Anticancer Activities Of Histone Deacetylase And DNA Methyltransferase Inhibitors On Pancreatic Cancer

Pancreatic cancer is one of the most malignant gastrointestinal tumors,characterized by a poor prognosis,absence of specific clinical symptoms and signs,prone to metastases and lack of response to conventional therapy.The morbidity and mortality of this disease are increasing in the whole worldwide.It is important to go on basic research and identify new therapeutic agents for fighting this devastating disease.There is growing evidence that gene expression govemed by epigenetic changes is crucial to the onset and progression of cancer.Epigenetics is the study of heritable changes in gene expression that are not coded in the DNA sequence itself.The best studied epigenetic mechanisms are DNA methylation and post-translational histone.DNA methylation and histone deacetylation are interconnected in gene silencing.Furthermore,a much more direct connection between DNA methylation and histone deacetylation exists by direct interactions between DNMTs and HDACs.HDAC and DNMT inhibitors have emerged as the accessory therapeutic agents for multiple human cancers,since they can block the activity of specific HDACs and DNMTs,restore the expression of some tumor suppressor genes and induce cell differentiation,growth arrest and apoptosis.The use of novel DNA methyltransferase inhibitors in clinical trials that allow monitoring of drug-induced DNA methylation changes should provide the foundation for improved epigenetic cancer therapies.This paper is to investigate the antitumor activity of HDACi(trichostatin A,TSA) and DNMTi (5-azadecitabine,5-AZA-dC) in pancreatic cancer.1.Expression of HDACI and DNMT1 proteins in Pancreatic Cancer TissuesObjective To detect the expression of HDAC1 and DNMT1 protein in pancreatic cancer and para-cancerous tissues,and investigate the correlation between HDAC1 and DNMT1 expression.Methods and materials Pancreatic carcinoma tumor tissue and paired para-cancerous tissues were obtained from 23 pancreatic carcinoma patients.Clinical data were collected. HDAC1 and DNMT1 protein expression was detected by streptavidin peroxidase immunohistochemistry.The correlation between HDAC1 and DNMT1 expression was analyzed.Results Positive cells were brown.Specific HDAC1 and DNMT1 staining were located in the nuclei of the cancer cells,while it was rarely seen in para-cancerous lesions. The positive rate of HDAC1 and DNMT1 protein expression in human pancreatic carcinoma tissues and para-cancerous tissues was 56.21%±10.26%vs 6.24%±5.28%and 54.83%±23.33%vs 6.30%±10.78%(P=0.000) respetively.Coefficient correlation of HDAC1 and DNMT1 expression between carcinoma tissues and para-cancerous tissues was significant at the 0.01 level(2 tailed).Conclusions In pancreatic carcinoma tissues HDAC 1 and DNMT1 protein was highly expressed,over-expression of HDAC1 protein and DNMT1protein might have an important role in the formation of tumor.2.Effects of TSA,5-AZA-dC on Proliferation and Apoptosis in Pancreatic Cancer Cell PaTu8988Objective To study the growth arrest and apoptotic effects as well as the inhibitory effects of TSA,5-AZA-dC in human pancreatic cancer cell line PaTu8988.Methods The growth suppression effect of TSA,5-AZA-dC on PaTu8988 were studied using Cell Count Kit(CCK-8);Apoptosis and cell life cycles were examined by Flow cytometry.Results 1.TSA and 5-AZA-dC inhibited the growth and proliferation of PaTu8988 cells in a concentration-dependent manner,which was showed by the growth curve.The proliferation ratios of cells treated with TSA(0.1,0.5,2.0μmol/L for 24h) were 95.12%±4.33%,77.84%±13.58%and 69.83%±7.04%respectively,5-AZA-dC(0.5,2.0, 10μmol/L for 72h) 94.91%±1.51%,88.19%±3.28%and 81.93%±4.89%respectively, therapeutic alliance groups(lower,mild and high dose) 86.69%±9.07%,72.86%±4.19%, 57.20%±9.67%respectively.2.The ratios of cell apoptosis was induced by TSA and 5-AZA-dC in a dose-dependent manner.The cells showed G2/M arrest after treated with TSA and 5-AZA-dC.Conclusions TSA and 5-AZA-dC inhibited PaTu8988 cell in the concentration -dependent manner.Increasing apoptosis of PaTu8988 cells may contribute to the anti-tumor properties of TSA and 5-AZA-dC.3.Expression of HDAC and DNMT genes in Pancreatic Cancer TissuesObjective To investigate the expression of p21,Cyclin D 1,Bax and Bcl-2 genes in as well as the inhibitory effects of TSA,5-AZA-dC in human pancreatic cancer cell line PaTu8988. Materials and Methods The expression of p21,Cyclin D1,Bax and Bcl-2 genes of cell lines PaTu8988 treated by TSA,5-AZA-dC was detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Protein was detected by western blot method,and the DNMT activity was detected by assay kit.Results 1.The relative quantitation(RQ) values of p21 and Bax genes of cells treated with TSA(0.1,0.5,2.0μmol/L for 24h) were obviously higher than control group,while Cyclin D1 and Bcl-2 were lower than control group.The expression of p21,Cyclin D1, Bax and Bcl-2 protein was at equal pace with mRNA.2.The DNMT activity of cell lines after treated with 5-AZA-dC was obviously decreased.Conclusions The inhibition and apoptosis of cell lines treated with TSA maybe caused by up-regulation of p21 and Bax genes expression or down-regulation of Cyclin D1 and Bcl-2 genes expression.4 Growth Suppression Effect of TSA and 5-AZA-dC on the Growth of Xenograft of Pancreatic Cancer in Nude MiceObjective To study the suppression effect of TSA and 5-AZA-dC on the pancreatic cancer xenograft models,in order to evaluate the potential role of HDACi and DNMTi as a novel anti-neoclassic agent for the treatment of pancreatic cancer.Methods 10 million PaTu8988 cells were injected into the back of BAL B/c nude mice.Once visible tumors were evidenced about 150mm~3,the animals were divided equally into 4 groups(6 animals/group) and treated with TSA,5-AZA-dC or control vehicle(PBS) by intraperitoneal injection once every 2 days for 5 times:Group A:control vehicle 0.2ml/animal;Group B:TSA 1mg/kg;Group C:5-AZA-dC 3mg/kg;Group D: TSA 1mg/kg and 5-AZA-dC 3mg/kg.The tumor volumes were measured every 5 days. The tumor xenografts and organs were removed,photographed and weighed.Using HE (hematoxylin-eosin) stain to show morphology,p21,Cyclin D1,Bax and Bcl-2 mRNA were measured by Real-time RT-PCR.Results 1.The growth of tumor xenograft was suppressed potently when administered with different drugs.Compared with control group,the mean tumor size in treated groups was reduced markedly at every point of test time.TSA caused profound tumor regression, some xenograft was even disappeared;5-AZA-dC can also decrease the tumor size,but the effects were not as remarkable as TSA.Antitumor result of combination TSA and 5-AZA-dC had much better effects than TSA or 5-AZA-dC.2.The relative quantitation (RQ) values of p21 and Bax genes of cells treated with TSA were obviously higher than control group,while Cyclin D1 and Bcl-2 were lower than control group.Conclusions TSA and 5-AZA-dC can attenuate the growth of pancreatic caner xenograft.The growth suppression effect of TSA in xenograft may partially due to its up-regulation the expression of p21 and Bax.TSA and 5-AZA-dC may exert a synergistic antitumor effect.