Phospholipase A2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells.
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.
Zhang
Fan
F
Department of Microbiology & Immunology, Institute of Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA.
Suarez
Giovanni
G
Sha
Jian
J
Sierra
Johanna C
JC
Peterson
Johnny W
JW
Chopra
Ashok K
AK
D017209:Apoptosis / Q000187:drug effects*; D016718:Arachidonic Acid / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D051152:Clusterin / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007378:Interleukins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D008954:Models, Biological; D054467:Phospholipases A2 / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
D051152:Clusterin; D053222:Fas Ligand Protein; C496971:IL32 protein, human; D007378:Interleukins; D011506:Proteins; D020543:Proteome; C055324:phospholipase A2-activating protein; D016718:Arachidonic Acid; D048031:JNK Mitogen-Activated Protein Kinases; D054467:Phospholipases A2; D053181:Caspase 8; D053453:Caspase 9; D013752:Tetracycline; D002945:Cisplatin