抗HSP90抗体通过内化eHSP90抑制人胃癌干细胞并提高顺铂的抗癌效果

Anti-HSP90 antibody promotes the anticancer effect of cisplatin by inhibiting human gastric cancer stem cells through internalizing eHSP90

目的:探索抗HSP90单克隆抗体28C10通过靶向肿瘤干细胞促进顺铂(cisplatin,DDP)对人胃癌细胞PAMC82恶性生物学行为的抑制效果及其可能的作用机制。方法:28C10单独或与DDP联合处理人胃癌细胞PAMC82,采用不同实验方法检测该细胞的无血清成球能力、迁移和侵袭能力与克隆形成能力,CCK-8法检测28C10对PAMC82细胞恶性生物学行为和协同DDP抗癌能力的影响。采用细胞免疫荧光及流式细胞术检测PAMC82细胞中HSP90及eHSP90(extracellular HSP90)的表达、定位、eHSP90^(+)亚群比例,以及28C10处理后对ALDH^(+)、CD44^(+)、eHSP90^(+)细胞亚群的影响。采用WB实验检测28C10作用后PAMC82细胞中HSP90、干性相关蛋白以及PI3K/AKT/mTOR信号通路蛋白表达的变化。结果:胃癌细胞PAMC82膜表面表达eHSP90,具有2%~3%的eHSP90^(+)细胞亚群,且eHSP90^(+)细胞多为与ALDH^(+)或CD44^(+)共阳性细胞。28C10处理能显著抑制PAMC82细胞的成球、克隆形成、增殖、耐药、迁移及侵袭能力,而且和DDP联用的效果更明显(P<0.05或P<0.01)。流式细胞术分析发现28C10处理显著抑制PAMC82细胞的eHSP90^(+)、ALDH^(+)和CD44^(+)亚群数量(均P<0.01)。免疫荧光实验发现28C10作用后eHSP90发生内吞,WB实验结果显示eHSP90、CD44、ALDH和干性相关蛋白OCT4、SOX2表达量均降低(P<0.05或P<0.01)。结论:抗HSP90单克隆抗体28C10可靶向胃癌PAMC82细胞的ALDH^(+)、CD44^(+)肿瘤干细胞相关亚群、内化eHSP90且降低细胞总HSP90的水平、抑制PI3K/AKT/mTOR信号通路,从而有效地抑制PAMC82细胞的干性、耐药和其他恶性生物学行为,协同DDP显著提高抗癌效果。

Objective:To explore the promotive effect of anti-HSP90 monoclonal antibody mAb 28C10 on Cisplatin(DDP)inhibiting malignant biological behavior of human gastric cancer PAMC82 cells by targeting tumor stem cells and the possible mechanisms of action.Methods:Human gastric cancer PAMC82 cells were treated with 28C10 alone or in combination with DDP.Then,the serumfree pellet-forming ability,clone-forming ability,migration and invasion ability of PAMC82 cells were detected by different experiments,and the effects of 28C10 on the malignant biological behaviors of PAMC82 cells and the synergistic anti-cancer effect of28C10 and DDP were detected by CCK-8 method.The expression,localization,e HSP90;subgroup ratio of HSP90 and eHSP90(extracellular HSP90)in PAMC82 cells and the effects of 28C10 on ALDH^(+),CD44^(+),eHSP90^(+)subgroups of PAMC82 cells were detected by cellular immunofluorescence and flow cytometry.The changes in HSP90,stemness-related proteins and PI3K/AKT/mTOR signaling pathway in PAMC82 cells treated with 28C10 were detected by WB.Results:e HSP90 was expressed on the membrane surface of gastric cancer PAMC82 cells,and there were about 2%-3%of eHSP90^(+)subgroup cells.eHSP90^(+)cells were mostly co-positive with ALDH^(+)or CD44^(+)cells.28C10 significantly inhibited the ability of pellet formation,clone formation,proliferation,drug resistance,migration and invasion of PAMC82 cells;moreover,the effect was more obvious when combined with DDP(all P<0.05or P<0.01).Flow cytometry analysis showed that 28C10 treatment significantly inhibited the number of eHSP90^(+),ALDH^(+)and CD44^(+)subgroups of PAMC82 cells(all P<0.01).Immunofluorescence assay showed that endocytosis of eHSP90 occurred after 28C10treatment.WB results showed that the expression levels of e HSP90,CD44,ALDH and the expression of stemness-related proteins OCT4 and SOX2 were decreased(P<0.05 or P<0.01).Conclusion:28C10 can target ALDH^(+)and CD44^(+)tumor stem cell-related subgroups of gastric cancer PAMC82 cells,internalize e HSP90,reduce the level of total HSP90,and inhibit PI3K/AKT/mTOR signaling pathway,thereby effectively inhibiting stemness,drug resistance and other malignant biological behaviors of PAMC82 cells and synergistically improving the anticancer effect of DDP.