An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines

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Abstract

A3 adenosine receptor agonists have been reported to influence cell death and survival. Here we report the effects of an A3 adenosine receptor agonist, IB-MECA, on the cell growth of human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB468 (estrogen receptor negative). Therefore, this study was aimed to investigate the expression and possible action of A3 receptor in the human breast cancer cell lines. IB-MECA, at 1–100 μM, resulted in a significant cell growth inhibition (P<0.05) which reached the maximum at 48 h, in the cell lines. In both cell lines, agonist-induced effects were antagonized by pretreatment with a selective A3 adenosine receptor antagonist, MRS1220. Using RT-PCR method, further confirmation was provided by the presence of mRNA of A3 receptor in the cells. In addition, IB-MECA was able to inhibit forskolin-stimulated cAMP levels, which indicate the functional form of A3 receptor on the cell surface of these breast cancer cell lines. These results suggest that the inhibitory effect of IB-MECA on the growth of human breast cancer cell lines is mediated through activation of A3 adenosine receptor.

Introduction

Adenosine, a purine nucleoside, acts as a primordial signaling molecule that has evolved to modulate physiological, pharmacological and biochemical responses in many organ systems. These specific functions are through G protein-coupled receptor subtypes. They are classified as A1, A2A, A2B and A3, which regulate the activity of adenylyl cyclase (Dixon, Gubitz, Sirinathsinghji, Richardson, & Freeman, 1996; Fredholm et al., 2000; Fredholm, Ijzerman, Jacobson, Klotz, & Linden, 2001; Olah & Stiles, 2000). Under normal conditions, adenosine is continuously produced intracellularly as well as extracellularly and activates A1 and A2A receptors (Fredholm et al., 2001; Olah & Stiles, 2000). However, at concentrations higher than normal, adenosine acts through other adenosine receptors, mainly A3AR (MacKenzie, Hoskin, & Blay, 1994; Ohana, Bar-Yehuda, Barer, & Fishman, 2001). A3AR had remained pharmacologically undetected until its identification by molecular cloning, first from rat and then from human. Its pharmacological characteristics were then clarified (Fredholm et al., 2000; Salvatore, Jacobson, Taylor, Linden, & Johnson, 1993). A3AR is involved in many biological functions such as: cardioprotection upon hypoxia (Shneyvays, Safran, Halili-Rutman, & Shainberg, 2000), neuroprotection in CNS by suppressing the release of neurotransmitters (Fredholm et al., 2001; Olah & Stiles, 2000), and anti-inflammation by inhibiting the cytokines production (Sajjadi, Takabayashi, Foster, Domingo, & Firestein, 1996), lymphocyte adhesion (MacKenzie, Hoskin, & Blay, 1994) and lymphocyte activity modulation (Priebe, Platsoucas, & Nelson, 1990). One of the major and important functions of adenosine, through A3 receptor, is stimulation of cell growth/proliferation and (or) activation of cell death (apoptosis/necrosis) in normal (Brambilla et al., 2000; Shneyvays, Nawrath, Jacobson, & Shainberg, 1998; Zhao, Kapoian, Shepard, & Lianos, 2002) and tumor cells (Fishman et al., 2001; Gao, Li, Day, & Linden, 2001; Gessi et al., 2001, Gessi et al., 2002; Kim et al., 2002; Kohno et al., 1996, Kohno et al., 1996; Merighi et al., 2002; Schneider, Wiendl, & Ogilvie, 2001; Yao et al., 1997). The cell proliferation or cell death effects are dependent on the cell type, the distribution of adenosine receptor subclasses and the levels on the cell surface (Brambilla et al., 2000, Merighi et al., 2002). Based on the studies reporting an elevation in the level of extracellular adenosine in the malignant tumors (Blay, White, & Hoskin, 1997; Spychala, 2000), this study was conducted to demonstrate the gene expression of A3AR as well as its biological effect on the cell survival in the breast cancer cell lines (MCF-7 and MDA-MB468) using a selective agonist, IB-MECA, and antagonist, MRS1220 (Jacobson, Moro, Kim, & Li, 1998).

Section snippets

Materials

Culture media and growth supplements were obtained from Gibco, flasks and plates from Nunc. IB-MECA, MRS1220, ADA, Ro-20-1724, Forskolin, and direct cAMP enzyme immouno assay kit (CA-200) purchased from Sigma, MTT from Merck and materials used for RT-PCR from Gibco.

Cell cultures

MCF-7 and MDA-MB468 breast cancer cell lines were obtained from the National Cell Bank of Iran (NCBI) and were grown in RPMI-1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a

A3 receptor gene expression

To confirm the presence of the receptor, the expression of A3AR was evaluated by investigating appropriate mRNA in two cell lines. RT-PCR analysis of total RNA prepared from the cells was performed. Primers were used to amplify specifically a fragment of the human A3AR cDNAs. As shown in Fig. 1 the amplified product from each cell line was of the appropriate size for A3 AR (156 bp).

The effect of A3 adenosine receptor on the cell growth

Treatment of MCF-7 and MDA-MB468 cell lines with A3AR agonist IB-MECA for 48 h resulted in a dose-dependent

Discussion

The present study has been performed to investigate the expression and function of adenosine receptors in the human breast cancer cell lines, MCF-7 (ER+) and MDA-MB468 (ER). Here the pharmacological and biochemical characterization of A3AR is described in these cells. The data, obtained from different approaches in the present study, attest to the existence of A3AR. The existence of A3AR was demonstrated on the breast cancer cell lines by the receptor activation upon exposure to A3 adenosine

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