阿里山十大功勞 ( Mahonia oiwakensis ) 對於人類肺癌細胞凋亡作用在活體外( in vitro )及活體內( in vivo ) 的研究

Abstract

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis ( MOE ), an endemic selected species in Taiwan, and investigated their effects on various human lung cells. Amount the four selected experimental cell lines, including A549, H1299, H1355 and H225, We found that A549 was the most sensitive cell line to MOE. The MTT assay showed decreased viability of all four kinds of malignant cells we chose in dose- dependent manner under treatment of MOE, but no influences on normal lung tissue cell line such as MRC-5. Interestingly, A549 is also the only normal expression of p53 in those four selected cell lines. So A549 was chosen to proceed following experiment. The data showed that MOE-induced apoptotic death in human A549 non-small cell lung carcinoma ( NSCLC ) in a dose- dependent manner. About the cell- cycle study, we treated with MOE also caused an increase in the sub-G1 fraction of cells, G1- phase arrest and decreased with S- phase population simultaneously. The morphology of MOE- treated A549 cells demonstrated the characteristics of the apoptotic procedures, such as sell shrinkage, cell surface blebbing, nuclear condensation and formation of apoptotic bodies. The mitochondrial- mediated pathway was implicated in this MOE-induced apoptosis as evidenced by activation of the caspase cascade 3,8 and 9, cleavage of poly ( ADP-ribose ) polymerase ( PARP ), disruption of mitochondrial membrane potential, and then release of cytochrome C in cytosol. A higher ratio of Bax/ Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced A549 cell apoptosis. Surprisingly, we surveyed if the extrinsic apoptotic pathway play a role in the MOE- induced apoptosis of A549 tumor cell. The Fas/ CD95 and Fas ligand were both increased in MOE- treated A549 cell line. However, the same reaction did not show in the TRAIL, DR4 and DR5 receptors. We speculated that MOE- induced apoptosis of A549 cells though both the intrinsic and extrinsic apoptotic pathway. Furthermore, we prescribed the pan- caspase inhibitor z-VAD-fmk to A549 cells before treating with MOE. The reversal of apoptosis was noted in our experiment. Then, in vivo experiment of A549 tumor-xenografted nude mice, MOE also restricted proliferation (P<0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P<0.05) and increased transferase-mediated dUTP nick-end labeling ( TUNEL )-positive staining (P<0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo though both intrinsic and extrinsic apoptotic pathway, suggesting that it may have therapeutic potential against human lung cancer.