Human Amnion Membrane Proteins Prevent Doxorubicin-Induced Oxidative Stress Injury and Apoptosis in Rat H9c2 Cardiomyocytes.

Abstract

Doxorubicin (DOX) is widely used as an effective chemotherapy agent in cancer treatment. Cardiac toxicity in cancer treatment with DOX demand urgent attention and no effective treatment has been established for DOX-induced cardiomyopathy. It has been well documented that human amniotic membrane proteins (AMPs), extracted from amnion membrane (AM), have antioxidant, anti-apoptotic, and cytoprotective properties. Therefore, in this study, we aimed to investigate the protective effects of AMPs against cardiotoxicity induced by DOX in cultured rat cardiomyocyte cells (H9c2). DOX-induced cell injury was evaluated using multi-parametric assay including thiazolyl blue tetrazolium bromide (MTT), the release of lactic dehydrogenase (LDH), intracellular Ca2+ , reactive oxygen species (ROS) levels, cellular antioxidant status, mitochondrial membrane potential (ΔΨm), malondialdehyde (MDA), and NF-κB p65 DNA-binding activity. Moreover, expression profiling of apoptosis-related genes (P53, Bcl-2, and Bax) and Annexin V by flow cytometry were used for cell apoptosis detection. It was shown that AMPs pretreatment inhibited the cell toxicity induced by DOX. AMPs effectively attenuated the increased levels of LDH, Ca2+ , ROS, and MDA and also simultaneously elevated the ΔΨm and antioxidant status such as superoxide dismutase (SOD) and Catalase (CAT) in pretreated H9c2 cardiomyocytes. Besides, the activity of NF-kB p65 was reduced and the p53 and Bax protein levels were inhibited in these myocardial cells subjected to DOX. These findings provide the first evidence that AMPs potently suppressed DOX-induced toxicity in cardiomyocytes through inhibition of oxidative stress and apoptosis. Thus, AMPs can be a potential therapeutic agent against DOX cardiotoxicity.