Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study

Yano N (2014) Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study. PLoS ONE 9(8): e104888. doi:10.1371/journal.pone.0104888 Metformin Protects Cardiomyocyte from Doxorubicin Induced Cytotoxicity through an AMP-Activated Protein Kinase Dependent Signaling Pathway: An In Vitro Study Laura C. Kobashigawa 0 Yan Chun Xu 0 James F. Padbury 0 Yi-Tang Tseng 0 Naohiro Yano 0 Miguel Lo pez, University of Santiago de Compostela School of Medicine - CIMUS, Spain 0 Department of Pediatrics, Women & Infants Hospital, The Warren Alpert Medical School of Brown University , Providence, Rhode Island , United States of America Doxorubicin (Dox) is one of the most widely used antitumor drugs, but its cumulative cardiotoxicity have been major concerns in cancer therapeutic practice for decades. Recent studies established that metformin (Met), an oral anti-diabetic drug, provides protective effects in Dox-induced cardiotoxicity. Met has been shown to increase fatty acid oxidation, an effect mediated by AMP activated protein kinase (AMPK). Here we delineate the intracellular signaling factors involved in Met mediated protection against Dox-induced cardiotoxicity in the H9c2 cardiomyoblast cell line. Treatment with low dose Met (0.1 mM) increased cell viabilities and Ki-67 expressions while decreasing LDH leakages, ROS generations and [Ca2+]i. The protective effect was reversed by a co-treatment with compound-C, an AMPK specific inhibitor, or by an over expression of a dominant-negative AMPKa cDNA. Inhibition of PKA with H89 or a suppression of Src kinase by a small hairpin siRNA also abrogated the protective effect of the low dose Met. Whereas, with a higher dose of Met (1.0 mM), the protective effects were abolished regardless of the enhanced AMPK, PKA/CREB1 and Src kinase activity. In high dose Met treated cells, expression of platelet-derived growth factor receptor (PDGFR) was significantly suppressed. Furthermore, the protective effect of low dose Met was totally reversed by co-treatment with AG1296, a PDGFR specific antagonist. These data provide in vitro evidence supporting a signaling cascade by which low dose Met exerts protective effects against Dox via sequential involvement of AMPK, PKA/CREB1, Src and PDGFR. Whereas high dose Met reverses the effect by suppressing PDGFR expression. - Funding: This work was supported by the National Center for Research Resources (SP20RR018728-10) and the National Institute of General Medical Science (8 P20 GM103537-10) from the National Institute of Health (to Y-TT and JFP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Doxorubicin (Dox), an anthracycline antibiotic, has been established as an agent against a wide range of cancers [1]. However, the severe cardiotoxicity of Dox is a major factor limiting its use in the treatment of many malignancies [2]. Intensive investigations of Dox-induced cardiotoxicity have been carried out. The different lines of evidence have provided putative mechanisms, but the precise mechanism underlying Doxinduced cardiotoxicity is not fully elucidated. Most studies favor the hypothesis that free radical-induced oxidative stress plays a pivotal role. This is supported by the chemical structure of Dox and its tendency to generate reactive oxygen species (ROS) during drug metabolism [35]. Recent findings indicate that endothelial nitric oxide synthase (eNOS) reductase domain converts Dox to an unstable semiquinone intermediate that favors ROS generation [5]. Although gaining less attention than ROS has received, a number of studies suggested that Dox-mediated alteration of Ca2+ homeostasis is another possible mechanism of cardiotoxicity. Recent studies have demonstrated that Dox-mediated ROS generation induces increase of intracellular Ca2+ ([Ca2+]i), which plays a critical role in damage of cardiomyocytes [6]. Metformin (Met) is an oral biguanide anti-hyperglycemic drug that is widely used for the management of type 2 diabetes mellitus. The therapeutic effects of Met have been attributed to a combination of improved peripheral uptake and utilization of glucose, decreased hepatic glucose output, decreased rate of intestinal absorption of carbohydrate, and enhanced insulin sensitivity [7,8]. Beyond its glucose lowering effects, Met has been shown to exhibit antioxidant properties in various tissues and acts to decrease lipid peroxidation, an effect that is independent of its effect on insulin sensitivity [9. 10]. Further, Met has been demonstrated to exert cardioprotective effects that could be due to its direct beneficial effects on cellular and mitochondrial function and therefore be independent of its insulin-sensitizing effect [11]. Through its activation of 59-adenosine monophosphate-activated protein kinase (AMPK), Met reduces the generation of ROS in cultured endothelial cells [12] and in animal models of heart failure [13,14] and protects cardiomyocytes from oxidative stress induced by H2O2 or TNFa [14,15]. However, the specific mechanism by which Met activates AMPK and the corresponding antioxidant effect has not been established. These antioxidant effects suggest that Met could offer a protection against the cardiotoxicity of Dox, although no data are available to support additional benefits of Met in patients being treated with the anthracycline. The present study was undertaken to delineate signaling pathways by which Met treatment evokes protective effects against the Dox induced cardiotoxicity. For this purpose, we studied Doxinduced in vitro toxicity in a fetal rat cardiomyoblast cell line, H9c2, human fetal cardiomyocyte cell line, RL-14 and rat neonatal primary cardiomyocyte. The results of this study provide evidence that the cardioprotective effects of Met are mediated by activation of the AMPK, PKA Src and platelet-derived growth factor receptor (PDGFR). Furthermore, the protective effects are suppressed with high dose Met (1 mM) treatment secondary to reduced cellular PDGF-receptor (PDGFR) expression. Materials and Methods Reagents and antibodies Unless otherwise specified, all materials were reagent grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-Ki67 antibody was obtained from BD Biosciences (San Jose, CA, USA). Alkaline phosphatase (ALP) conjugated horse anti-mouse IgG antibody was obtained from Vector Laboratory (Burlingame, CA, USA). Antiphosphorylated/total AMPKa, anti-phosphorylated/ total acetyl-CoA carboxylase (ACC) and anti-phosphorylated PDGF receptor b (PDGFRb) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-phosphorylated/total CREB1, c-Src and total PDGFR-b antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphorylated tyro (...truncated)