Anti-hepatoma Activities And Mechanisms Study Of 5,6-dihydroxy-3,7,4’-trimethoxyflavonol

Hepatocellular carcinoma (HCC) is one of the most common primary tumors in adults with a high mortality rate and poor prognosis worldwide. Chemotherapy, radiotherapy and surgery are the common strategy against hepatocarcinoma currently. Due to the rapid proliferation of cancer cells, frequent acquisition of drug-resistant phenotypes and the occurrence of secondary malignancies, chemotherapy sometimes is failure. Therefore, the development of novel therapeutic agents becomes greatly in need. Natural plants products are very valuable sources of anticancer agents. 5,6-dihydroxy-3,7,4’-trimethoxyflavonol (AH5) is isolated from the Chinese herb, Aster himalaicus, which has long been used as Chinese folk medicine to treat snakebites, fever, cold, tonsillitis and pneumonia. The study was undertaken to investigate the effects and mechanisms that underlie the anti-cancer activity of 5,6-dihydroxy-3,7,4’-trimethoxyflavonol in HCC cells.We first assessed the growth inhibitory effect of AH5 using an MTT colorimetric assay.AH5 exhibited growth inhibitory effects in all cell lines tested, but lower cytotoxicity in normal cells with higher IC50 values in comparison to those of the HCC cells. The exposure of HCC cells to AH5 decreased the viability in a dose- and time-dependent manner.Then the flow cytometic analysis showed that the proportion of cells arrested in G2/M phase was remarkably increased in HCC cells treated with AH5. The activity of the cyclin B/cdc2 complex is controlled in G2/M phase and is required for entry into mitosis in eukaryotes. AH5 decreased the expression of cyclin B and induced the phosphorylation of cdc2 in HepG2 cells.We performed further tests to evaluate whether the AH5-induced cytotoxic effect was related to apoptosis. Apoptotic characteristics were observed by DAPI staining in HepG2 cells. The morphological changes were obvious, including the formation of apoptotic bodies and nuclear condensation. Meanwhile, the quantitative analysis of hepatocarcinoma cells apoptosis were monitored throng Annexin V-FITC and PI double staining by flow cytometry. The analysis revealed that the proportion of cells stained with Annexin V increased in a dose-dependent manner in all three AH5-treated cell lines. Moreover, the cleaved forms of caspase-9 and -3 were notably increased in AH5-treated HepG2 cells, confirming that AH5 induces apoptosis in hepatocarcinoma cells. HepG2 cells which expressed wild-type p53 was sensitive to AH5.Then we study the anti-cancer mechanisms of AH5 in HepG2 cells. Western blot and RT-PCR analysis were used to investigate the expression of p53 and p21 Waf1/CiP1 AH5 increased the mRNA and protein levels of p53 and its target gene p21 Waf1/CiP1 in HepG2 cells. Moreover, the G2/M phase arrest induced by AH5 in HepG2 cells could partially reverse by PFT-α, a specific transcriptional inhibitor of p53. Above results showed that, p53-mediated cell cycle arrest was involved in anti-cancer mechanisms of AH5.Apoptosis can be induced by two distinct pathways:the mitochondria-and the death receptor-mediated pathways. In the present study, results showed that AH5 induced a substantial loss of mitochondrial membrane potential in HepG2 cells. Moreover, cytochrome c was released from mitochondria upon AH5 treatment as revealed by western blotting. AH5 treatment also led to the activation of caspase-9 and the downstream effector caspase-3. All of these results indicated that AH5 can activate the mitochondria-mediated apoptotic pathway in HepG2 cells.Many studies have demonstrated that p53 is an important nuclear transcription factor and transactivates multiple genes involved in apoptosis. The pro-apoptotic protein Bax is one of the critical downstream mediators of p53 signaling. P53 can activate the transcription of Bax gene and locate to mitochondria to direct enhance the activation of Bax. The imbalance between the Bax and Bcl-2 contributes to the mitochondria-mediated apoptosis. Here we demonstrate that AH5 induced the mRNA and protein expression of p53 and Bax. The expression of Bcl-2 was also repressed by AH5. The levels of both p53 and Bax were increased and accumulated in the mitochondria. Moreover, the reduction of cell viability, the loss of mitochondrial membrane potential and the apoptotic ratio induced by AH5 could be partially reversed by specific inhibitors of p53. Neither PFT-α nor PFT-μ. fully suppressed AH5-induced cell death and apoptosis. All these findings indicated that AH5-induced apoptosis in HepG2 cells requires p53 activation and both the transcription-dependent and -independent mechanisms of p53 are involved.In conclusion, AH5 inhibits HepG2 cells proliferation through p53-mediated G2/M arrest and mitochondria-mediated apoptosis, which is associated with both p53-mediated transcription-dependent and -independent pathways. The current status and future advancement of AH5 will be useful for the development of chemotherapeutic agents against liver cancer.