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目的:观察5’-氮杂-脱氧胞苷酸(5’-Aza-2’-deoxycytidine,5’-Aza-dC)联合他莫昔芬(tamoxifen,TAM)对乳腺癌耐药相关蛋白(breast cancer resistance protein,BCRP)介导的雌激素受体α(estrogen receptor alpha,ERα)阴性乳腺癌细胞多柔比星(adriamycin,ADR)耐药的影响。方法:5’-Aza-dC联合不同浓度TAM处理乳腺癌MDA-MB-435s细胞后,应用甲基化特异性PCR法检测ERα基因启动子区甲基化状态,蛋白质印迹法检测ERα与BCRP蛋白的表达,MTT法检测ADR半数抑制浓度(half inhibitory concentration,IC50)。结果:5’-Aza-dC可浓度依赖性去除MDA-MB-435s细胞ERα基因启动子区甲基化并恢复ERα蛋白的表达,0.5μmol/L5’-Aza-dC可获得较高水平ERα蛋白的表达;0.5μmol/L5’-Aza-dC或100μmol/LTAM单独处理细胞,对BCRP蛋白的表达水平无明显影响;0.5μmol/L5’-Aza-dC联合1、10和100μmol/LTAM处理细胞后,BCRP蛋白的相对表达量分别降低了18.3%、41.1%和75.3%;0.5μmol/L5’-Aza-dC联合10和100μmol/LTAM处理后,细胞对ADR敏感度分别增强至阴性对照组的1.4和4.2倍。结论:在5’-Aza-dC恢复ERα表达的前提下,TAM可显著下调ERα阴性乳腺癌细胞BCRP蛋白的表达并增强细胞对ADR的敏感度,2者联合可能成为逆转ERα阴性乳腺癌细胞多药耐药的一种途径。
OBJECTIVE: To observe the effect of 5’-Aza-deoxycytidine (5’-Aza-dC) and tamoxifen (TAM) on breast cancer resistance- cancer resistance protein (BCRP) -mediated estrogen receptor alpha (ERα) -negative breast cancer cell line adriamycin (ADR). Methods: Methylation-specific PCR was used to detect the methylation status of ERα gene promoter region after treatment with 5’-Aza-dC and different concentrations of TAM in breast cancer MDA-MB-435s cells. Western blotting was used to detect ERα and BCRP protein The half inhibitory concentration (IC50) of ADR was detected by MTT assay. Results: 5’-Aza-dC could down-regulate the promoter region of ERα gene in MDA-MB-435s cells and restore the expression of ERα protein in a concentration-dependent manner. Higher levels of ERα protein were obtained with 0.5μmol / L 5’- 0.5μmol / L 5’-Aza-dC or 100μmol / LTAM alone had no effect on the expression of BCRP protein. After treated with 0.5μmol / L 5’-Aza-dC in combination with 1,10 and 100μmol / , The relative expression of BCRP protein decreased by 18.3%, 41.1% and 75.3% respectively. After treated with 0.5μmol / L 5’-Aza-dC in combination with 10 and 100μmol / LTAM, the sensitivity of cells to ADR increased to 1.4 And 4.2 times. CONCLUSION: TAM can down-regulate the expression of BCRP protein and enhance the sensitivity of cells to ADR under the premise that 5’-Aza-dC restored the expression of ERα. The combination of these two factors may reverse the ERα-negative breast cancer cells A way of drug resistance.