Investigation Of The Synergistic Cytotoxicity And Mechanism Of Histone Deacetylase Inhibitors TSA Or DP Combine With Paclitaxel On Non-small Cell Lung Cancer Cell Lines

Objective: To observe the synergistic cytotoxicity of histone deacetylase inhibitors TSA or DP with paclitaxel on the NSCLC cell lines, and to elucidate the possible mechanism.Methods: 1. The growth inhibition effects of TSA, DP and paclitaxel on 3 NSCLC cell lines A545, H1299 and H322 were detected by MTT assay. The H1299 and H322 cells were seeded in 96 wells plates and treated as follows: (1)untreated control, (2)paclitaxel 96h (TAX), (3) DP(25ng/ml) 12h(DP), (4)Afer exposing to DP 12h then followed exposing to paclitaxel for 84h (DF), (5)cells exposed to DP and paclitaxe at same time, DP treated for 12h, paclitaxel treated for 96h(DTST), (6)cells continuous exposed to paclitaxel for 84h, then followed with DP12h (DB), (7)TSA(300nM)12h, (8)cells exposed to TSA 12h then continuous exposed to paclitaxel 84h (TF), (9)cells exposed to TSA and paclitaxel at same time, TSA treated 12h, paclitaxel treated 96h(TTST), (10)cells continuous exposed to paclitaxel 84h, then treated with TSA12h(TB). 2. H1299 and H322 cells were seeded into 6 wells plates and combine treated as described above for 24h. Cell cycle and apoptotic cell death were determined by flow cytometry assay. 3. After differented treatment, the protein expression level of HER2, p21, survivin, phosphorylated-ERK and PARP were determined by Western blot analysis. 4. Fluoromicroscope was used to observe apoptosis, cell death and the nucleolus change stained by Hoechst 33258 and PI.Results: 1. The HER2 expression levels of 3 NSCLC cell lines were different. The cell line H322 was classified with high level of HER2 expression, while the cell line H1299 with the low level expression. 2. The histone deacetylase inhibitors TSA and DP showed significant growth inhibition effect on NSCLC cell lines A549, H1299 and H322, which weretime and dose- dependent and were not correlated with the HER2 expression level. A549 cells (p53 wild type) seems to be more sensitive with TSA and DP treatment than H322(p53 mutant type) or H1299(p53 null). 3. Paclitaxel showed significant growth inhibitive effect on NSCLC cell lines A549, HI299 and H322, which were not correlated with the HER2 expression level. 4. TSA and DP showed good synergistic effects with paclitaxel on cell growth inhibition. The methods of co administration TF and DF (as described in method 1) had better synergistic effect. 5. TSA, DP and paclitaxel all enhanced p21 protein expression in HI299 and H322 cells. In H1299 eels, in groups DF, DB, DTST, TF, TB co administrated TSA with paclitaxel increased more p21 expression level than in group TAX, but in group TTST, the p21 expression level decreased and less than that of in group TAX. In H322 cell line in groups DF, DB, the p21 expression level increased and more than that of in group TAX. But in groups TF, TTST, TB and DTST co administrated TSA or DP with paclitaxel caused the p21 expression level decreasing, and p21 expression level of those was less than that of group TAX. 6. After exposing to TSA or DP for 12h, cell cycle of the cell lines H322 and HI299 arrested at G0/G1 phase. In groups DF, DB, TF, TB co administrated TSA with paclitaxel resulted in cell cycle of the cell lines H322 and H1299 arrested dominantly at G0/G1 phase, but had the tendency of shift to G2 phase. In groups TTST, DTST, cell cycle arrested dominantly at G2/M phase. 7. Cleaved PARP could be detected in H322 cell line after co-administrated TSA or DP with paclitaxel, but not in HI299 cell line. The percentage of apoptosis increased after co administrated TSA or DP with paclitaxel in H322 cell line, but which was low in HI299 cell line. 8. The survivin protein expression level increased significantly in both cell lines after exposed to paclitaxel for 24h. while as TSA and DP can decrease survivin protein expression either treated alone or co administrated with paclitaxel. 9. The phosphorylated-ERK protein expression level increased in both cell lines after exposed to paclitaxel 24h. TSA and DP decreased survivin protein expression levels of both cell lines either treated alone or co administrated with paclitaxel.Conclusion: 1. The histone deacetylase inhibitors TSA and DP significantly inhibited cell growth on NSCLC cell lines A549, H1299 and...