Zhang Fan, Suarez Giovanni, Sha Jian, Sierra Johanna C, Peterson Johnny W, Chopra Ashok K
Department of Microbiology & Immunology, Institute of Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA.
Cell Signal. 2009 Jul;21(7):1085-99. doi: 10.1016/j.cellsig.2009.02.018. Epub 2009 Mar 1.
Phospholipase A(2) (PLA(2))-activating protein (PLAA) is a novel signaling molecule that regulates eicosanoid production and participates in inflammatory responses. In our current study, we revealed that PLAA production was induced by the chemotherapeutic drug cisplatin in HeLa cervical carcinoma cells. To determine the potential pro-apoptotic effects of PLAA induction by cisplatin, we utilized HeLa (Tet-off) cells overexpressing the plaa gene (plaa(high)) and compared them with control (plaa(low)) cells, which produce endogenous plaa from the chromosome. Cisplatin-stimulated plaa(high) cells contained significantly higher levels of DNA fragmentation, caspase 3, 8 and 9 activities, PLA(2) enzyme activity, and cytochrome c leakage from mitochondria than did the cisplatin-stimulated plaa(low) cells. Importantly, siRNA against PLAA (siRNA-PLAA) reduced the levels of cisplatin-induced PLAA, DNA fragmentation, and PLA(2) activation, while promoting cell viability in both plaa(high) and plaa(low) cells. Cisplatin-induced-cytochrome c leakage in plaa(high) cells was reduced by siRNA-PLAA and restored by the addition of exogenous arachidonic acid (AA), suggesting to us that PLAA induction by cisplatin promoted cytochrome c leakage/mitochondrial damage partially by accumulating AA. In addition, cisplatin-stimulated plaa(high) cells produced less cytoprotective clusterin than did the cisplatin-stimulated plaa(low) cells, and siRNA-PLAA promoted clusterin production from both plaa(high) and plaa(low) cells. We showed that clusterin reduced DNA fragmentation in cisplatin-stimulated plaa(high) and plaa(low) cells, which is consistent with the notion that clusterin promotes cancer chemoresistance. Furthermore, cisplatin-stimulated plaa(high) cells produced more IL-32 (a pro-apoptotic protein) than did cisplatin-stimulated plaa(low) cells, and siRNA-PLAA reduced IL-32 production from both plaa(high) and plaa(low) cells. Finally, our proteomic analysis revealed that cisplatin-stimulated plaa(high) cells contained higher levels of phosphorylated JNK/c-Jun and FasL than did plaa(low) cells treated the same way. In summary, our data indicated that PLAA induction enhanced cisplatin-induced-apoptosis through four pathways, namely by: 1) accumulation of AA and mitochondrial damage, 2) downregulation of the cytoprotective clusterin, 3) upregulation of the pro-apoptotic IL-32, and 4) induction of JNK/c-Jun signaling and FasL expression.
磷脂酶A(2)（PLA(2)）激活蛋白（PLAA）是一种新型信号分子，可调节类花生酸的产生并参与炎症反应。在我们当前的研究中，我们发现化疗药物顺铂可诱导人宫颈癌HeLa细胞产生PLAA。为了确定顺铂诱导PLAA产生的潜在促凋亡作用，我们利用过表达plaa基因的HeLa（Tet-off）细胞（plaa(high)），并将其与从染色体产生内源性plaa的对照细胞（plaa(low)）进行比较。与顺铂刺激的plaa(low)细胞相比，顺铂刺激的plaa(high)细胞中DNA片段化水平、半胱天冬酶3、8和9的活性、PLA(2)酶活性以及线粒体细胞色素c泄漏水平显著更高。重要的是，针对PLAA的小干扰RNA（siRNA-PLAA）降低了顺铂诱导的PLAA水平、DNA片段化和PLA(2)激活，同时提高了plaa(high)和plaa(low)细胞的活力。siRNA-PLAA减少了顺铂诱导的plaa(high)细胞中的细胞色素c泄漏，并通过添加外源性花生四烯酸（AA）得以恢复，这向我们表明顺铂诱导的PLAA通过积累AA部分促进了细胞色素c泄漏/线粒体损伤。此外，与顺铂刺激的plaa(low)细胞相比，顺铂刺激的plaa(high)细胞产生的细胞保护蛋白簇集素较少，而siRNA-PLAA促进了plaa(high)和plaa(low)细胞中簇集素的产生。我们发现簇集素减少了顺铂刺激的plaa(high)和plaa(low)细胞中的DNA片段化，这与簇集素促进癌症化疗耐药性的观点一致。此外，与顺铂刺激的plaa(low)细胞相比，顺铂刺激的plaa(high)细胞产生的白细胞介素32（一种促凋亡蛋白）更多，而siRNA-PLAA减少了plaa(high)和plaa(low)细胞中白细胞介素32的产生。最后，我们的蛋白质组学分析表明，与以相同方式处理的plaa(low)细胞相比，顺铂刺激的plaa(high)细胞中磷酸化JNK/c-Jun和FasL水平更高。总之，我们的数据表明，PLAA的诱导通过四种途径增强了顺铂诱导的凋亡，即：1）AA积累和线粒体损伤；2）细胞保护蛋白簇集素的下调；3）促凋亡白细胞介素32的上调；4）JNK/c-Jun信号传导和FasL表达的诱导。
