| Summary: | 碩士 === 國立臺灣大學 === 藥學研究所 === 92 === In this study, we have carried out an anticancer screening test in numerous components derived from several natural sources by sulforhodamine B (SRB) assay. We found that the prostanoid-like extract from Clavularia viridis displayed effective anti-proliferative and cytotoxic activities in human hepatocellular carcinoma Hep3B cells. Bromovulone Ⅲ, Chlorovulone Ⅱand Chlorovulone Ⅲ were the most effective compounds with 50% inhibition of growth (GI50) of 0.55 µM, 1.73 µM, and 0.83 µM, respectively. The anticancer effects of several prostaglandins (PG) were compared while only PGA1 and PGD2 showed modest effect by SRB assay, indicating the superiority of marine prostanoid-like extracts from Clavularia viridis. The prostanoid-induced DNA condensation was identified by Hoechst33342 staining technique in Hep3B cells. The data of electron microscopy also show the apoptotoc body formation. By using flow cytometry to detect the progression of cell cycle, we found that the population in subG1 phase dramatically increased in response to Bromovulone Ⅲ for 48 hours. All of these results reveal that Bromovulone Ⅲ is able to induce apoptotic cell death in Hep3B cells. By using Western Blotting analysis to investigate the expressions of Bcl-2 family of proteins and caspase cascades, the data showed that Mcl-1 but not the other Bcl-2 family proteins was up-regulated in response to Bromovulone Ⅲ stimulus. Furthermore, the caspase-related signaling pathways were also examined in this study. The data demonstrated that Bromovulone III had little influence on caspase-3, -6, -8 and -9, while significantly induced the expression of XIAP and activation of m-calapin, caspase-7 and -12 in Hep3B cells. It is worth noting that Bromovulone III could also induce the increase of expression of the transcription factor, GADD153/CHOP. Since caspase-12 and GADD153/CHOP are two biological markers activated in cells under endoplasmic reticulum (ER) stress, it is suggested that the apoptotic death in Hep3B cells induced by Bromovulone III is mediated through the ER stress. In this study, four other apoptotic signals were also examined, such as cytoplasmic calcium distribution, mitochondrial membrane potential and mass and plasma membrane potential. Besides, mitochondria swelling and ER-stress were detected by using electron microscopy methods. The data showed that Bromovulone Ⅲ could induce the increase of cytoplasmic calcium concentraction and mitochondrial mass, the depolarization of mitochondrial membrane potential and the hyperpolarization of plasma membrane potential. Taken together, it is suggested that Bromovulone Ⅲ can effectively induce the apoptotic death in Hep3B cells through ER stress-mediated activation of m-calapin, caspase-7 and -12, and the increase of protein expression of GADD153/CHOP.
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