The Anti-Tumor Effect of LJ-529, a Novel Agonist to A3 Adenosine Receptor, in Estrogen Receptor-Negative Human Breast Cancer Cells via Inactivation of Akt and Wnt Pathways

Abstract

Synthetic agonists to the Gi-protein-coupled A3 adenosine receptor (A3AR) have recently been suggested as a possible therapeutic agent to various cancers. Because the more aggressive estrogen receptor (ER)-negative breast cancers are resistant to widely used hormonal therapies, the need for the development of an effective therapeutic agent is well recognized. Thus we tested the anti-tumor effect of a novel A3AR agonist, generically known as LJ-529, in ER-negative breast cancer cell lines, MDA-MB-231 and SK-BR-3, which overexpress epidermal growth factor receptor (EGFR) and/or c-ErbB2. Anchorage-dependent in vitro cell growth was attenuated by LJ-529. Such inhibitory effect of LJ-529 was partially due to enhanced apoptosis that the drug-treated cells not only accumulated in sub-G1 phase but also resulted in nuclear fragmentation as evidenced by flow cytometry and DAPI staining, respectively. Consistent with the apoptotic effect of LJ-529, proteolytic cleavages of Caspase 3 and c-PARP were also observed. Furthermore, Akt and the Wnt signaling pathways were down-regulated and p27kip was induced by LJ-529. In c-ErbB2-overexpressing SK-BR-3 cells, the expression of c-ErbB2 and its downstream extracellular signal-regulated kinase (ERK) pathway were down-regulated by LJ-529. Finally, oral administration of LJ-529 for 28 days in a xenograft model, using nude mice bearing MDA-MB-231 and SK-BR-3, gave significantly reduced tumor sizes. Taken together, the A3AR agonist LJ-529 exhibited anti-tumor effect in ER-negative breast cancer cells, which are resistant to hormonal therapies and tend to be a more devastating form or malignancy, both in vitro and in vivo and involved the activation of pro-apoptotic proteases and the down-regulation of the Akt, Wnt, and ERK signaling pathways. However, such effect of LJ-529 acted independently of its receptor, because no endogenous A3AR was detected by RT-PCR in both cell lines tested.