Specific A3 adenosine receptor (A3AR) agonist, 2-chloro-N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (2-Cl-IB-MECA), demonstrates anti-proliferative effects on various types of tumor. In the present study, the cytotoxicity of 2-Cl-IB-MECA was analyzed in a panel of tumor and non-tumor cell lines and its anticancer mechanisms in JoPaca-1 pancreatic and Hep-3B 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 β-catenin, patched1 (Ptch1) and glioma-associated oncogene homolog zinc finger protein 1 (Gli1), which are components of the Wnt/β-catenin and Sonic hedgehog/Ptch/Gli transduction pathways. In concordance with these findings, the protein expression levels of cyclin D1 and c-Myc were reduced. Using a luciferase assay, it was revealed for the first time a decrease in β-catenin transcriptional activity, as an early event following 2-Cl-IB-MECA treatment. In addition, the protein expression levels of multidrug resistance-associated protein 1 and P-glycoprotein (P-gp) were reduced and the P-gp xenobiotic efflux function was also reduced. Next, the enhancing effects of 2-Cl-IB-MECA on the cytotoxicity of conventional chemotherapy was investigated. It was found that 2-Cl-IB-MECA enhanced carboplatin and doxorubicin cytotoxic effects in the JoPaca-1 and Hep-3B cell lines, and a greater synergy was found in the highly tumorigenic JoPaca-1 cell line. This provides a novel in vitro rationale for the utilization of 2-Cl-IB-MECA 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 2-Cl-IB-MECA. Notably, experiments with A3AR-specific antagonist, N-[9-Chloro-2-(2-furanyl)(1,2,4)-triazolo(1,5-c)quinazolin-5-yl]benzene acetamide, revealed that 2-Cl-IB-MECA had antitumor effects via both A3AR-dependent and -independent pathways. In conclusion, the present study identified novel antitumor mechanisms of 2-Cl-IB-MECA in pancreatic and hepatocellular carcinoma in vitro that further underscores the importance of A3AR agonists in cancer therapy.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc 77900 Olomouc Czech Republic

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