The Action Mechanism Of Trichostatin A Sensitizes Cisplatin-resistant Human Lung Adenocarcinoma Cell Lines To Apoptosis Research

BackgroundLung cancer is one of the most common cancers worldwide. There are about 80% of lung cancers are defined as non-small cell lung cancer (NSCLC). While surgery remains to be the primary treatment at the early stage, many stageⅡandⅢpatients will progress within few months after tumor resection. For most NSCLC patients, they are diagnosed at local or more often distant advanced stage, chemotherapy is the leading way to lung cancer.The use of platinum-based combination chemotherpy remains the standard treatment for non-small lung cancer,However, the resistance to platinum limits further treatment clinically. For this reason, it is important to search for an appropriate and effective way to reverse cisplatin resistance in lung cancer.The mechanism of cisplatin-resistance has been associated with multi-factors including different cellular accumulation and detoxification of the drug, inhibition of apoptosis and DNA repairs. In treating lung cancers, the chemo-sensitivity of cisplatin is affected by the changes of gene expression, including those known to be associated with cell cycle regulation and apoptosis.Epigenetic alterations, such as the histone acetylation and DNA methylation in the promoter of genes, contribute to the changes of gene expression. The dys-regulation in the epigenetics can lead the onset and progression of cancer, increasingly more reports have demonstrated that it also plays an important role in drug resistance. Histone deacetylase (HDAC) inhibitors have been known to promote transcription of genes required for cell differentiation and apoptosis; therefore,they emerged as a class of cancer therapeutic agents. Trichostatin A (TSA) is one of such HDAC inhibitors. It was originally isolated from Streptomyces and identified as a fungicidic antibiotic and inhibited all class I and II HDACs. TSA can act as a chemo-sensitizer in ovarian cancer, gastric cancer, erythroleukemia cells, the molecular mechanisms of TSA-sensitized cytotoxicity of chemotherapeutic drugs remain largely unknown.In this study, we investigate the apoptosis-inducing effect of TSA in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP cell line) and examine whether TSA can enhance the sensitivity to cisplatin treatment and the underlying molecular mechanisms.Objective To investigate the apoptosis-inducing effect of TSA in the human lung adenocarcinoma cisplatin-resistant cell line A549/CDDP and to examine whether TSA can enhance the sensitivity to cisplatin treatment. Methods 1.A549 and A549/CDDP Cell lines were cultured with DMEM medium. Neutral Red assay Cell viability was performed to evaluate the resistance of A549/CDDP cells. 2. The A549/CDDP cells were treated with different concentrations of TSA.3. Apoptosis was studied using Hoechst 33258 staining and flow cytometry analysis. cell cycle and mitochondrial membrane potential were analyzed by flow cytometry.4. The A549/CDDP cells were treated with the combined treatment of cisplatin and TSA. Results 1.The resistant index of A549/CDDP cell was 12.8.2. TSA was 446.59±27.32 nmol/L,The growth curve showed the ratio of growth decreased with the increase of concentration of TSA.3.TSA induced apoptosis in A549/CDDP cells. morphologic changes including nuclear chromatin condensation fluorescence strength was observed with fluorescence microscope. Apoptosis was measured as the percentage of sub-G1 DNA content by flow cytometry.Treated by TSA, mitochondrial membrane potential was descreased and cells were arrested at S phase.4. Treated by the combined treatment of cisplatin and TSA(31.25 nmol/L and 62.5 nmol/L), the reverse multiple was 1.52,1.70. Conclusions 1.Compared with A549 cells, A549/CDDP cells have good resistance.2. TSA induced apoptosis in A549/CDDP cells.3. Treated by TSA, mitochondrial membrane potential was descreased and cells were arrested at S phase.4 TSA enhances the sensitivity of CDDP-resistant A549 cells to cisplatin.Objective To investigate whether DAP kinase contributes to cisplatin resistance and the underlying molecular mechanisms. Methods Different proteins was detected by Western blot method. To confirm the role of Death-associated protein kinase (DAPK) in the TSA-induced apoptosis in A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has higher expression level of DAPK expression,and the DAPK activity was inhibited by C-terminal fragment of DAPK and RNA interference.Results TSA induced apoptosis in A549/CDDP cells, along with the concomitant DAPK up-regulation. When DAPK is over-expressed, CDDP-resistant A549 cells become sensitive to both TSA and cisplatin. Moreover, the cytotoxicity of TSA can be alleviated when DAPK activity is inhibited by the expression of a recombinant C-terminal fragment of DAPK or RNA interference. Conclusions DAPK might involve in cisplatin-resistance of the A549/CDDP cells,the up-regulation of DAPK is one of the mechanisms mediating TSA action to sensitize cisplatin-resistant cells to apoptosis.Objective To investigate the mechanism of TSA induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Different proteins were analyzed by Western blot method, Colorimetric method was used to measure Caspase-8 activities. Results Treated by TSA,Western blotting analyses showed that the levels of cFLIP decreased, procaspase-8 decreased, and it resulted in the activation of Caspase-8 and Bid cleavage. The levels of procaspase-3 and PARP decreased, the cleavage of the activated PARP molecules would undergo more rapid apoptosis. Caspase-8 activity increase and cFLIP level decrease were regulated by TSA in a dose and time-dependent manner. Conclusion TSA induce A549/CDDP cell apoptosis by decreasing cFLIP level, activating caspase-8.Objective To investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Different proteins were analyzed by Western blot method, A549/CDDP cells were transfected by Bcl-2 expression Vector or endogenous Bcl-2 was downregulated by siRNA. Results Western blot analyses showed that the levels of Bcl-2 decreased, while expression of Bax increased. Simultaneously Caspase-3 was activated.Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis, while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA.Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.