Neferine ameliorates cardiomyoblast apoptosis induced by doxorubicin: possible role in modulating NADPH oxidase/ROS-mediated NF\u03baB redox signaling cascade

Lohanathan Bharathi Priya,Viswanadha Vijaya Padma,Chih-Yang Huang,Rathinasamy Baskaran

doi:10.1038/s41598-017-12060-9

Lohanathan Bharathi Priya, Viswanadha Vijaya Padma + Show 2 more

Open Access

https://doi.org/10.1038/s41598-017-12060-9

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Abstract

Doxorubicin (DOX) mediated cardiomyopathy is a major challenge in cancer chemotherapy. Redox-cycling of doxorubicin by flavoenzymes makes the heart more vulnerable to oxidative stress leading to cardiac dysfunction. The present study evaluates the role of neferine, a bisbenzylisoquinoline alkaloid, in curbing the molecular consequences of DOX-exposure in H9c2 cardiomyoblasts. Neferine pre-treatment increased cell viability upon DOX-exposure. DOX activates NADPH oxidase subunits, (p22phox, p47phox, gp91phox) as the primary event followed by peak in [Ca2+]i accumulation by 2 h, ROS by 3 h and activated ERK1/2 and p38 MAPKinases, time dependently along with the activation and translocation of NFκB and up-regulated COX2 and TNF-α expressions. Neferine pre-treatment modulated NADPH oxidase/ROS system, inhibited MAPKinases and NFκB activation, reduced sub G1 cell population and concomitantly increased cyclin D1 expression reducing DOX-mediated apoptosis. The study demonstrates for the first time, the molecular sequential events behind DOX toxicity and the mechanism of protection offered by neferine with specific relevance to NADPH oxidase system, MAPKinases, inflammation and apoptosis in H9c2 cells. Our data suggests the use of neferine as a new approach in pharmacological interventions against cardiovascular disorders as secondary complications.

Highlights

Anthracycline antineoplastic drug, doxorubicin (DOX) is a widely used chemotherapeutic agent in the treatment of different types of cancer including solid tumors, leukemias, lymphomas and breast cancer[1]
DOX activates pro-inflammatory gene, Nuclear Factor-κB (NFκB) through Reactive Oxygen Species (ROS) and toll-like receptor 2 (TLR2) mediated signaling, leading to cytokine production and apoptosis, which results in cardiac dysfunction[11]
Results of TUNEL assay confirmed the apoptotic event in DOX-treated H9c2 cells, which is evident from the formation of DNA breaks and apoptotic body formation (Fig. 6A,B) with a significantly decreased expression of the anti-apoptotic protein, Bcl-2 and phosphorylated Bad

Summary

Introduction

Anthracycline antineoplastic drug, doxorubicin (DOX) is a widely used chemotherapeutic agent in the treatment of different types of cancer including solid tumors, leukemias, lymphomas and breast cancer[1]. In cardiomyocytes and endothelial cells, ROS generated through NADPH oxidase has been reported to interfere with redox signaling[9]. DOX activates pro-inflammatory gene, Nuclear Factor-κB (NFκB) through ROS and toll-like receptor 2 (TLR2) mediated signaling, leading to cytokine production and apoptosis, which results in cardiac dysfunction[11]. Finding an ideal candidate with multiple actions for alleviating DOX-induced cardiotoxicity by modulating NADPH oxidase, ROS generation and apoptosis is important. The precise molecular mechanism of action of neferine on DOX-induced apoptosis has not been reported so far. Based on the available data, the present study was designed to elucidate the modulatory role of neferine on NADPH oxidase mediated redox signaling through MAPKinases and NFκB pathway in DOX-induced toxicity in H9c2 cells

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