低氧对肺腺癌多药耐药细胞耐药性的影响及其机制

Effects of hypoxia on drug sensitivity of pulmonary adenocarcinoma multidrug-resistance cell line and its mechanism

目的观察低氧对肺腺癌多药耐药细胞A549/CDDP耐药性的影响并探讨可能的机制。方法分别在常氧(21%O_2)及低氧(3%O_2)条件下,应用四甲基偶氮唑盐比色(MTT)法检测A549/CDDP对7种化疗药物的耐药性,应用Annexin V-PI染色流式细胞术检测细胞的凋亡及坏死,应用MTT法绘制生长曲线,PI染色流式细胞术检测细胞周期分布;罗丹明123排出实验检测细胞药物外排效率;测量各组细胞培养基pH值的变化情况,流式细胞术检测多柔比星在细胞内的累积情况,荧光显微镜/流式细胞仪观察ROS特异性探针DCFDA荧光强度。结果低氧条件下,A549/CDDP对多柔比星、VP-16、长春新碱、顺铂明显耐受,对丝裂霉素、5-FU、泰素帝敏感;增殖能力有所增强;药物外排效率无明显变化;培养基呈现明显酸化;细胞内多柔比星的浓度下降;细胞内ROS浓度降低。结论低氧显著影响肺腺癌多药耐药细胞的耐药特性。微环境酸化引起细胞内药物浓度的不同可能是导致这一现象的原因之一。同时,增殖能力的改变,ROS的产生可能也参与其中。

Objective To investigate the efficacy of seven cytotoxic drugs used in pulmonary adenocarcinoma multidrug resistant cell line A549/CDDP under hypoxia （3% O2 ） and to explore the possible mechanisms for the change of efficacy. Methods The efficacy of seven kinds of cytotoxic drugs for A549/ CDDP cell lines under hypoxia was analyzed by MTT assay. Then the results from MTT assay were affirmed by flow cytometry for Annexin-V-PI double staining. The proliferation of A549/CDDP was determined by MTT assay and flow cytometry for PI staining. After that, Rhodamine123 exclusion assay was employed to explore the efficacy of drug exclusion mediated by P-gp. The generation of ROS was detected by fluorescence microscopy/flow cytometry. At last, the relationship between pH of culture medium and the intracellular concentration of daunorubicin was investigated. Results The efficacy of cytotoxic drugs under hypoxia was different from that under normoxia. Proliferation of A549/MDR cells was enhanced under hypoxia but no close correlation was found between proliferation and cytotoxic effects. Under hypoxia, the efficacy of rhodamine123 efflux was not changed, culture medium became more acidic and the generation of ROS decreased. Furthermore, the intracellular fluorescence intensity of daunorubicin was much lower in this acidic microenvironment. Conclusion These results indicated that the drug susceptibility was greatly influenced by hypoxia and different intracellular concentrations of drugs induced by microenvironment acidification may be the main cause of the change of drug efficacy. In addition, proliferation may cause the change of drug resistance under hypoxia for A549/MDR cells. The decreased generation of ROS may be another reason for the resistance of A549/CDDP cell line to dannorubicin under hypoxic condition. Moreover, the drug exclusion mediated by P-gp may not be the key reason.