组蛋白去乙酰化酶抑制剂与紫杉醇联合应用对肺癌细胞的毒性作用

The cytotoxicity of histone deacetylase inhibitor plus paclitaxel to lung cancer cells

目的评价组蛋白去乙酰化酶(HDAC)抑制剂曲古抑菌素A(TSA)联合紫杉醇(TAX)对肺癌细胞株H1299的抑制作用。方法以不同浓度TSA和(或)TAX处理H1299细胞,MTT法检测细胞存活率,计算TAX的半数抑制浓度(IC50)并绘制细胞生长曲线。随后将H1299细胞分成4组:对照组,常规培养细胞36h;TAX组,TAX(10nmol/L)处理细胞24h;TSA组,TSA(300nmol/L)处理细胞12h;TF组,TSA(300nmol/L)处理细胞12h后,加入TAX(10nmol/L)处理24h。采用流式细胞术观察细胞周期分布和细胞凋亡率;对细胞进行Hochest33342染色后,在荧光显微镜下观察细胞核形态的改变;Western blotting检测人多聚ADP核糖聚合酶(PARP)剪切蛋白和p21蛋白表达。结果TSA预先作用12h可以使TAX对H1299细胞的IC50由110.6±38.7nmol/L下降至63.7±11.8nmol/L(P<0.05)。与对照组比较,TAX和TF组细胞存在明显的G0/G1期阻滞,但4组的细胞凋亡率均很低(P>0.05)。TSA、TAX及TF组可见以死亡为主的细胞核改变现象,凋亡小体很少。4组均未检测到PARP剪切蛋白。与对照组比较,TSA、TAX及TF组的p21蛋白表达均有明显增加(P<0.05);与TAX组比较,TSA组和TF组的p21蛋白明显增加(P<0.05),且后两组间无显著差异(P>0.05)。结论联合HDAC抑制剂TSA可提高TAX对肺癌细胞H1299的毒性,促进细胞死亡,但其机制与细胞凋亡无关。

Objective To evaluate the cytotoxicity of trichostatin A （TSA）,a histone deacetylase inhibitor,plus paclitaxel to H1299 strain lung cancer cells. Methods H1299 cells were exposed to TSA and/or paclitaxel in different concentrations in different ways. The proliferation rates were determined by MTT assay,the 50% inhibition concentration （IC50） of paclitaxel was calculated and the growth curve was plotted. The H1299 cells were then divided into 4 groups：control group （cells were normally cultivated for 36h）,TAX group （cells were treated with 10nmol/L of paclitaxel for 24h）,TSA group （cells were treated with 300nmol/L of TSA for 24h） and TF group （cells were exposed to TAX for 24h after being treated with TSA）. Cell cycle and apoptosis were determined by flow cytometry. The morphological changes in nuclei as stained with Hoechst 33342 were observed by fluorescence microscopy. The protein expression levels of p21 and cleaved poly-ADP ribose polymerase （PARP） were determined by Western blotting. Results TSA significantly enhanced the inhibition of paclitaxel in lung cancer cell lines H1299. When combined with TSA,the IC50 of paclitaxel decreased significantly from 110.6±38.7nmol/L to 63.7±11.8nmol/L in H1299 cells （P0.05）. In contrast with the control group,obvious G1/G0 retardation in growth curve was found in TAX and TF groups. Necrosis,not apoptosis,could be observed primarily under fluorescence microscopy in each group. Expression of p21 was up-regulated in TAX,TSA and TF groups when compared with that in control group,and was higher in TSA and TF groups than in TAX group （P0.05）,while no difference was found between TSA and TF group （P0.05）. Conclusion The HDAC inhibitor TSA,combined with TAX,may enhance the cytotoxicity of paclitaxel to,and promote the death of,lung cancer cell line H1299,which may not be related to apoptosis.