Effects Of Trichostatin A Combined With Docetaxel On Apoptosis ANDα-Tubulin Of Lung Adeno-Carcinoma A549Cells

Objective:To characterize the effects involved in apoptosis and α-tubulin following administration of trichostatin A(TSA) and Docetaxel(Doc) to lung adenocarcinoma cells A549, and the expression of caspase-3and survivin.Methods:The A549cells were cultured in RPMI-1640, divided into four groups:1、control;2、TSA250nmol/L;3、Docetaxel10μ/mL;4、TSA250nmol/L+Docetaxel10μg/mL, and treated with drugs for24hours. Cellular morphological changes were observed under the light microscope. Cell inhibition ratio was evaluated using the MTT assays; Hoechst33258staining and flow cytometry analysis, and cell cycle was detected by flow cytometry analysis; The protein expression Acetyl-α-tubulin was detected by immunofluorescence and Western blot. The protein expression of caspase-3and survivin are also detected by Western blot. All the data above were expressed by x±s, and were recorded and analyzed using χ2test, t test with SPSS15software. The statistic significance were defined as p<0.05.Results:1. Cellular morphological changes were observed under the light microscope①Control:Cell adherence was aequalis, various cell divisions were observed and floating dead cells were not detected in the control group.②TSA:TSA-treated cells became enlarged (indicated by open arrows) or asteroidal (indicated by solid arrow), the cell gap increased, and floating dead cells can be seen in cultures with TSA.③Docetaxel:Docetaxel-treated cells became deflated and shrinked (indicated by arrowheads), and floating dead cells can be seen cultures with TSA.④TSA+Docetaxel:In cultures with TSA and Docetaxel, cell density was reduced and dead cells were observed compared to control and singal drug group. 2. Cell inhibition ratio in different groups were detected by methyl thiazolyl tetrazoliumAfter treated by drugs for24h, Docetaxel inhibited the growth of A549cells in dose-dependent manner; The combined group treatment with TSA and Docetaxel induced more severe apoptosis. The inhibition rates of Docetaxel10μg/mL and TSA250nmol/L were respectively (30.6±2.5)%、(10.5±1.2)%; Docetaxel10μg/mL+TSA250nmol/L were (65.6±3.5)%, much higher than the group treated by drug alone (p<0.01).3. The changes of nucleus were assessed using Hoechst33258stainingAfter treated by drugs in different groups for24h, the changes of nucleus were dectected by immunofuorescence. The condensed and fragmented nuclei were increased in cells treated with TSA, Docetaxel-treated cells became condensed and assembled. Compared with control and drug alone, the strongest effect was observed in cells treated with TSA and Docetaxel.4. Cell apoptosis and cell cycle were detected by flow cytometry analysisAnnexin V/PI method showed that:①Control:(1.2±0.5)%②TSA250nmol/L:(17.6±1.8)%③Docetaxel10μg/mL:(39.2±3.7)%④TSA250nmol/L+Docetaxel10μg/mL:(64.2±4.2)%The combined group treated with TSA and Docetaxel induced more severe apoptosis, and the cell cycle was markedly arrested in G2/M phase (p<0.05) to the single drug.5. The expression of Acetyl-α-tubulin were determined by immunofluorescenceTSA(250nmol/L)、Docetaxel (10μg/mL) alone or combined treated A549cells for24h. The expression of Acetyl-α-tubulin were determined by immunofluorescence. Under the same exposure, the fluorescence intensity was increased in cells treated with TSA, Docetaxel compared with the control group. The strongest effect was observed in cells treated with TSA and Docetaxel.6. The expression of Acetyl-α-tubulin、caspase-3and survivin were detected by Western blotting treated with TSA and Docetaxel.TSA(250nmol/L)、Docetaxel (10μg/mL) alone or combined treated A549cells for24h. Compared with single drug or control, Acetyl-α-tubulin and the active caspase-3increased, along with concomitant survivin down-regulation in the combined group.Conclusions:TSA induced sensitivity to Docetaxel treatment in A549cells. Promoted cells apoptosis and the cell cycle were markedly arrested in G2/M phase. The down-regulation of survivin and up-regulation of Acetyl-α-tubulin and promote caspase-3activity are possible mechanisms mediating sensitization to Docetaxel treatment in A549cells.