蛋白酶体抑制剂MG-132及其与顺铂联合应用对喉癌Hep-2细胞株的影响

Effect of the proteasome inhibitor MG-132 alone and combined with cisplatin on human laryngeal squamous cell carcinoma Hep-2 cell line

目的研究蛋白酶体抑制剂MG-132对喉癌Hep-2细胞增殖和凋亡的影响,及其与顺铂(DDP)联合应用对喉癌细胞的毒性作用。方法以Hep-2细胞为实验对象,分别用0、1、2.5、5、10、20μmol/L的MG-132干预Hep-2细胞48h;另用2.5μmol/L MG-132分别干预细胞0、12、24、48、72h。将细胞分为4组:对照组(不做处理)、MG-132组(加入终浓度1μmol/L的MG-132)、单用DDP组(分别加入终浓度为10、20、40、80μmol/L的DDP)及MG-132+DDP组(加入1μmol/LMG-132和10、20、40、80μmol/L的DDP),各组干预细胞48h。利用四甲基偶氮唑蓝(MTT)法、流式细胞术(FCM)检测细胞增殖抑制率及凋亡率的变化。结果MTT检测结果显示MG-132可有效抑制Hep-2细胞的增殖,呈浓度(r=0.944,P<0.01)及时间依赖性(r=0.945,P<0.01),48h时的半数抑制量(IC50)=2.50μmol/L;与单用DDP比较,MG-132联用DDP后对细胞的增殖抑制作用明显增强(P<0.05)。DDP的IC50由62.22μmol/L下降至27.79μmol/L。FCM检测结果显示:细胞凋亡率随MG-132作用时间的延长而上升,具有时间依赖性(r=0.888,P<0.01);与单用DDP组(16.40%)及MG-132组(25.63%)凋亡率比较,MG-132与DDP联用后诱导细胞凋亡的作用显著增强(37.00%,P<0.01)。结论蛋白酶体抑制剂MG-132可有效抑制人喉鳞癌Hep-2在体外的增殖并诱导其凋亡,从而显著增强DDP对喉鳞癌Hep-2细胞株的毒性作用。

Objective To study the effect of proteasome inhibitor MG-132 on proliferation and apoptosis of human laryngeal squamous cell carcinoma Hep-2 cell line, and to explore if cisplatin （DDP） is combined with MG-132, can enhance the cytotoxicity to laryngocarcinoma cells. Methods Hep-2 cells were treated with different doses （0, 1, 2.5, 5, 10, 20 μmol/L） of MG-132 for 48h. In another experiment, cells were treated with 2.5μmol/L MG-132 for different length of time （0, 12, 24, 48, 72h）. Cells were further divided into 4 groups： control group without any treatment, MG-132 group cells were treated with 1μmol/L MG-132 alone, DDP group were treated with 10, 20, 40 and 80μmol/L DDP alone, and MG-132＋DDP group were given 1μmol/L MG-132 combined with 10, 20, 40, 80μmol/L DDP for 48h. Cell growth was assessed by methyl thiazolyl tetrazolium （MTT） assay, and apoptosis was determined with flow cytometry （FCM）. Results MTT assays showed that the MG-132 can effectively inhibit the proliferation of Hep-2 cell in a dose-（r=0.944, P0.01） and time-dependent manner （r=0.945, P0.01）. At 48h the 50% inhibition concentration （IC50）=2.50μmol/L. MG-132 combined with DDP could significantly enhance the inhibition ratio of the cells compared with DDP group （P0.05）, the IC50 of DDP declined to 27.79μmol/L from 62.22μmol/L. FCM demonstrated that the apoptotic rate was increased accompanied with prolongation of treatment time in a time-dependent manner （r=0.888, P0.01）. Combination of MG-132 and DDP could markedly increase the apoptotic rate of Hep-2 cells （apoptotic rate 37.00%） compared with DDP group （16.40%） and MG-132 group （25.63%, P0.01）. Conclusions The proteasome inhibitor MG-132 can inhibit proliferation and induce apoptosis of human laryngeal squamous cell carcinoma Hep-2 cell line. The combination of MG-132 and DDP may enhance the anticancer effect on Hep-2 cells.