【摘要】 背景与目的:西米特酸是从我国南海分布广泛的黄色海绵Rhabdastrella sp.中发现的一种异臭椿三萜类化合物。前期研究表明,西米特酸可抑制多种肿瘤细胞的增殖且较安全。本研究探讨西米特酸对人急性白血病细胞HL-60凋亡的影响,及其可能的促凋亡机制。方法:MTT法检测不同浓度西米特酸作用后HL-60细胞的增殖情况,琼脂糖凝胶电泳检测DNA条带,荧光显微镜观察细胞形态学变化。Westernblot法检测西米特酸作用后,细胞中Caspase-3、Poly(ADP-ribose)polymerase(PARP)、Bcl-2、Bax、P73蛋白变化;基因芯片检测凋亡相关基因的表达,逆转录-聚合酶链反应(RT-PCR)对一些凋亡相关基因进行验证。结果:不同浓度西米特酸作用72h可抑制HL-60细胞的增殖,IC50为(0.64±0.21)μg/ml。1μg/ml西米特酸处理36h后,HL-60细胞出现核染色质浓集和DNA梯形条带,并且Caspase-3和PARP出现明显的断裂片段。凋亡相关基因芯片检测发现,P73、JunD、TNFAIP3、GADD45A、PPP1R15A等基因表达增强,MAP2K5、IGF2R等基因表达降低。RT-PCR法检测发现P73、JunD表达上调,MAP2K5、IGF2R表达下调。Westernblot法进一步检测发现P73蛋白表达增高,而Bcl-2和Bax蛋白无明显改变。结论:西米特酸可诱导HL-60细胞凋亡,其机制可能与P73、JunD等基因表达上调和MAP2K5、IGF2R等基因表达下调有关。更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Rhabdastrellic acid-A is an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata from South China Sea. Our previous study indicated that rhabdastrellic acid-A can inhibit the proliferation of many types of tumor cells with minor toxicity. This study was to investigate the apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its possible mechanisms. METHODS: Inhibitory effect of rhabdastrellic acid-A on the proliferation of HL-60 cells was evaluated by MTT assay. DNA fragmentation was analyzed by agarose electrophoresis. Cell morphology was observed under fluorescent microscope. The protein levels of Caspase-3, poly(ADP-ribose) polymerase (PARP), P73, Bcl-2 and Bax were analyzed by Western blot. The expression profile of apoptosis-related genes was analyzed by gene microarray. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to confirm some altered genes identified by gene microarray. RESULTS: Rhabdastrellic acid-A inhibited the proliferation of HL-60 cells and the 50% inhibition concentration (IC50) was (0.64±0.21) μg/ml. When treated with 1 μg/ml rhabdastrellic acid-A for 36 h, condensation of nuclear chromatin of HL-60 cells was observed under fluorescent microscope and DNA fragmentation was observed by agarose electrophoresis. Also, rhabdastrellic acid-A induced cleavage of PARP and Caspase-3. The mRNA levels of 44 genes, including p73, JunD, TNFAIP3 and GADD45A, were up-regulated and the mRNA levels of 16 genes, including MAP2K5 and IGF2R, were down-regulated. The results were further confirmed by RT-PCR. The protein level of P73 was up-regulated after rhabdastrellic acid-A treatment. CONCLUSION: Rhabdastrellic acid-A could induce the apoptosis of HL-60 cells which may be related to the up-regulation of apoptosis-related genes such as p73 and JunD, and the down-regulation of MAP2K5 and IGF2R.更多还原