Phospholipase A 2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells

Abstract

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.