Elsevier

Biochemical Pharmacology

Volume 70, Issue 7, 1 October 2005, Pages 1066-1078
Biochemical Pharmacology

Inhibitory effects of rosmarinic acid on adriamycin-induced apoptosis in H9c2 cardiac muscle cells by inhibiting reactive oxygen species and the activations of c-Jun N-terminal kinase and extracellular signal-regulated kinase

https://doi.org/10.1016/j.bcp.2005.06.026Get rights and content

Abstract

Rosmarinic acid (RA) is a naturally occurring polyphenolic and is found in several herbs in the Lamiaceae family, such as, Perilla frutescens. ADR is a potent anti-tumor drug, but is unfortunately potently cardiotoxic. This study was undertaken to investigate the inhibitory effect of RA on ADR-induced apoptosis in H9c2 cardiac muscle cells at a mechanistic level. In vitro, ADR significantly decreased the viabilities of H9c2 cells, and this was accompanied by apoptotic features, such as a change in nuclear morphology and caspase protease activation. RA was found to markedly inhibit these apoptotic characteristics by reducing intracellular ROS generation and by recovering the mitochondria membrane potential (Δψ). In addition, RA reversed the downregulations of GSH, SOD and Bcl-2 by ADR. In the present study, ADR was found to activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), transcriptional factor-activator-protein (AP)-1. We found that c-fos, Jun-B, Jun-D and p-c-Jun were super shifted by ADR, indicating that these proteins have an important role in the ADR-induced AP-1 activation. The inhibitions of JNK and ERK using appropriate inhibitors or dominant negative cell lines reduced ADR-induced apoptosis in H9c2 cardiac muscle cells. Taken together, these results suggest that RA can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells by inhibiting ROS generation and JNK and ERK activation. Thus, we propose that RA should be viewed as a potential chemotherapeutic that inhibits cardiotoxicity in ADR-exposed patients.

Introduction

ADR is a quinone-containing anti-cancer drug that is widely used to treat different types of human neoplastic diseases and a wide range of solid tumors, including those of the breast, lung and thyroid [1], [2], [3]. However, the clinical usefulness of this drug is severely restricted because of the development of severe cardiomyopathy or congestive heart failure years after ADR therapy [4], [5]. Significant efforts have been directed toward developing an adjunctive therapy that reduces ADR-induced cardiotoxicity and enhances its therapeutic efficacy. The putative mechanism of ADR-induced cardiotoxicity involves its redox activation to a semi-quinone intermediate and the formation of ROS, the latter of which induces myocyte apoptosis [6]. The involvement of ROS is evidence by the finding that ROS scavengers can inhibit ADR-induced cardiomyocyte apoptosis [7]. Pharmacological and clinical attempts to reduce ADR cardiotoxicity have only been partially successful, potential species identified include, ROS scavengers such as superoxide dismutase (SOD), catalase and mannitol [8], [9].

Rosmarinic acid (RA), a natural phenolic is found in many Lamiaceae herbs, such as Perilla frutescens, sage, basil and mint. RA has been reported to inhibit complement-dependent inflammatory processes [10] and may have therapeutic potential [11]. Moreover, the medicinal value of RA has been well recognized, especially in regard to its anti-oxidant and anti-inflammatory activities [12], [13]. However, the mechanism underlying these RA-induced effects on the cardiovascular system is unknown. Therefore, in this study we examined the effects of RA on ADR-damaged cardiac muscle cells in the hope of elucidating its mode of action (Fig. 1).

Section snippets

Reagents

RA was isolated from L. lucidus (Labiatae) [14]. Dulbecco's Modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin and other tissue culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). Bicinchoninic acid (BCA) protein assay reagents were from Pierce; ADR was from Sigma; the annexin V-FITC apoptosis detection kits (6993KK) were from PharMingen (San Diego, CA) and 3,3′-diexyloxocarbo-cyanine iodide (DiOC6(3)) was from Molecular Probes (Eugene, Oregon). All

Rosmarinic acid-induced cell resistance to ADR-induced cytotoxicity and apoptosis in H9c2 cells

In order to determine if RA modifies ADR-induced responses in H9c2 cells, cells were exposed to RA (20 μg/mL) and then stimulated with ADR (1, 2 or 4 μM). Fig. 2A shows the effect of treatment of RA on the cytotoxicity exerted by ADR in this cell line over a period of 30 h. At concentrations >0.1 mg/mL RA was found to inhibit against ADR-induced cell death (Fig. 2B). Maximum RA inhibition of ADR-induced toxicity (at an ADR concentration 2 μM) was observed at 10 or 20 μg/mL by Hoechst staining. The

Discussion

This study shows that RA effectively protects cardiac muscle cells from ADR-induced cell death. Several studies have indicated that apoptosis occurs in the human heart during end stage cardiac failure or acute myocardial infarction, which suggests that apoptosis is involved in cardiovascular disease [23]. We expected at the beginning of this study that apoptosis is involved in the genesis of ADR-induced cardiomyopathy.

Bcl-2 is regarded as an important cellular component that guards against

Acknowledgments

This research was supported by the Korean Ministry of Science and Technology through the Korean Center for Healthcare Technology Development.

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