Effects Of Trichostatin A Combined With Cisplatin On Apoptosis Of Lung Adenocarcinoma A549 Cells

In recent years, the importance of epigenetic alterations has been appreciated in cancer development, including the role of abnormal DNA methylation and histone acetylation on aberrant silencing of multiple tumor suppressor genes in a diversity of human cancers. Histone deacetylase (HDAC) inhibitors, which interfere with the function of histone deacetylase, are emerging as potent anticancer agents due to their effective anti-proliferative activity in a wide variety of tumors, mediated by mitotic defects through the aberrant acetylation of histone and non-histone proteins It has been reported that trichostatin A (TSA), a potent specific inhibitor of HDAC, is able to lead to cell growth arrest, differentiation, and/or apoptosis in a number of cancers. Evidence also suggests that TSA may have a promising therapeutic effect on cancer cells when combined with radiotherapy or chemotherapy.Cisplatin is a kind of anticancer drug containing platinum. It is a broad anticancer spectrum, whose strong function, synergistic effect with various antitumor drugs, and no cross resistance, make it one of the most commonly used drugs in combination chemotherapy. Cisplatin places two links with single chain DNA or cross links with double chain DNA, and inhibits the process of DNA replication in cancer cells, so leading to cell apoptosis. Cisplatin belongs to cell cycle non-specific drugs, and causes damage to the cell membrane structure. But after long-term use of cisplatin, most patients will have resistance to cisplatin. At the same time, cisplatin also has relatively more side effects.Studies have found that, TSA can enhance the sensitivity of cisplatin resistant lung cancer cells to cisplatin. Another study found that, in prostate cancer, breast cancer, ovarian cancer and gastrointestinal malignant tumors, TSA combined with cisplatin has a significantly higher inhibitory effect on cell proliferation than single drug treatment groups. Compared with single drug groups, the histone deacetylase inhibitor combined with cisplatin has increased acetylation level. Then the following questions are raised:Are there synergistic effects when TSA is combined with cisplatin in lung carcinoma cells? Can TSA enhance the sensitivity of lung carcinoma cells to cisplatin, so as to reduce the clinical dosage of cisplatin?In this light, the present study was designed to investigate antitumor activity by combining TSA with cisplatin on lung adenocarcinoma A549 cells, and its inner mechanism. Our results demonstrated that TSA in combination with cisplatin could be valuable in the treatment of lung cancer.Objective:To characterize the effects in apoptosis of lung adenocarcinoma cells A549 following administration of trichostatin A(TSA) and Cisplatin, and the expression change of cFLIP and caspase-8 protein in A549 cells.Methods:1、Culture the A549 cells in RPMI-1640 medium, and divide the cells into four groups: 1)、Control group; 2)、TSA 250nmol/L group; 3)、Cisplatin 10μg/mL group; 4)、 TSA250nmol/L+ Cisplatin 10μg/mL group. Treat the four group of cells with drugs for 24 hours. Observe the cellular morphological changes under the light microscope.2、Divide the cells into ten groups:1)、Control group 2)、TSA 250nmol/L group 3)Cisplatin 5.3μg/mL group 4) TSA250nmol/L+ Cisplatin 5.3μg/mL group 5) Cisplatin 9μg/m group 6) TSA250nmol/L+Cisplatin 9μg/mL group 7) Cisplatin 13μg/mL group 8) TSA250nmol/L+ Cisplatin 13μg/mL group 9) Cisplatin 20μg/mL group 10) TSA250nmol/L+ Cisplatin 20μg/mL group。 Evaluate the cell inhibition ratio using the MTT assays.3、Divide the cells into four groups as method 1, observe apoptosis of cells using Hoechst33258 staining.4、 Divide the cells into four groups as method 1, detect the protein expression of cFLIP Pro-caspase-8 and Caspase-8 using Western blot. All the data above were recorded and analyzed by using χ2 test and t test in software SPSS 15. The statistic significance was defined as p<0.05.Results:1. Observed the cellular morphological changes using light microscope①Control group:Cells had clear boundary, arranged regularly, adherent tightly, and we did not found floating dead cells in control group.②TSA 250nmol/L group:Compared with the control group, the cells became larger or finger like change, with widened cell gaps, and in cultures we found floating dead cells.③ Cisplatin 10μg/mL group:Cisplatin-treated cells became small, round and shrinkage, and floating dead cells can be seen.④TSA25 Onmol/L+Cisplatin 10μg/mL group:In cultures with TSA and Cisplatin, the cell density decreased significantly, and death cells increased obviously.2. Detected the inhibitory effects of different concentrations of cisplatin on cells by methyl thiazolyl tetrazolium methodIn this work, The cell apoptosis rate increased with the increase of Cisplatin concentration. And the inhibitory rates increased when combined Cisplatin with TSA. The inhibitory rates of cisplatin 5.3μg/mL and cisplatin 5.3μg/mL+TSA250nmol/L were 13.68%、26.42%. The inhibitory rates of cisplatin 9μg/mL and cisplatin 9μg/mL+ TSA250nmol/L were 28.73%、46.72%。The inhibitory rates of cisplatin 13μg/mL and cisplatin 13μg/mL+TSA250 nmol/L were 44.58%、49.65%。The inhibitory rates of cisplatin 20μg/mL and cisplatin 20μg/mL+TSA250nmol/L were 51.12%、61.92%。Notably, TSA in combination with cisplatin caused a greater inhibitory effect on A549 cells than single-drug therapy (P<0.05).3. Assessed the changes of apoptosis using Hoechst33258 staining methodAfter treated by different drugs in the four groups for 24h, the changes of apoptosis were dectected by Hoechst33258 staining method. Cisplatin, TSA, alone and in combination led to marked morphological changes such as chromatin condensation, nuclear fragmentation and apoptotic bodies, which were in accordance with typical morphological characteristics of cells undergoing apoptosis. Compared with single treatment, the combination of TSA with cisplatin elicited much more evident occurrence of apoptosis.4. TSA in combination with cisplatin decreased the expression of cFLIPTo investigate whether the treatment with cisplatin or TSA alone and the combination increased cleavage of cFLIP known to be involved in the apoptotic cascade, Western blot analysis was used to examine the expression of cFLIP in A549 cells following these interventions. The results showed that either single treatment or combination treatment resulted in decrease of cFLIP. The level of cFLIP protein in combination treatment cells was higher than that in single treatment cells (Fig.4).5. TSA in combination with cisplatin reduced Pro-caspase-8 and increased the expression of Caspase-8Lastly, we assessed the level of Pro-caspase-8 and Caspase-8 in A549 cells treated with TSA, cisplatin alone and their combination for 24 h. Either single treatment or combination treatment resulted in decrease of Pro-caspase-8 and increase of Caspase-8. The level of Caspase-8 protein in combination treatment cells was higher than that in single treatment cells.Conclusions:Combine TSA to Cisplatin can increase the effect of Cisplatin in the treatment of A549 cells. The occurrence of apoptosis in response to these stimuli was through a caspase-dependent signal pathway.