榄香烯乳逆转肺腺癌A549/DDP细胞耐药性及可能作用机制的研究

The efect of elemene reversing the multidurg resistance of A549/DDP lung cancer cells

目的 :探讨榄香烯乳(elemene)逆转人肺腺癌耐顺铂(cisplatin,DDP)细胞A549/DDP的耐药性及可能的作用机制。方法 :采用MTT法检测榄香烯乳单药作用时的细胞毒作用及与DDP联合作用对A549/DDP细胞耐药的逆转作用;Hoechst 33342法进行细胞核染色,荧光显微镜下观察细胞形态的改变;FCM检测细胞凋亡率;蛋白质印迹法检测细胞质中细胞色素C(cytochrome C,cyt C)、pro-caspase-3、caspase-3和抗凋亡因子B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)家族蛋白表达的情况。结果 :不同浓度榄香烯乳对A549/DDP细胞均有一定的抑制作用,呈时间-剂量依赖性效应,联合DDP能提高耐药细胞株A549/DDP对DDP的敏感性而逆转其耐药性。Hoechst 33342法和FCM法检测结果显示,榄香烯乳联合DDP能更大程度上诱导A549/DDP细胞的凋亡,提高耐药细胞的凋亡率,同时还上调A549/DDP细胞质中cyt C、caspase-3和Bad蛋白的表达,而下调pro-caspase-3和Bcl-2蛋白的表达。结论 :榄香烯乳能逆转A549/DDP细胞耐药性可能与其损伤线粒体膜,使线粒体释放cyt C到细胞质、同时活化caspase-3、上调促凋亡蛋白Bad和下调抗凋亡蛋白Bcl-2表达以启动凋亡路径有关。

Objective： To investigate the effect of elemene on the drug resistance of cisplatin （DDP）- resistant human lung adenocarcinoma A549/DDP cells, and its possible mechanism. Methods： The growth inhibition of A549/DDP cells treated with elemene alone or combined with different concentrations of DDP was detected by M-I-l assay. The morphological changes of apoptosis of A549/DDP cells treated with different concentrations of elemene （20 and 40 pg/mL） for 24 h were observed by Hoechst 33342 staing under fluorescence a microscope. The apoptosis rate of A549/DDP cells treated with different concentrations of elemene （20 and 40 μg/mL） after 24 h were detected by FCM （flow cytometry）. The expressions of cytochrome C, pro-caspase-3, caspase-3 and the Bcl-2 family proteins were measured by Western blotting analysis. Results： MI-I result showed that different concentrations elemene could inhibit the proliferation of A549/DDP cells in a time- and dose- dependent manner. Intriguingly, elemene plus DDP enhanced the sensitivity of A549/ DDP cells to DDP and reversed the resistance of A549/DDP cells. Elemene was also a strong inducer of apoptosis in this model system, and a synergistic effect on induction of cell death was observed when the tumor cells were treated with both agents. The result showed that elemene could enhance A549/DDP cell apoptosis. Furthermore, the combination of elemene and DDP also enhanced the protein expressions of cytochrome C, caspase-3 and Bad, and reduced the protein levels of Bcl-2 and pro-caspase-3 in the A549/DDP cells. Conclusion： The reversal of the multi-drug resistance of A549/DDP cell line by elemene may result from its effect on induction of apoptosis. Elemene treatment can impaire mitochondrial membrane, release cytochrome C into cytoplasm, activate caspase-3, up-regulate pro-apoptotic protein Bad and down-regulate anti-apoptotic protein Bcl-2, and finally caused apoptosis.