RESUMO
Specific A3 adenosine receptor (A3AR) agonist, 2chloroN6(3iodobenzyl)5'Nmethylcarboxamidoadenosine (2ClIBMECA), demonstrates antiproliferative effects on various types of tumor. In the present study, the cytotoxicity of 2ClIBMECA was analyzed in a panel of tumor and nontumor cell lines and its anticancer mechanisms in JoPaca1 pancreatic and Hep3B hepatocellular carcinoma cell lines were also investigated. Initially, decreased tumor cell proliferation, cell accumulation in the G1 phase and inhibition of DNA and RNA synthesis was found. Furthermore, western blot analysis showed decreased protein expression level of ßcatenin, patched1 (Ptch1) and gliomaassociated oncogene homolog zinc finger protein 1 (Gli1), which are components of the Wnt/ßcatenin and Sonic hedgehog/Ptch/Gli transduction pathways. In concordance with these findings, the protein expression levels of cyclin D1 and cMyc were reduced. Using a luciferase assay, it was revealed for the first time a decrease in ßcatenin transcriptional activity, as an early event following 2ClIBMECA treatment. In addition, the protein expression levels of multidrug resistanceassociated protein 1 and Pglycoprotein (Pgp) were reduced and the Pgp xenobiotic efflux function was also reduced. Next, the enhancing effects of 2ClIBMECA on the cytotoxicity of conventional chemotherapy was investigated. It was found that 2ClIBMECA enhanced carboplatin and doxorubicin cytotoxic effects in the JoPaca1 and Hep3B cell lines, and a greater synergy was found in the highly tumorigenic JoPaca1 cell line. This provides a novel in vitro rationale for the utilization of 2ClIBMECA in combination with chemotherapeutic agents, not only for hepatocellular carcinoma, but also for pancreatic cancer. Other currently used conventional chemotherapeutics, fluorouracil and gemcitabine, showed synergy only when combined with high doses of 2ClIBMECA. Notably, experiments with A3ARspecific antagonist, N[9Chloro2(2furanyl)(1,2,4)triazolo(1,5c)quinazolin5yl]benzene acetamide, revealed that 2ClIBMECA had antitumor effects via both A3ARdependent and independent pathways. In conclusion, the present study identified novel antitumor mechanisms of 2ClIBMECA in pancreatic and hepatocellular carcinoma in vitro that further underscores the importance of A3AR agonists in cancer therapy.