乳腺癌多药耐药DEDD作用及机制研究

目的 死亡效应结构域DNA结合蛋白(death effector domain DNA-binging protein,DEDD)可抑制乳腺癌的生长和转移,但在乳腺癌多药耐药中的作用尚不明确.本研究将探讨DEDD在人乳腺癌多药耐药中的作用及机制.方法 通过转染DEDD-shRNA建立稳定敲低DEDD表达的MCF-7人乳腺癌细胞株,MTS实验检测多柔比星、紫杉醇对MCF-7 WT(Wild type,野生型)、MCF-7 control shRNA和MCF-7 DEDD-shRNA细胞活力的影响,Annexin-V和碘化丙啶(propidium iodide,PI)双染后流式细胞仪检测细胞凋亡.蛋白质印迹法检测乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)在药物干预后的蛋白表达水平.结果 多柔比星干预后,半数抑制浓度IC50的多因素方差分析结果显示,不同处理(F=89.49,P＜0.001)、不同时间(F=50.81,P＜0.001)组间比较差异有统计学意义.多柔比星干预24或48 h后,MCF-7 DEDD-shRNA的半数抑制浓度IC50分别为(0.97±0.15)和(0.62±0.09) μmol/L,明显高于MCF-7 WT(0.46±0.05)和(0.26±0.03) μmol/L和MCF-7 control shRNA细胞(0.39±0.05)和(0.25±0.03)μmol/L,P＜0.001.紫杉醇干预后,半数抑制浓度IC50的多因素方差分析结果显示,不同处理(F=15.94,P＜0.001)、不同时间组间(F=129.70,P＜0.001)比较差异有统计学意义.紫杉醇干预24或48 h后,MCF-7 DEDD-shRNA的半数抑制浓度IC50为(16.74±2.26)和(9.88±1.47) μmol/L,明显高于MCF-7 WT(13.39±1.44)和(7.69±0.92) μmol/L(P=0.001)和MCF-7 control shRNA细胞(12.58±1.15)和(7.17±1.12) μmol/L(P＜0.001).多柔比星干预后24 h后流式细胞凋亡结果显示,不同处理(F=131.46,P＜0.001)、不同浓度组间(F=160.95,P＜0.001)比较差异有统计学意义.正常培养状态下,MCF-7 control shRNA细胞和MCF-7 DEDD-shRNA细胞的凋亡率分别为(14.32±1.47)％和(11.58±1.63)％,而给予0.5μmol/L和1 μmol/L多柔比星作用24 h后,MCF-7DEDD-shRNA细胞的凋亡率分别为(21.62±1.97)％和(24.39±2.36)％,明显低于MCF-7 control shRNA细的(36.26±1.87)％和(38.23±1.46)％,P＜0.001.同样,紫杉醇干预后24 h,不同处理(F=124.81,P＜0.001)、不同浓度组问(F=172.56,P＜0.001)细胞凋亡率比较差异有统计学意义.给予9.37和18.74 μmol/L紫杉醇作用24 h后,MCF-7DEDD-shRNA细胞的凋亡率分别为(30.26±1.63)％和(32.18±2.16)％,明显低于MCF-7 control shRNA组的(53.84±2.17)％和(58.27±2.16)％,P＜0.001.多柔比星作用后不同时间BCRP蛋白质印迹检测统计分析结果显示,不同处理(F=14.67,P＜0.001)、不同时间(F=6.39,P=0.006)组间BCRP蛋白表达比较差异有统计学意义.给予0.5 μmol/L多柔比星作用24 h后,3组细胞的BCRP相对表达水平分别为0.87±0.04、1.06±0.02和5.25±0.18.MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.多柔比星作用48 h后,3组细胞的BCRP相对表达水平分别为1.06±0.01、0.97±0.04和2.98±0.13.MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平出现回落,但仍显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.同样,紫杉醇作用后不同时间BCRP蛋白质印迹检测统计分析结果显示,不同处理(F=7.26,P=0.004)、不同时间(F=8.32,P=0.002)组间BCRP蛋白表达比较差异有统计学意义.给予9.37 μmol/L紫杉醇作用24 h后,3组细胞的BCRP相对表达水平分别为0.81±0.05、2.25±0.10和1.78±0.14.其中MCF-7 control shRNA组细胞BCRP表达水平高于MCF-7 WT (P＜0.001)和MCF-7 DEDD-shRNA (P=0.002)组.而紫杉醇作用48 h后,3组细胞的BCRP相对表达水平分别为0.73±0.04、1.73±0.05和3.12±0.12,MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平继续增高,且显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.结论 敲低DEDD表达可增强乳腺癌细胞的抗肿瘤药耐药性,BCRP的表达增加可能是低表达DEDD乳腺癌细胞多药耐药的机制之一.

Effects and meschanisms of death effector domain DNA-binging protein on multiple drug resistance of breast cancer

OBJECTIVE Our previous study highlighted that DEDD (death effector domain DNA-binging protein) could inhibit the growth and metastasis of breast cancer.However,the effect of DEDD on multiple drug resistance is still unkown.In this study,we aimed to explore the effects and meschanisms of DEDD on multiple drug resistance of breast cancer.METHODS Stable knockdown DEDD cells were established by transfecting DEDD shRNA into MCF-7 cells.MTS assay was used to evaluate the cell viability of MCF-7 cells (Wild type/WT,MCF-7 control shRNA and DEDD shRNA) after treated with doxorubicin or paclitaxel for indicated times.Cell apoptosis was measured by flow cytometric analysis after Annexin V/PI double staining.The expressions of BCRP (breast cancer resistance protein) were detected by immunobloting.RESULTS After treatment with doxorubicin,MTS results showed that there were significant differences between the different cells (F=89.49,P＜0.001) or different treatment time (F-50.81,P＜0.001) groups.Tthe IC50 values of doxorubicin in MCF-7 DEDD-shRNA cells were (0.97±0.15) and (0.62±0.09) μmol/L respectively after 24 h or 48 h treatment with doxorubicin,which were significantly higher than that of MCF-7 WT cells (0.46 ± 0.05) μmol/L,(0.26±0.03) μmol/L] and MCF-7 control shRNA cells (0.39±0.05) μmol/L,(0.25±0.03) μmol/L,P＜0.001].After treatment with paclitaxel,there were significant differences in IC50 between the different cells (F=15.94,P＜ 0.001) or different treatment time (F=129.70,P＜0.001) groups.The IC50 valus of paclitaxel in MCF-7 DEDD-shRNA cells were (16.74±2.26) and (9.88±1.47) μmol/L respectively after treatment for 24 h or 48 h,which were significantly higher than that of MCF-7 WT cells (13.39±1.44 μmol/L,(7.69±0.92) μmol/L,P=0.001] and MCF-7 control shRNA cells (12.58±1.15) μmol/L,(7.17±1.12) μmol/L,P＜0.001].After treatment with doxorubicin,apoptosis assay showed that there were significant differences between the different cells (F=131.46,P＜0.001) or different concentrations (F=160.95,P＜0.001) groups.Under normal culture conditions,the apoptotic rates of MCF-7 cells were as follows:MCF-7 control shRNA cells (14.32 ± 1.47)％,MCF-7 DEDD-shRNA cells (11.58 ± 1.63)％.After treatment of 0.5 μmol/L or 1μmol/L doxorubicin for 24 h,the apoptotic rates of MCF-7 DEDD-shRNA cells were (21.62± 1.97)％ and (24.39 ± 2.36)％,which were significantly lower than that of MCF-7 control shRNA cells (36.26±1.87)％ and (38.23±1.46)％,P＜0.001.Similarly,after treatment of 9.37 μmol/L or 18.74 μmol/L paclitaxel for 24 h,there were significant differences between the different cells (F=124.81,P＜0.001) or different concentrations (F=172.56,P＜0.001) groups.The apoptotic rates of MCF-7 DEDD-shRNA cells were (30.26 ± 1.63)％ and (32.18 ± 2.16)％,which were significantly lower than that of MCF-7 control shRNA cells (53.84 ± 2.17)％,P＜ 0.001.Immunobloting results showed that there were significant differences in BCRP levels after treatment with doxorubicin between the different cells (F=14.67,P＜0.001) or different treatment time (F=6.39,P=0.006) groups.After treatment with 0.5 μmol/L doxorubicin for 24 h,the relative levels of BCRP in three kinds of cells were as follows:MCF-7 WT 0.87 ± 0.04,MCF-7 control shRNA 1.06 ± 0.02 and MCF-7 DEDD-shRNA 5.25 ± 0.18.The levels of BCRP in MCF-7 DEDD-shRNA cells were significantly higher compared with MCF-7 WT and MCF-7 control shRNA cells (P＜0.001).After treatment with 0.5 μmol/L doxorubicin for 48 h,the relative levels of BCRP in three kinds of cells were 1.06±0.01,0.97±0.04 and 2.98±0.13.The levels of BCRP in MCF-7 DEDD-shRNA cells showed a deceasing,but still higher that MCF-7 WT and MCF-7 control shRNA cells (P＜0.001).Similarly,there were significant differences in BCRP levels after treatment with paclitaxel between the different cells (F=7.26,P=0.004) or different treatment time (F=8.32,P=0.002) groups.After treatment of 9.37 μmol/L paclitaxel for 24 h,the relative levels of BCRP in three kinds of cells were 0.81±0.05,2.25±0.10 and 1.78±0.14.The levels of BCRP in MCF-7 control shRNA cells were higher than that of MCF-7 WT and MCF-7 DEDD-shRNA cells (P＜0.001).However,after treatment of 9.37 μmol/L paclitaxel for 48 h,the levels of BCRP in MCF-7 DEDD-shRNA increased continuously,and which were significantly higher than that of MCF-7 WT and MCF-7 control shRNA cells (P＜0.001).CONCLUSIONS In this study,we demonstrate that DEDD knockdown in MCF-7 cells showed characteristic multiple drug resistance to conventional chemotherapy drugs.The increasing of BCRP levels was a potential mechanism of multidrug resistance in DEDD lower expression breast cancer cells.