[关键词]
[摘要]
目的 确定小叶莲中graphislactone A的制备分离方法;探讨graphislactone A诱导人乳腺癌多药耐药细胞MCF-7/ADR凋亡的作用机制。方法 小叶莲药材采用醋酸乙酯超声提取,提取物经过硅胶柱色谱法富集目标成分,再用重结晶法纯化。采用MTT法检测graphislactone A对乳腺癌细胞MCF-7和乳腺癌多药耐药细胞MCF-7/ADR的细胞毒活性。采用流式细胞术、RT-PCR技术和Western blotting法分别检测graphislactone A对乳腺癌多药耐药细胞MCF-7/ADR凋亡、凋亡相关基因及P-糖蛋白(P-gp)蛋白表达的影响。结果 建立了小叶莲中graphislactone A的制备分离方法。Graphislactone A对乳腺癌细胞MCF-7和乳腺癌多药耐药细胞MCF-7/ADR具有细胞毒活性,IC50值分别为2.38、9.35μmol/L,且能够上调乳腺癌多药耐药细胞MCF-7/ADR中p53、bax、caspase-3基因的表达,下调bcl-2基因和P-gp的蛋白表达。结论 Graphislactone A为首次从高等植物中发现的酚类化合物,该制备分离方法效率高、样品回收率高,得到的目标成分纯度高。与阳性药依托泊苷相比,Graphislactone A对乳腺癌细胞MCF-7和乳腺癌多药耐药细胞MCF-7/ADR具有较强的细胞毒活性,且能够诱导其凋亡,其机制可能与p53的表达水平上调、激活caspase-3通路、调节bax和bcl-2的相对比例,下调P-gp的蛋白表达有关。
[Key word]
[Abstract]
Objective To establish the preparative isolation technique of grahislactone A from the fruits of Sinopodophyllum hexandrum, and to investigate the action mechanism for apoptosis induction of graphislactone A in MCF-7/ADR cell line (human breast adencarcinoma cell line). Methods The fruits of S. hexandrum were extracted under ultrasound by EtOAc. Graphislactone A was enriched by silica gel column chromatography, and then purified by recrystallization. The cytotoxic activities of graphislactone A were tested against MCF-7 and MCF-7/ADR cell lines by MTT. The apoptosis of MCF-7/ADR cell line was evaluated by flow cytometry. RT-PCR was used to detect the expression of apoptosis-related genes in MCF-7/ADR cell line. Expression of P-gp in MCF-7/ADR cell line was investigated by western blotting. Results Graphislactone A was isolated from the fruits of S. hexandrum, which showed the potent cytotoxic activities against MCF-7 and MCF-7/ADR cell lines, with IC50 values of 2.38 and 9.35 μmol/L, respectively. With the increased dose of graphislactone A, the expression levels of p53, bax, caspase-3 genes were up-regulated, while bcl-2 and P-gp down-regulated in MCF-7/ADR cell line. Conclusion Graphislactone A as phenolic compoundswas firstly found in higher plants. The preparative isolation has the advantages of high-efficiency, high sample recovery, and high-purity compared with etoposide. Graphislactone A showed strong cytotoxic activities and apoptosis induction action in MCF-7 and MCF-7/ADR cell line.
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[基金项目]
国家自然科学基金资助项目(31300284);河南中医药大学省属高校基本科研业务专项资金项目(2014KYYWF-QN26);河南省高等学校青年骨干教师资助计划(2015GGJS-096);河南中医药大学科技创新人才支持计划(2016XCXRC01)