自噬在多发性骨髓瘤细胞系RPMI8226阿霉素耐药中的作用

Autophagy is involved in doxorubicin induced resistance of human myeloma cell line RPMI8226

目的探讨自噬在多发性骨髓瘤（MM）细胞阿霉素（DOX）耐药中的作用。方法用浓度递增法诱导多发性骨髓瘤细胞系RPMI8226细胞建立DOX耐药株RPMI8226／DOX；用M1Tr法测定RPMI8226／DOX细胞耐药倍数；分别用透射电镜、抗LC3一FITC抗体免疫荧光、WesternbIot检测MM细胞自噬情况；AnnexinV／P1标记流式细胞术检测羟氯喹、3-甲基腺嘌呤（3-MA）抑制自噬对DOX诱导RPMI8226／DOX细胞凋亡的影响。结果通过浓度梯度递增法诱导的RPMI8226／DOX细胞，耐药倍数约为10．8，透射电镜下RPMI8226／DOX细胞出现较多自噬体或自噬溶酶体；荧光显微镜下见RPMI8226／D0x细胞出现荧光颗粒；WesternbIot检测结果显示RPMI8226／DOX细胞LC3-Ⅱ蛋白表达水平较RPMI8226细胞升高；流式细胞术检测结果显示：单用8ixmoL／L羟氯喹和10mmoI／L3-MA作用24h后RPMI8226／DOX细胞凋亡率分别为（3．24±1．08）％和（2．81±0．80）％，与空白对照组的（2．12±1．24）％比较，差异无统计学意义（P〉0．05）；2、4、6μmol／LDOX作用24h后RPMI8226／DOX细胞凋亡率为（9．75±2．15）％、（24．36±2．16）％、（40．51±3．14）％；在此基础上加入8μmol／L羟氯喹后细胞凋亡率分别上升为（16．56±1．89）％、（36．4±2．91）％和（62．68±3．75）％，与单用DOX相比差异有统计学意义（P〈0．05）；而加用10mmol／L3-MA后细胞凋亡率分别上升为（15．47±1．85）％、（39．28±3．06）％和（55．46±4．07）％，与单用DOX相比差异亦有统计学意义（P〈0．05）。结论自噬在耐药骨髓瘤细胞中被活化，抑制自噬可部分逆转DOX诱导的骨髓瘤细胞耐药，提示自噬可能为骨髓瘤细胞耐药机制之一。

Objective To explore the role of autophagy in doxorubicin （ DOX）-induced resistance of human myeloma cell line RPMI8226. Methods We established doxorubicin induced resistant subline of my- eloma cell line RPMI8226/DOX by drug concentration step-elevation method. Resistant index of DOX was measured by MTY assay. Autophagy of myeloma cell lines RPMI8226/s and RPMI8226/DOX was detected by transmission electron microscopy, immunofluorescence （LC3-FITC） and western blot respectively. Apoptosis of RPMI8226/DOX cells induced by DOX combined with autophagic inhibitor hydroxychloroquine or 3-MA was identified by Annexin V-FITC/PI double fluorescence dyeing. Results Resistant index of RPMI8226/ DOX was approximately 10.8 fold of that of RPMI8226/S. Electron microscopic studies revealed that most of RPMI8226/DOX cells displayed viable attributes and contained numerous autophagic vacuoles. Fluorescent images of RPMI8226/DOX cells showed a punctuate distribution in LC3 protein. Increased LC3- II protein in RPMI8226/DOX cells was determined by immunoblotting. There were no differences among 8 μmol/L HCQ （ 3.24± 1.08 ）%, 10 mmol/L 3-MA （ 2.81±0.80） % or control [ （2.12 ±1.24） % ] （ P 〉 0.05 ） in terms of Annexin V-FITC/PI double fluorescence dyeing; Compared with apoptosis of （9. 75 ± 2. 15 ）%, （24.36±2.16）% and （40.51±3.14）% of RPMI8226/DOX cells under 2, 4 and 6 μmol/L DOX, apoptosis increased signifcantly after 24 h incubation under 2, 4 and 6μmol/L DOX combined with 8 μmol/L HCQ as of [ （ 16.56±1.89） %, （36.44±2.91 ） % and （62.68±3.75 ） %, respectively ], or under 2, 4and 6 txmol/L DOX combined with 10 mmol/L 3-MA as of ~ （ 15.47±1.85 ） %, （ 39.28 ± 3.06 ） % and （55.46 ±4.07 ） %, respectively I （ P 〈 0.05 ）. Conclusion Autophagy was involved in doxombicin-in- duced resistance of myeloma cell line RPMI8226, DOX resistance in myeloma cells was reversed partly by au- tophagy inhibitor hydroxyehloroquine or 3-MA, and autopahgy may be one of mechanisms for drug resistance.