Researches On The Novel Histone Deacetylase Inhibitor And The Anti-tumor Molecular Mechanisms

Guided by the epigenetic theories, we screened a series of synthesized compounds which might be novel HDAC inhibitors. We evaluated the inhibition on HDAC and HDAC-1of the compounds and investigated the anti-proliferation effects on different tumor cell lines of them. We discovered a HDAC inhibitor with effective inhibition of HDAC and HDAC-1, which might be used for novel and potent antitumor treatment.The Elisa and fluorescent substrate assays were used for the evaluation on the inhibition of HDAC and HDAC-1of the compounds. The MTT assay was used to observed the anti-proliferation on different tumor cell lines such as MCF-7、HCT116、A549、Hela and K562. The results indicated that part of the compounds inhibited the activity of HDAC or HDAC-1and exhibited the anti-tumor activity depending on the chemical structures.Combined with the results on HDAC inhibition and anti-proliferation, we selected16#.22#and29#which effective inhibited the activity of HDAC and the proliferation of tumor cell lines for further investigation. We found all of16#,22#and29#could arrest the cell cycle and induced apoptosis in Hela cells, and the total apoptosis of16#was the highest among the3compounds. Mechanism researches exhibited that the antitumor effects of16#,22#and29#were relative to the up regulation of p21cip/WAF1.We investigated the effects of DWP0016on glioma cell line U251and neuroblastoma cells SH-SY5Y. The MTT results showed that DWP0016effectively inhibited the proliferation of U251and SH-SY5Y cells but not affect the proliferation of normal cells L-02, which indicated the anti-glioma potential of DWP0016. The Hoechst staining. DMA fragmentation and flowcytometry results showed that DWP0016induced apoptosis in U251and SH-SY5Y cells, arrested cells at G1/S phase phase. The caspase-3dependent apoptosis was identified by caspase activity test. Real time PCR and western blotting results showed that the cell cycle block was relative to the activation of p21cip/WAF1and the suppression of cyclin D and cyclin E. Anti-tumor gene p53was found to play an important role in the mechanisms of cell cycle arrest and apoptosis induced by DWP0016in U251cells. DWP0016up-regulated the transcription and expression of p53and PUMA, companied by the acetylation on p53. Moreover, DWP0016triggered the mitochondria apoptosis pathway as result of p53activation. After silencing p53by specific siRNA, the inhibition of growth, cell cycle arrest and apoptosis induced by DWP0016were totally blocked. These results indicated that p53was a dominant target gene in DWP0016-treated U251cells. Collectively, DWP0016induced G1/S phase cell cycle and apoptosis by p53activation in U251glioma cells.Mechanisms research showed that DWP0016didn’t affect the expression of p53but significantly up regulated the expression of PTEN. Moreover, DWP0016stimulated the activity of the PTEN promoter, which was relative to the up-regulation on PTEN transcriptor Egr-1. In addition, the up-regulation of PTEN led to the inhibition on the phosphorylation of PI3K and Akt, which directly inhibited PI3K/Akt cell signal transduction pathway. The key role of PTEN in SH-SY5Y cells was identified by using gene silencing technique, which resulted in total block of the inhibition of growth, cell cycle arrest and apoptosis induced by DWP0016treatment. Collectively, DWP0016induced phase cell cycle and apoptosis by PTEN activation, companied by the inhibition on PI3K/Akt pathway in SH-SY5Y neuroblastoma cells.Then we constructed xenograft animal models to observe the antitumor activity in vivo. The results displayed that DWP0016significantly inhibited the growth of tumor in the xenograft animal models, which exhibited better efficacy than SAHA. Importantly, the treatment by DWP0016exhibited no general toxicity. Immunochemistry results showed DWP0016down-regulated the expression of HDAC-1and up-regulated the expression of Ac-H3and PTEN in the tumor specimens from animal models, which indicated the inhibition of HDAC&HDAC-1and activation of PTEN in vivo.In summary, we screened a series of novel HDAC inhibitor, made a study of the antitumor activity of typical compounds and investigated the molecular regulation mechanisms in U251cells and SH-SY5Y cells. These studies explored the effects on different genes and pathways by HDAC inhibitors in vitro and in vivo, which facilitated further optimization and application for the development of novel HDAC inhibitors.