Study Of Histone Deacetylase Inhibitor Effects And Its Mechanism Of Human Liver Carcinoma

Backgroud: The acetylation of histones plays very important role in expression of gene. Histones are a kind of nucleoprotein which are tightly combined with DNA. One of the most important mechanisms determining the activity of target genes is the posttranslational modification of the N-terminal tails of core histones by acetylation and subsequent changes in chromatin structure. Therefore, histones not only pack the DNA, but also enlarge the message of gene. Acetylation of histones holds vital part of epigenetics theory. By opinion of epigenetics theory, the changes of gene expression can take place without a variety of DNA structure.The acetylation of histones has connection immediacy with the induction of gene expression. There are many spots where can be acetylated or deacetylated. But the target genes have fixed location with the acetylation or deacetylation of histones. So we can say the acetylation of histones happens on target genes.The study found that there was unbalance between histones acetylation and deacetylation in many soild tumor such as lung cancer, prostate cancer, glioma and so on. The increase of HDACs and the decrease of HATs lead to that. The potential benefit of HDAC inhibition has been established by the use of enzyme inhibitor in vitro. In lung cancer, cervical cancer, prostate cancer, galactophore cancer, head and neck cancer, the therapeutic value ofHDACs inhibition has been tested. Local remodeling of chromatin and dynamic changes in nucleosomal packaging of DNA are key steps in the regulation of gene expression and consequently affect proper cell function, differentiation and proliferation. Controlling and regulation histones acetylation is new attempt as a therapeutic target of tumor. In 1998,Warrell used NaPB and RA in a patient of APL and had clinical success, which approved the effect of HDACs inhibition on tumor for the first time.From then on, as HDACs inhibition, NaPB was used for induction and differention in malignant cells. With the study accumulating, datam sufficiently evidenced the effect of NaPB on solid tumour in vitro and in vivo. Furthermore clinical success had been gained in glioma, lung cancer and prostate cancer. Being compared with other traditional chemical agents, NaPB has particular advantages: low toxic, broad growth inhibitory properties, novel differentiation inducer. Otherwise, NaPB can boost up effects of other chemical agents when they are used together.As known as well, liver carcinoma is one of familiar malignant tumour which has low diagnosis rate and high metastasis chance. Liver carcinoma is short of chemistry drugs sensitivity. As a result, liver carcinoma is difficult to cure in clinic and has high death rate. Therefore it is emergent for us to do something. To investigate effective measure of dealing with liver carcinoma has vital value both in science and society. In our country there had a little datum about effect of HDACs inhibitor on tumour, maybe because investigators did not pay much attention to this field. We selected human liver carcinoma cell lines Bel-7402 and HepG2, then treated with HDACs inhibitor NaPB. In vitro experiments we observed the effect of NaPB on growth arrest, cellular differentiation, inducement, apoptpsis and probed intothe mechanism. We were interested in evaluating the effect of NaPB on liver carcinoma. Intended to estimate the possibility and foreground of NaPB in treating liver carcinoma. We wished to supply new attempt and to offer potent method.Objective: To investigate the influence of HDACs inhibitor NaPB on inducement of liver carcinoma cell lines and detect the probable mechanism. Observed the changes in expression of anti-oncogene (tumor suppressor gene), factors of apoptpsis and histones acetylation after being treated with NaPB in liver carcinoma cell lines. Our purpose was in confirmation of relation between histones acetylation and liver carcinoma. To afford evidences of NaPB's effect on liver carcinoma. The first part: To explore the effects of NaPB which is the inhibitor of the histone deacetylase on proliferation and phase growth arrest in human liver carcinoma cell lines. Observed the directly influence on configuration of liver carcinoma cell lines in vitro. The second part: To study the mechanism of NaPB on restraining proliferation and inducing apoptosis in human liver carcinoma cell lines. Intented to seek the relation between antioncogene and NaPB. To argue the route of apoptosis in human liver carcinoma cell lines induced by NaPB. The third part: To explore the effects of sodium phenylbutyrate which is the inhibitor of the histone deacetylase on histones acetylation and its significance in human liver carcinoma cell lines. To investigate the mRNA expression of HDACl and HDAC4, furthermore to analyze their function in human liver carcinoma cell lines.Methods: The first part: Carcinoma cells Bel-7402 and HepG2 were treated with sodium phenylbutyrate at different concentration (2,4,6,8mM). Cells not treated with NaPB were used as controls. MTT assay was adoptedto describe the proliferation of carcinoma cells. Light microscopy was used to find morphological changes in carcinoma cells. Further evaluation for cell cycle and early apotosis was determined by flow cytometry. Untreated and cells treated with NaPB were labeled with propidium iodide and annexin V-fluorescein isothiocyanate and evaluated for induction of apoptosis. The second part: Carcinoma cells Bel-7402 and HepG2 were treated with Sodium phenylbutyrate at different concentration (2,4,6,8mM). Expression of p21WAF1/CIP1, p27 and p53 was determined by RT- PCR and cellimmunochemistry. The level of protein including Bax, P21WAF1/CIP1, P27 andP53 was determined by cell immunochemistry. The third part: Carcinoma cells Bel-7402 and HepG2 were treated with Sodium phenylbutyrate at different concentration. RT-PCR was adopted to study the mRNA expression of HDAC1 and HDAC4 in carcinoma cells. Western blot was used to inspect acetylation of histone H4(lys5).Results: The first part: We found that carcinoma cells Bel-7402 andHepG2 treated with 2mM concentrations of NaPB for 96h underwent a low-grade decrease in cell growth. Higher concentration of NaPB resulted in more pronounced growth inhibition at earlier time points. The cells grew slowly with long spindle connection between adjacent cells. Cells were then counted. The number of cells in treated group was significantly less than the number of cells in the control group. NaPB treatment caused a time- and dose-dependent inhibition in carcinoma cells Bel-7402 and HepG2 proliferation. Using flow cytometry, we found a significant increase in the number of cells in the G1 phase and a decline in the fraction of S-phase cells by 24h in Bel-7402 and HepG2 cells treated with 4mM concentrations of NaPB. A detectable increase in apoptosis rate of HepG2 and Bel-7402 wasfound too. The power of forming clone in Bel-7402 and HepG2 cells was reduced by NaPB treatment for 1 week (93%, 37%; 96%,44%). The second part: NaPB treatment led to increasing mRNA expression of P21 WAF1/CIP1 and P27 but had no affection on the expression of P53in human liver carcinoma cells Bel-7402 and HepG2 with control cells. NaPB inhanced synthsizing protein of p21WAF1/CIP1,P27 and Bax in human liver carcinoma cells Bel-7402 and HepG2 NaPB could induce the expression of p21WAF1/CIP1 independent of the P53 route in human liver carcinoma cells Bel-7402 and HepG2. The third part: The mRNA expression of HDAC1 and HDAC4 in carcinoma cells Bel-7402 and HepG2 was high. NaPB treatment did not cause a decline in the mRNA expression of HDACl and HDAC4 in carcinoma cells Bel-7402 and HepG2. NaPB treatment caused an increase of histones acetylation in carcinoma cells Bel-7402 and HepG2.Conclusions: NaPB treatment could inhibit proliferation of human liver carcinoma cells Bel-7402 and HepG2 , cause partial differentiation and led to partial apoptosis through inducing the expression of P21 WAF1/CIP1, p27 and Bax . NaPB affected differential antioncogene, such as p21WAF1/cip1 and P27.But the NaPB had little impact of P53. Sodium phenylbutyrate treatment could not inhibit mRNA expression of HDACl and HDAC4 in carcinoma cells. At the same time NaPB treatment inhanced histones acetylation.