钠氢交换蛋白抑制剂Cariporide对MCF-7/ADR细胞化疗敏感性影响机制研究

目的 肿瘤细胞膜内外pH值的改变对于肿瘤的侵袭、转移密切相关,通过细胞内外的H+/Na+交换维持细胞内环境的稳态,细胞的凋亡水平与化疗药物耐药的发生密切相关.本研究观察钠氢交换蛋白1(sodium hydrogen exchange 1,NHE-1)抑制剂 Cariporide(CP)对人乳腺癌细胞MCF-7/ADR增殖、凋亡和对多柔比星(adriamycin,ADR)敏感性影响,并探讨其相关作用机制.方法 以终浓度分别为3、6、9、12、60和100 μg/mL的CP处理MCF-7和MCF-7/ADR细胞48 h,以终浓度分别为1、5、50和100 μg/mL的ADR以及2种药物联用分别处理MCF-7/ADR细胞24、48 h,CCK8法检测其对细胞的增殖影响,Graphpad prism 5分别计算单用及药物联用时的IC50,CompuSyn软件计算2种药物的联用指数(CI);采用流式细胞术检测药物单用及联用后对细胞凋亡率的影响;RT-PCR法检测MDR-1、NF-κB等相关核转录因子的表达;蛋白质印迹法检测CD44、MDR-1、NF-κB p65、Caspase-3/9和Bcl-2的表达.结果 CP对乳腺癌细胞有增殖抑制作用,呈剂量及时间依赖性,CP与ADR联用可以明显降低ADR对MCF-7/ADR细胞的IC50值,其值由(82.45±5.86) μg/mL下降至(42.2±2.03) μg/mL,t=7.57,P<0.05.流式细胞术检测结果显示,对照组、单用ADR组、单用CP组、药物联用组细胞的凋亡率分别为(4.02±0.19)%、(6.34±0.39)%、(9.55±0.47)%和(15.12±0.65)%,F=666.2,P<0.001.荧光定量PCR结果表明,单用CP及CP联用ADR组MDR-1、NF-κB mRNA 表达均下降.蛋白质印迹法结果显示,单用CP及CP联用ADR耐药相关蛋白CD44、MDR-1表达下调,NF-κB p65表达下调,抑凋亡蛋白Bcl-2表达下降,凋亡相关蛋白cleaved Caspase-3和cleaved Caspase-9表达上调.结论 CP可以增加MCF-7/ADR细胞对ADR的敏感性,可能是通过下调耐药相关蛋白的表达,活化凋亡相关蛋白,诱导细胞凋亡,从而有效逆转肿瘤细胞的耐药.

Mechanism of NHE1 inhibitor on the sensitivity of MCF-7/ADR cells to chemotherapeutic drug resistance

OBJECTIVE The change of pH value in tumor cell membrane was closely related to the invasion and metastasis of tumor, intracellular and extracellular H+/Na+ exchange could maintain the homeostasis of intracellular environment, and apoptosis of tumor cells was closely related to drug resistance to chemotherapy.The objective of this study was to explore the proliferation and apoptosis effects of NHE-1 inhibitor (Cariporide, CP) on human breast cancer MCF-7/ADR cells and the relative signal pathway.METHODS MCF-7 and MCF-7/ADR cells was cultured in vitro and treated with CP and ADR respectively (3、6、9、12、60、100 μg/mL) and (1、5、50、100 μg/mL), the proliferation rates were determined by CCK8 assay, the value of IC50 was analyzed by Graphpad prism 5,and the combination index(CI) was counted by CompuSyn.Apoptosis was determined with flow cytometry (FCM).The protein expressions of MDR-1, NF-κB p65, CD44, cleaved caspase3/9 and Bcl-2 were detected by western blot.The levels of MDR-1 and NF-κB mRNA were analyzed by RT-PCR.RESULTS CCK8 assay showed that the inhibition was positively correlated to the concentration of drugs and time when the MCF-7 and MCF-7/ADR cells were exposed respectively 3-100 μg/mL CP and 1-100 μg/mL ADR.CP combined with ADR can significantly reduce the IC50 value, which was decreased from (82.45±5.86) μg/mL to (42.2±2.03) μg/mL, t=7.57, P<0.05.Flow Cytometry showed that the apoptosis rate of MCF-7/ADR cells stimulated by none drugs、only ADR、only CP、ADR and CP for 48 h respectively were (4.02±0.19)%、(6.34±0.39)%、(9.55±0.47)%、(15.12±0.65)%,F=666.2,P<0.001.The RNA levels of MDR-1 and NF-κB were decreased.The expression of the protein and apoptosis protein such as MDR-1、NF-κB p65、CD44、Bcl-2 were down regulated, while cleaved caspase3 and cleaved caspase-9 were up regulated.CONCLUSIONS NHE-1 inhibitor (cariporide) may significantly inhibit the proliferation of human breast cancer cells and increase the sensitivity of MCF-7/ADR cells to adriamycin, which may be related to the down regulation of drug resistance related proteins, the activation of apoptosis related proteins and apoptosis induction.These may effectively reverse the multidrug resistance of cancer cells.