Lin Meng-Liang, Chen Shih-Shun, Ng Sue-Hwee
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, R.O.C.
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan, R.O.C.
Anticancer Res. 2015 Oct;35(10):5359-68.
The endoplasmic reticulum chaperone glucose-regulated protein 78 (GRP78) is selectively expressed on the surface of cancer cells, and contributes to the survival of cancer cells by forming complexes with p85α and promoting phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling. Hereιin we report that 2'-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) induces apoptosis of human nasopharyngeal carcinoma (NPC) cells, as characterized by morphological changes, DNA fragmentation, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase. Using cell surface biotinylation, flow cytometric analysis, co-immunoprecipitation, and ectopic expression of GRP78, we demonstrated that the attenuation of the cell surface localization and complex formation with p85α of GRP78 by CHM-1 was involved in the inhibition of PI3K-AKT signaling and the induction of apoptosis. CHM-1 treatment induced phosphorylation on Thr 69 of B cell lymphoma 2 and inhibited phosphorylation of Ser 136 on Bcl-2-associated death promoter, that were reversed by overexpression of GRP78. We further observed that loss of mitochondrial membrane potential and increase in reactive oxygen species content, release of mitochondrial cytochrome c, caspase-9 activation, and apoptotic cell death induced by CHM-1, were suppressed by treatment with cyclosporine A, and by the overexpression of constitutively active AKT1 or GRP78. These results indicate that CHM-1 induces NPC cell apoptosis by suppressing the formation of the cell surface-associated GRP78-PI3K-AKT signaling complex, likely through inhibition of the formation of cell surface-associated GRP78-p85α complexes.
内质网伴侣葡萄糖调节蛋白78（GRP78）在癌细胞表面选择性表达，并通过与p85α形成复合物并促进磷脂酰肌醇3激酶-蛋白激酶B（PI3K-AKT）信号传导来促进癌细胞存活。在此我们报告，2'-氟-6,7-亚甲基二氧基-2-苯基-4-喹诺酮（CHM-1）可诱导人鼻咽癌（NPC）细胞凋亡，其特征为形态变化、DNA片段化、半胱天冬酶-3激活以及聚（ADP-核糖）聚合酶的裂解。通过细胞表面生物素化、流式细胞术分析、免疫共沉淀以及GRP78的异位表达，我们证明CHM-1对GRP78细胞表面定位及其与p85α复合物形成的减弱参与了对PI3K-AKT信号传导的抑制和凋亡的诱导。CHM-1处理诱导了B细胞淋巴瘤2第69位苏氨酸的磷酸化，并抑制了Bcl-2相关死亡促进因子第136位丝氨酸的磷酸化，而GRP78的过表达可逆转这些变化。我们进一步观察到，CHM-1诱导的线粒体膜电位丧失、活性氧含量增加、线粒体细胞色素c释放、半胱天冬酶-9激活以及凋亡细胞死亡，均被环孢素A处理以及组成型活性AKT1或GRP78的过表达所抑制。这些结果表明，CHM-1可能通过抑制细胞表面相关的GRP78-p85α复合物的形成，来抑制细胞表面相关的GRP78-PI3K-AKT信号复合物的形成，从而诱导NPC细胞凋亡。
