高压氧联合阿霉素对阿霉素耐药神经胶质瘤细胞株U87敏感性的影响及其分子机制

Effects of hyperbaric oxygen combined with adriamycin on the sensitivity of adriamycin drug-resistant glioma U87 cells and possible mechanism involved

目的探讨高压氧(HBO)联合阿霉素(ADR)对ADR耐药神经胶质瘤细胞株U87(U87/ADR)敏感性的影响及其分子机制。方法建立ADR耐药神经胶质瘤U87细胞(U87/ADR)株;按照实验设计分成4组,对照组、ADR组、HBO组、HBO+ADR组。用MTT实验检测HBO+ADR对U87/ADR细胞增殖能力的影响;利用Annexin V-FITC/PI双染法和流式细胞术检测HBO+ADR对U87/ADR细胞凋亡的影响;利用Western blotting检测U87/ADR细胞凋亡相关蛋白及JNK通路蛋白的表达水平。结果ADR浓度为40、60、80μg/ml时,HBO+ADR能够有效逆转U87/ADR细胞对ADR耐药作用(P<0.01);Hochest33324染色实验显示蓝色荧光强度显著升高,细胞核由于浓集而呈亮蓝色,细胞呈明显凋亡形态;流式细胞术和凋亡蛋白的检测结果进一步表明,HBO+ADR后促进U87/ADR细胞凋亡率(P<0.01);HBO+ADR可激活JNK信号通路并抑制U87/ADR细胞中P-gp、MRP蛋白的表达(P<0.01)。结论HBO与ADR联用增强U87/ADR细胞对ADR的敏感性并诱导细胞凋亡,其机制可能与联用HBO后调控JNK信号通路和耐药相关蛋白有关。

Objective To explore the effects of hyperbaric oxygen(HBO)combined with adriamycin(ADR)on the sensitivity of adriamycin drug-resistant glioma U87 cells and possible mechanism involved.Methods An adriamycin-resistant glioma U87/ADR cell line was established.In accordance with the experimental design,the subjects were divided into 4 groups;i.e.the control group;the ADR group;the HBO group and the HBO+ADR group.The effects of HBO combined with ADR on the proliferation of U87/ADR cells were detected by MTT assay;the effects of HBO+ADR on the apoptosis of U87/ADR cells were detected by Annexin V-FITC/PI double staining and flow cytometry and the expression levels of apoptosis-related U87/ADR and JNK pathway proteins were detected by Western blotting.Results With ADR levels at 40,60 and 80μg/ml,HBO+ADR could effectively reverse the drug resistance of U87/ADR cells to ADR(P<0.01).Hochest33324 staining experiment showed that the blue fluorescence intensity significantly increased,and the nucleus was bright blue due to concentration,displaying a distinct apoptotic morphology.Flow cytometry and apoptotic protein detection further indicated that HBO+ADR treatment could enhance apoptosis rate of U87/ADR cells(P<0.01).HBO+ADR could activate JNK signaling pathway and inhibit the expression levels of P-gp and MRP proteins in the U87/ADR cells(P<0.01).Conclusion The combined use of HBO and ADR could enhance the sensitivity of U87/ADR cells to adriamycin and induce apoptosis,the mechanism of which might be related to the regulation of JNK signaling pathway and drug resistance-related proteins.