积雪草甙诱导肿瘤细胞凋亡及增强长春新碱的抗肿瘤作用

Asiaticoside Inducing Apoptosis of Tumor Cells and Enhancing Anti-tumor Activity of Vincristine

背景与目的:中药积雪草的三萜类成分积雪草甙(asiaticoside,ATS)具有促进胶原蛋白合成作用,对创伤愈合有较好的疗效。近年有文献报道积雪草甙有一定的抗肿瘤活性。本文研究积雪草甙诱导肿瘤细胞凋亡及其与长春新碱(vincristine,VCR)的协同作用。方法:采用MTT法检测积雪草甙单用或合用VCR对KB、KBv200、MCF-7和MCF-7/ADM细胞的增殖抑制作用,流式细胞术分析KB细胞周期和凋亡率的变化,琼脂糖凝胶电泳、荧光显微镜和Westernblot方法观察KB细胞凋亡和细胞周期相关性质改变。结果:积雪草甙对KB、KBv200、MCF-7和MCF-7/ADM细胞的IC50分别为(1.11±0.13)、(1.82±0.08)、(1.58±0.15)mg/ml和(3.25±0.46)mg/ml,且KBv200和MCF-7/ADM细胞对积雪草甙表现出与相应的亲本细胞相近的药物敏感性。经积雪草甙处理后的KB细胞表现出凋亡细胞特征性的改变。积雪草甙与VCR合用对多种肿瘤细胞均有显著协同作用,合并用药组KB细胞凋亡率、Bcl-2蛋白磷酸化水平明显高于单用组,且仅有合用组检测到活化后的caspase-3蛋白。积雪草甙与VCR合用组KB细胞阻滞于S-G2/M期的比率和CyclinB1蛋白的表达水平高于单用组,P34cdc2蛋白表达略有降低。结论:积雪草甙可诱导肿瘤细胞凋亡并与长春新碱显示协同作用,有可能作为生化调节剂应用于肿瘤化?

BACKGROUND &OBJECTIVE: Asiaticoside (ATS), a triterpene extracted from Centella asiatica (L.) Urban, a traditional Chinese herb,possesses good wound healing activities because of its stimulative effect on collagen synthesis. Recently, the anti tumor effect of asiaticiside has been reported. This study was to examine the induction of apoptosis in cancer cells, and the enhancement of vincristine (VCR) cytotoxicity by asiaticoside. METHODS: MTT assay was used to evaluate inhibitory effect of asiaticoside combined with vincristine on proliferation of several cancer cell lines, including KB, KBv200, MCF 7, and MCF 7/ADM. Cell cycle, and apoptosis of KB cells were analyzed by flow cytometry; apoptosis induction was also proved by electrophoresis,and morphologic assessment; the expression of apoptosis , and cell cycle related proteins were determined by Western blot. RESULTS: The IC50 values of asiaticoside for KB, KBv200, MCF 7, and MCF 7/ADM cells detected by MTT assay were (1.11±0.13) mg/ml, (1.82±0.08) mg/ml, (1.58±0.15) mg/ml, and (3.25±0.46) mg/ml, respectively. Multidrug resistant KBv200, and MCF 7/ADM cancer cells displayed similar sensitivity to asiaticoside as their parental counterparts (KB, and MCF 7 cells). Moreover, asiaticoside induced apoptosis in KB cells. At sub cytotoxicity concentration, asiaticoside showed synergistic effect with vincristine in these 4 cell lines. The apoptosis rates were much higher in asiaticoside plus vincristine groups than in vincristine or asiaticoside groups. Bcl 2 phosphorylation levels were higher in the combination groups than in vincristine or asiaticoside alone groups. The activated caspase 3 protein was only presented in the combination groups. Asiaticoside plus vincristine enhanced S G2/M arrest, up regulated Cyclin B1 protein expression, and down regulated P34cdc2 protein expression in KB cells. CONCLUSION: Asiaticoside,as a biochemical modulator,may induce apoptosis,and enhance anti tumor activity of vincristine in cancer cells, might be useful in cancer chemotherapy.