Abstract C227: Doxorubicin induces AKT phosphorylation through activation of the aryl hydrocarbon receptor in the heart

Abstract

Abstract Doxorubicin (DOX) is one of the most effective anticancer drugs in treating different types of tumors. However, cumulative cardiotoxicity is a significant side effect of this therapy resulting in clinical heart failure through the production of reactive oxygen species (ROS). However, the protective responses of the heart to DOX treatment are still unknown. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is primarily involved in the detoxification of xenobiotic agents that generate ROS. Activation of AhR results in its migration to the nucleus where it dimerizes with ARNT1 to form a transcription factor for genes coding many phase I drug metabolizing enzymes, such as the cytochrome P450, CYP1A1. We hypothesize that DOX treatment activates AhR and triggers Akt phosphorylation as a protective mechanism against oxidant stress. H9C2 cells incubated with 2.5 M DOX for 4 hours showed a significant increase of AhR protein levels in the nuclear fraction, which then decreased to baseline levels by 24 hours of treatment. The same pattern of change was seen for CYP1A1 expression. Immunoprecipitation of the nuclear protein fraction with AhR antibodies and subsequent immunoblotting with ARNT1 antibodies showed increased binding between these proteins after 4 hours of DOX treatment. Similar results were shown when ARNT1 immunoprecipitation and AhR immunoblotting were performed. Increased phosphorylation of Akt at Ser473 was detected at 4 hours of DOX treatment, which decreased by 24 hours. A similar temporal pattern of phosphorylation was identified for known downstream targets of Akt (pSer9-GSK3 and pSer136-BAD). Transfection with either AhR or ARNT1 siRNAs blocked the phosphorylation of Akt following DOX treatment in H9C2 cells, increased ROS production as well as cytotoxicity. DOX treatment of isolated adult rat cardiomyocytes showed similar pattern of changes in Akt phosphorylation and its downstream targets as well as increased oxidative stress. In mice injected with 10 mg/kg DOX, AhR translocated into the nucleus by 4 hours after injection, which was associated with ARNT1 binding and increased CYP1A1 expression. Phosphorylation of Akt, GSK3 and BAD increased by 24 hours after DOX injection. Oxidative stress was significantly higher in mice injected with DOX after 4 hours. We conclude that the early cardiac response to DOX treatment involves Akt phosphorylation through AhR activation.Moreover, silencing of AhR might lead to higher oxidative stress and increased cellular toxicity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C227.