Program

PO3-7-22

Phospholipase C-related catalytically inactive protein enhances cisplatin-induced apoptosis

[Speaker] Satoshi Asano:1
[Co-author] Yuka Maetani:1, Yasuka Ikura:1, Takashi Kanematsu:1
1:Department of Cellular and Molecular Pharmacology, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Japan

Background
Cisplatin is one of the most widely used cancer chemotherapy agents. It induces DNA damage and consequent caspase-9, -7 and -3-dependent apoptosis. However, clinical responses to cisplatin are variable, and many tumors develop resistance over time. Winograd-Katz and Levitzki reported that cisplatin induced internalization of epidermal growth factor receptor followed by AKT phosphorylation, indicating the relation between cisplatin resistance and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway, an important signaling for X-linked inhibitor of apoptosis protein (XIAP)-mediated anti-apoptotic activity.
Phospholipase C-related catalytically inactive protein (PRIP) is a novel protein that negatively regulates PI3K-AKT signaling pathway by controlling conversion of PI(4,5)P2 to PI(3,4,5)P3. Here, we investigate whether PRIP enhances cisplatin-induced apoptosis.

Methods
PRIP1 and PRIP2 double knockout mouse embryonic fibroblast (PRIP DKO MEF) was isolated from PRIP DKO mouse. EGFP-PRIP or empty vector plasmids were stably transfected in MCF-7 cells which are one of human breast carcinoma, and these cell survivals were examined by using a cisplatin-induced apoptosis assay. Cisplatin-induced cytotoxicity was evaluated using a MTT assay and counting apoptotic nuclei. A western blot analysis was performed to examine apoptosis signaling.

Results
Viability of MCF-7 cells transfected with PRIP1 was significantly lower than that of control cells. Conversely, PRIP DKO MEFs survived in the presence of cisplatin compared with wild-type MEFs. The number of apoptotic nuclei was increased in MCF-7 cells transfected with PRIP1. In contrast, it was decreased in PRIP DKO MEFs. Cleaved caspase 7, 9 and cleaved poly ADP ribose polymerase were greater PRIP1-expressing MCF-7 cells than those in control cells in the presence of cisplatin. XIAP, which is stabilized by phosphorylated AKT and inhibits caspase activity, was decreased in the PRIP1-expressing MCF-7 cells.

Conclusions
Our findings demonstrated that PRIP promotes XIAP degradation and enhances the cisplatin-induced apoptotic cell death. Therefore, exogenously expressed PRIP can render cancer cells more sensitive to anti-cancer therapy using cisplatin.
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