Establishment of DDP Resistant Variant of Triple Negative Breast Cancer Cell Line MDA-MB-231/DDP and Its Appraisal
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摘要: 目的 建立人三阴性乳腺癌细胞MDA-MB-231顺铂（DDP）耐药细胞株，并对其耐药机制进行初步分析。方法 采用逐步增加顺铂浓度、间歇诱导的方法，建立人三阴性乳腺癌MDA-MB-231/DDP体外耐药细胞模型；MTT法检测MDA-MB-231/DDP的耐药特性；高效液相色谱法检测耐药细胞内DDP药物浓度的改变；实时荧光定量PCR法分析两种细胞耐药相关基因MDR1、BCRP、MMP7及 GST-π表达差异。结果 MTT显示MDA-MB-231/DDP的顺铂、5-氟尿嘧啶及环磷酰胺的耐药指数分别为9.80、4.43及2.21；高效液相检测显示2种细胞分别与6 μg/ml的DDP接触24 h后，MDA-MB-231细胞内DDP的浓度为（47.10±2.37）ng/（5×104） cells，而MDA-MB-231/DDP细胞则为（6.30±1.64）ng/（5×104）cells（P<0.01）；MDA-MB-231/DDP细胞中MDR1、BCRP、MMP7及GST-π基因 mRNA的表达量分别是亲本细胞的15.39、13.73、22.52及44.90倍（P<0.01）。结论 逐步增加浓度、间歇诱导的方法建立了稳定、耐药性较高的MDA-MB-231/DDP细胞株，其耐药机制可能与ABC转运蛋白表达增加等多种机制相关。Abstract: Objective To establish a triple negative breast cancer cell line MDA-MB-231 with the characterization of cisplatin (DDP) resistance, and investigate its biological mechanism of drug resistance. Methods A DDP-resistant human triple negative breast cancer cell line MDA-MB-231/DDP was obtained discontinuously by gradually increasing doses of DDP. The drug resistance of MDA-MB-231/DDP cells was evaluated by MTT assay. The variation of intracellular concentration of DDP in MDA-MB-231/DDP cells was evaluated by HPLC. The expression of MDR1, BCRP, MMP7 and GST-π were detected by Real-time PCR. Results The cisplatin, 5-Fu and CTX were acting on MDA-MB-231/DDP and the resistance fold (RF) were 9.80, 4.43 and 2.21. After treated with 6μg/ml DDP for 24h, the concentration of DDP in MDA-MB-231 and MDA-MB-231/DDP cells were (47.10±2.37)ng/(5×104) cells and (6.30±1.64) ng/(5×104) cells (P<0.01). The expression of MDR1, BCRP, MMP7 and GST-π mRNA in MDA-MB-231/DDP cells were 15.39, 13.73, 22.52 and 44.90 times the expression of those in the parent cells. Conclusion A drug-resistant cell line MDAMB-231/DDP with high resistance fold is established by discontinuous induction and gradually increasing doses of DDP. The resistance mechanism may be associated with the increased expression of ABC transporter
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