干细胞样SP细胞(side population cell)比例;Western blotting检测细胞凋亡、周期、侵袭迁移及肿瘤干细胞相关蛋白表达变化。结果:MTT、克隆形成抑制和细胞划痕实验结果表明,EM-3可以显著抑制鼻咽癌细胞的增殖,随着药物浓度的增大,细胞克隆数逐渐减少,体积逐渐变小,而且能够显著抑制鼻咽癌细胞的迁移;流式细胞术结果表明随着药物浓度的增加,凋亡率逐渐增加,并且G_2/M期细胞比例逐渐增加;超速流式分选细胞仪结果表明,EM-3可以显著降低CNE2-S18肿瘤干细胞样SP细胞的比例;Western blotting结果表明,随着药物浓度的增加,x IAP、Bcl-2、Cyclin D1、MMP2(药物高浓度)、MMP9、p-Met、Oct4(药物高浓度)及Sox2蛋白表达减少,而Cyclin B1、Bax蛋白表达增多,并伴随Caspase-9、Caspase-3活化及多聚ADP核糖聚合酶PARP酶切失活。结论:EM-3通过抑制Stat3通路诱导鼻咽癌细胞发生凋亡,并诱导G_2/M期阻滞。此外,EM-3经MMPs途径抑制鼻咽癌细胞迁移,同时可以有效降低CNE2-S18肿瘤干细胞样SP细胞干性。
Objective: To study the anti-tumor molecular mechanism of ethanol extract (EM-3) from Elephantopus mollis H.B.K. Methods: The effect of EM-3 on the proliferation and migration of nasopharyngeal carcinoma cells was detected by MTT assay, inhibition of cell cloning and cell scratch assay. Cell apoptosis was detected by Annexin V-FITC / PI double staining. Flow cytometry was used to detect the proportion of CNE2-S18 tumor-bearing side population cells. Western blotting was used to detect the changes of apoptosis, cell cycle, invasion and migration and the expression of tumor stem cell related proteins. Results: The results of MTT, clonogenic inhibition and cell scratch assay showed that EM-3 could significantly inhibit the proliferation of NPC cells. With the increase of drug concentration, the number of cell clones decreased, the volume became smaller, The results of flow cytometry showed that the apoptotic rate increased gradually with the increase of drug concentration, and the proportion of cells in G 2 / M phase increased gradually. The results of ultracentrifugation flow cytometry showed that EM-3 Western blotting results showed that x IAP, Bcl-2, Cyclin D1, MMP2 (high drug concentration), MMP9, p-Met, Oct4 (High concentration of drug) and Sox2 protein expression, while Cyclin B1, Bax protein expression increased, accompanied by Caspase-9, Caspase-3 activation and poly ADP ribose polymerase PARP enzyme inactivation. Conclusion: EM-3 can induce apoptosis of NPC cells by inhibiting Stat3 pathway and induce G 2 / M arrest. In addition, EM-3 inhibits the migration of nasopharyngeal carcinoma cells through the MMPs pathway, and at the same time can effectively reduce the stem cell-like SP cells of CNE2-S18 tumor.