Abstract 3859: WNT1 Inducible Signaling Pathway Protein-1 Blocks Doxorubicin-mediated Cardiomyocyte Death by Suppressing p53, Bax and Bcl-XS, and Activating PI3K-Akt-p70S6K-Survivin Signaling
Introduction: Doxorubucin (DOX) is a potent chemotherapeutic agent, but exerts both acute and chronic cardiotoxic effects. Since the secreted matricellular protein WNT1 Inducible Signaling Pathway Protein-1 (WISP1) exerts both cytoprotective and growth-promoting effects in cardiomyocytes, we hypothesized that WISP1 will antagonize DOX cardiotoxicity, and established the underlying molecular mechanisms.
Methods: C57Bl/6 neonatal mouse cardiomyocytes were treated with rhWISP1 (10 ng/ml) prior to DOX (1 μg/ml). Cell death was analyzed by MTT viability assay and quantitation of fragmented DNA in the cytoplasm; pro and anti-apoptotic gene expression by RT-qPCR, Northern blotting and immunoblotting; PI3K, Akt, and ERK activations by ELISA, immunoblotting and immunecomplex kinase assays. Signal transduction pathways were identified using pharmacological inhibitors, antisense oligonucleotides (ODN), and adenoviral transduction of wild type and mutant proteins.
Results: DOX activated pro-apoptotic p53, Bax, Bcl-XS, caspase 3 and PARP, inhibited anti-apoptotic Bcl-2 and Bcl-XL expressions, stimulated cytochrome c release, and induced cardiomyocyte death. Pre-treatment with WISP1 reversed DOX-induced activation and nuclear translocation of p53 and Bax, and inhibition of Bcl-2 and Bcl-XL expressions. Overexpression of Bcl-2 (Ad.Bcl-2) mimicked WISP1 prosurvival effects, whereas transfection with Bcl-2/Bcl-XL chimeric anti-sense ODN augmented the proapoptotic effects of DOX. Further, WISP1 activated the prosurvival factors Akt and ERK. Overexpression of constitutively active Akt (Ad.MyrAkt) recapitulated WISP1 cell survival effects. Furthermore, while DOX suppressed the anti-apoptotic survivin, WISP1 restored its expression via PI3K-Akt-p70S6 signaling. Moreover, forced expression of survivin (Ad.BIRC5) antagonized DOX-induced cardiomyocyte death.
Conclusions: WISP1 antagonizes DOX-mediated cardiomyocyte death. WISP1 inhibits pro-apoptotic p53, Bax, Bcl-xs and caspases, activates Akt and ERK, and induces survivin. Our results provide important insights into the cytoprotective effects of WISP1 in cardiomyocytes, and suggest a potential therapeutic role for WISP1 in drug-induced cardiotoxicity.