内质网应激诱导人乳腺癌MDA-MB-231细胞系凋亡

ER stress induces apoptosis of human breast cancer cell line MDA-MB-231

目的探讨内质网应激(ERS)致雌激素受体阴性(ER-)人乳腺癌MDA-MB-231细胞系凋亡及钙激活中性蛋白酶2(calpain2)在凋亡效应中的作用。方法实验设空白对照组(DMEM)、对照组(DMEM+DMSO)和实验组,用不同浓度衣霉素(TM)诱导乳腺癌细胞不同时间,MTT法和流式细胞术检测细胞增殖抑制率及凋亡率;Western blot法检测葡萄糖调节蛋白78(GRP78)表达量,以GRP78表达最高点确定ERS模型建立,检测凋亡相关蛋白caspase4和CHOP表达以及calpain及其内源性抑制酶calpastatin的表达,用ERS抑制剂(4-PBA)和calpain抑制剂(calpeptin)分别预处理细胞2 h,观察对上述效应的影响。结果 9、12和18μmol/L TM诱导ERS对该细胞增殖有明显抑制效应,抑制率分别为33.88%±1.32%、51.51%±8.85%和55.77%±2.61%,细胞凋亡率分别为9μmol/L TM组16.70%±0.46%和12μmol/L TM组28.1%±1.09%,与对照组相比有明显差异(P<0.05);9μmol/L TM诱导细胞24 h的GRP78表达上调最高(P<0.01);ERS还可明显上调caspase4、CHOP和calpain表达,下调calpastatin表达(P<0.01或P<0.05),上述效应均能被4-PBA和calpeptin减弱或阻断(P<0.05)。结论 ERS可诱导MDA-MB-231细胞凋亡,calpain2参与调控凋亡发生。

Objective To investigate endoplasmic reticulum（ER） stress-induced the apoptosis of estrogen receptor negative （ ER - ） breast cancer cells ; moreover, the effect of calpain2 on ER stress-induced the apoptosis is explored as well. Methods Experimental group （ DMEM）, control group （ DMEM ＋ DMSO） , and experimental group were set up. Tunicamycin ？ of various concentrations were added to cultures to act on cells for different durations, MT＇F method and flow cytometry were used for detecting the cell proliferation inhibition rate and apoptosis rate; Western blot was used for the expression of the marker protein glucose regulated protein of ERS, and based on the highest point of GRP78 expression, the drug concentration and timing of TM established by ERS were determined, determining the expressions of apoptosis proteins caspase4 and CHOP, as well as the expression of calpain and its endogenous inhibition enzyme calpastatin; ERS inhibitor （4-PBA） and calpain inhibitor （calpeptin） were respectivelysubject to pretreat cells, and the influences of the above effects observed. Results ERS inhibited the cells＇ prolifer- ations and 9, 12, 18 μmol/L were respectively 33.88% ± 1.32%, 51.51% ±8.85% and 55.77% ±2. 61% ,and in comparison with the controls （ P 〈 0.01 ） ; 9 and 12 μmol/L TM＇s cell apoptosis rates induced by ERS were re- spectively 16. 70% ±0. 46% and 28.10% ± 1.09%, in comparison to the controls （P 〈0. 05） ; 9 μmol/L TM＇s action on cells 24 h GRP78 yielded the highest expression and showed statistical significance in comparison to the controls（P 〈 0. 05 ） , and those of 12 μmol/L were obviously down-regulated; ERS obviously up-regulated the ex- pressions of caspase4, CHOP and calpain, and the expression of calpastatin down-regulated （P 〈 0. 01 or 0. 05 ）. The effects can be reduced or blocked by 4-PBA and calpeptin （ P 〈 0. 05 ）. The expressions of CHOP all down- regulated among them 4-PBA and the treated group（P 〈 0. 05）. Conclusions The strong ER stress can induce ap- optosis of breast cancer MDA-MB-231 cells; expect that, calpain2 may be engaged in its mechanism.