Role Of Histone Deacetylase 1 In Human Lung Adenocarcinoma Carcinogenesis

Lung cancer is known to be the leading cause of cancer death for both man and woman in the word.Lung adenocarcinoma is 30%of all the patients with lung cancer. Most of the lung adenocarcinoma patients are in the late stage of the disease when first diagnosed and the the survival rate stays on the platform.New therapeutic approaches in treatment of lung adenocarcinoma are anxiously needed considering the poor cure rate and high cost and toxicity of chemotherapy treatment used currently.Detailed exploration of the molecular biology of lung adenocarcinoma is also required for understanding this fatal malignant disease profoundly.The condition of acetylation are regulated by histone acetyltransferases(HATs)and histone deacetylases(HDACs).The dynamic balance between histone acetyltransferases and histone deacetylases controls the structure of chromatin and expression of genes.The chaos of HATs and HDACs is one of the important molecular mechanism of carcinogenesis.HATs and HDAC are two enzymes with opposing effects that regulate the transcription of genes by controlling the acetylation status of histones.The acetylation atε-amino group of specific lysines in the N-terminus of histones neutralizes the positive charge on lysine.This is thought to reduce the affinity of histone complexes to DNA chains,thus enhancing the access of transcriptional factors to DNA.On the other hand,deacetylation results in a more condensed chromatin.HDACs which belong to histone deacetylases superfamily have been classified into four distinct families:TypeⅠ,Ⅱ,ⅢandⅣ.The members of TypeⅠHDACs include HDAC1,HDAC2,HDAC3 and HDAC8.Overexpression of HDAC1 in human cells can not only increase the proliferation of cancer cells but also enhance the migration or invasion abivility of cells through extracellular matrix.Overexpression of HDAC1 mRNA and protein has been observed in human gastric and prostate tumors.Gastric carcinomas,colorectal carcinomas,cervical dysplasias and endometrial stromal sarcomas all overexpress HDAC2 as compared to their normal counterparts.It has been reported that specific siRNAs targeting HDAC3 and HDAC1 produced a concentration-dependent inhibition of histone hyperacetylation in HeLa cells and can impede the cells proliferation as well as inducing apoptosis.Senese et al showed that using RNA interference-mediated HDAC1 protein knockdown can cause cell cycle arrest at either G1 phase or G2/M phase, resulting in the loss of mitotic cells,cell growth inhibition,and an increase in the percentage of apoptotic cells.On the contrary,HDAC2 knockdown had no effect on cell proliferation unless HDAC1 was knocked down concurrently.These data demonstrated that TypeⅠHDACs,such as HDAC1,are important in the regulation of proliferation and survival in cancer cells.HDAC inhibitor(HDACi) is a new kind of anticancer agents which can cause cell cycle arrest,differentiation,and/or apoptosis of tumours by blocking deacetylation function of HDACs.The modificative effects of HDACi seem to be dependent upon the type of tumour cell rather than the specific HDAC inhibitors used.For NSCLC cells in culture,these compounds lead to growth arrest,cells differentiation,or apoptosis. Although the involvement of HDACs in the development of cancer has been proven,the detailed mechanisms underlying the regulation of NSCLS cell proliferation,apoptosis and cell cycle by individual HDACs are still unclear.The inhibitory effects of different members of HDACi are nonspecific for different HDAC isoforms.Also,the regulation of these enzymes during cancer growth is largely unknown.It significance of new drug screening and synthesis to define target of HDACi is considerable.This study is about to detect the expression of HDACs mRNA and protein in lung adenocarcinoma and latero-cancer tissues to exploring the effects of HDACs in lung adenocarcinoma.The role of HDAC1 in tumor cell proliferation and apoptosis is investigated using RNA interference technology in two different human lung adenocarcinoma cell lines(A549 and NCI-H1299).The involvments of Bax,Bcl-2,cyclin B1 and p21 in the effects of HDAC1 on lung adenocarcinoma are also determined in our study.1.The expression of HDAC1 mRNA and protein in human lung adenocarcinomaObjective:To detect the expression of HDAC1 mRNA and protein in human lung adenocarcinoma and paracancerous tissues and explore its role in lung adenocarcinoma.Methods and materials:Six cases of lung adenocarcinoms tissue samples and matched latero-cancer tissues were collected and detected HE staining to confirm their adenocacinoma characteristics.The mRNA and protein levels of HDAC1 of the samples were detected by Real-time PCR and Western blotting analysis,respectively.The location of HDAC1 expression is determined by streptavidin peroxidase immunohistochemistry techniques.Results:The results of Realtime-PCR showed that the average level of HDAC1 mRNA expressoion in adenocarcinoma tissues are significant higher than that in matched latero-cancer tissues(2.30 vs 1,P=0.03),which was cofirmed by Western blotting analysis. Immunohistochemistry showed HDAC1 protein is localized in cell nucleus.Conclusions:The expression of HDAC1 mRNA and protein levels were both significant higher in human lung adenocarcinoma tissues than in latero-cance tissues,indicating its possible involvement in lung adenocarcinoma carcinogenesis.2.Effects of RNAi knockdown of HDAC1 and trichostatin A(TSA) treatment on the growth of lung adenocarcinoma cell linesObjective:To evaluate the role of RNAi knockdown of HDAC1 in tumor cell proliferation by comparing with TSA treatment.Methods and materials.The role of HDAC1 in tumor cell proliferation was investigated using specific RNA interference targeting HDAC1 and TSA treatment in A549 and NCI-H1299 human lung adenocarcinoma cell lines.Cells were classified into three groups:the control group,negative siRNA group,and HDAC1 siRNA group. Realtime-PCR and Western blotting analysis were performed to detect the mRNA and protein expression of HDAC1 and Ac-H4,its acetylated product,in each group.The effects on cells proliferation,cell cycle progression and apoptosis were determined by flow cytometry,annexin-V staining and MTT assay,respectively.Results:The expression of HDAC1 mRNA and protein in lung adenocarcinoma cell lines were significantly suppressed after specific HDAC1 siRNA transfection.Western blotting analysis revealed an increased acetylation level of a subset of histones H4 after suppression of HDAC1.The growth of two cell lines with HDAC1 knockdown were both significantly inhibited when compared with negative siRNA controls.The survival rates of both A549 and NCI-H1299 cells after HDAC1 siRNA transfection significantly decreased (69%and 83%,respectively),while after TSA treatment the survival rates of these two cells decreased to a similar extent(63%and 76%,respectively).Either RNAi knockdown or TSA treatment resulted in increased early apoptotic rate of A549(13.45%vs 5.22%) and NCI-H1299(11.76%vs 8.09%).G2/M cyclin arrest was also observed in two cell lines with suppressed HDAC1 expression when compared with the controls.Conclusions:Transfection of specific HDAC1 siRNA effectively inhibits the proliferation of lung adenocarcinoma cells.The suppression of HDAC1 expression resulted in both G2/M cell cycle arrest and increased cells apotosis.These data suggest that inhibition of HDAC1 may provide a valuable approach for lung adenocarcinoma treatment. It is worthwhile evaluating HDAC1 as a potential candidate for anticancer therapy in lung adenocarcinoma.3.Effect of RNAi knockdown of HDAC1 on the expresison of Bax,Bcl-2,p21 and cyclin B1 gene in lung adenocarcinoma cell linesObjective:To evaluate the role of transient transfection of HDAC1 siRNA inducing gene concerning about proliferation and apotosis in lung adenocarcinoma cell line.Methods and materials:Transient transfection of A549 and NCI-H1299 cell lines with specific HDAC1 siRNA were performed.The level of mRNA and protein expression on Bcl-2,Bax,p21 and cyclinB1 were detected by Realtime-PCR and Western blotting analysis.Results:The levels of Bcl-2 and CyclinB1 mRNA in A549 cells were significantly decreased after HDAC1 siRNA transfection compared to negative control(37%vs 57%).On the contrary,Bax and p21 expression in A549 cells were significantly increased(4.82 and 3.38 folds, respectively).In NCI-H1299 cells the mRNA expression of CyclinB1 were significantly decreased (49%) after HDAC1 siRNA transfection,while Bax and p21 mRNA levels were increased(3.88 and 3.91 folds,respectively).The results of western blotting analysis were coincident with those of Realtime-PCR analysis.Conclusions:Specific HDAC1 siRNA transfection can inhibit the expression of Bcl-2 and cyclin B1 gene while increase the expression of Bax and p21.Based on the above experiments,the final conclusions were as followings:1.The expression of HDAC1 mRNA and protein levels were higher in human lung adenocarcinoma tissues than that in latero-cance tissues,indicating possible involvement of HDAC1 in carcinogenesis of lung adenocarcinoma.2.Transfection of specific HDAC1 siRNA can effectively inhibit the proliferation of lung adenocarcinoma cell lines.RNAi knockdown of HDAC1 in cells resulted in G2/M cycle arrest and increased cells apoptosis,indicating the potential of HDAC1 as a candidate for anticancer therapy in lung adenocarcinoma.3.RNAi knockdown of HDAC1 can inhibit the expression of Bcl-2 and cyclin B1 gene while increasing the expression of Bax and p21 gene.In summary,we found in this study that RNA interference for HDAC1 induces cell cycle arrest and apoptosis in lung adenocarcinoma.We suppose that HDAC1 may play an important role in carcinogenesis of lung adenocarcinoma and may be a valuable new target for cancer treatment.The role of other member of HDACs should be further studied.