ABSTRACT
Ursolic acid (UA), a naturally occurring pentacyclic triterpene, is a potent in-vitro anticancer agent, acting through control of growth, apoptosis and differentiation. As the mechanism of its proapoptotic effects on human hepatocellular carcinoma cells has not been extensively studied, we performed an in depth evaluation of the effects of UA on apoptosis in human HepG2 cells. UA was found to inhibit the proliferation of HepG2 cells in a concentration and time-dependent manner. After treatment, cells showed evidence of activation of apoptosis, including the presence of apoptotic bodies and DNA fragmentation. UA-induced apoptosis was accompanied by a significant decrease in bcl-2 and survivin expression, with the corresponding ratio of bax/bcl-2 increased. The treatment with UA also increased the protein level and enzymatic activity of caspase-3. Z-DEVD-fmk, a specific caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and the DNA fragmentation induced by UA, demonstrating the requirement for caspase-3 activity in UA-induced apoptosis. Inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway was also involved, as inhibition of PI3K by LY294002 significantly increased UA-induced apoptosis. Kinetic experiments indicated that UA downregulated PI3K/p85 subunit (PI3K/p85) and phospho-Akt, before downregulating survivin. The further results also confirmed that LY294002 not only downregulated survivin alone, but considerably enhanced the repression of survivin combined with UA. UA therefore seemed to downregulate the expression of survivin by blocking PI3K/Akt. Taken together, the data suggest that the proapoptotic effect of UA on HepG2 cells is mediated by activation of caspase-3, and is highly correlated with inactivation of PI3K/Akt/survivin pathway.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Microtubule-Associated Proteins/drug effects , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Caspase 3/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Humans , Inhibitor of Apoptosis Proteins , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Microtubule-Associated Proteins/metabolism , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Survivin , Time Factors , Triterpenes/administration & dosage , Ursolic AcidABSTRACT
We previously reported that over-expression of SMYD3, a histone H3-K4 specific di- and tri-methyltransferase, plays a key role in cell viability, adhesion, migration and invasion. In this study, we investigated the mechanisms underlying these phenomena and found that knocking down SMYD3 expression in tumor cells significantly reduced the biological function of HGF and inhibited carcinoma cells migration and invasion. Due to the fact that the proto-oncogene c-Met encodes the high-affinity receptor for HGF, and the HGF-c-Met signaling plays a critical role in the tumor genesis, we further identified the partial correlation between SMYD3 and c-Met. The results showed that high expression of c-Met accompanied with over-expression of SMYD3. Silencing SMYD3 expression in tumor cells by specific shRNAs down-regulated c-Met gene transcription, while over-expressing SMYD3 induced c-Met transcription. Moreover, we demonstrated here that two SMYD3 binding sites within the c-Met core promoter region were significant in the transactivation of c-Met. The present findings provide significant insights into the epigenetic regulatory mechanisms of oncogene c-Met expression, and develop the strategies that may inhibit the progression of cancer migration and invasion.