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.

• Keywords
• 2‑Cl‑IB‑MECA, adenosine A3 receptor, chemosensitivity, hepatocellular carcinoma, multidrug resistance, pancreatic carcinoma,
• MeSH
• Adenosine analogs & derivatives   MeSH
• Cell Line MeSH
• Drug Resistance MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Liver Neoplasms * drug therapy   MeSH
• Pancreatic Neoplasms * genetics   MeSH
• Cell Proliferation MeSH
• Zinc Finger Protein GLI1 genetics   metabolism   MeSH
• Hedgehog Proteins MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide MeSH Browser
• Adenosine MeSH
• Zinc Finger Protein GLI1 MeSH
• Hedgehog Proteins MeSH

A key objective in immuno-oncology is to reactivate the dormant immune system and increase tumour immunogenicity. Adenosine is an omnipresent purine that is formed in response to stress stimuli in order to restore physiological balance, mainly via anti-inflammatory, tissue-protective, and anti-nociceptive mechanisms. Adenosine overproduction occurs in all stages of tumorigenesis, from the initial inflammation/local tissue damage to the precancerous niche and the developed tumour, making the adenosinergic pathway an attractive but challenging therapeutic target. Many current efforts in immuno-oncology are focused on restoring immunosurveillance, largely by blocking adenosine-producing enzymes in the tumour microenvironment (TME) and adenosine receptors on immune cells either alone or combined with chemotherapy and/or immunotherapy. However, the effects of adenosinergic immunotherapy are not restricted to immune cells; other cells in the TME including cancer and stromal cells are also affected. Here we summarise recent advancements in the understanding of the tumour adenosinergic system and highlight the impact of current and prospective immunomodulatory therapies on other cell types within the TME, focusing on adenosine receptors in tumour cells. In addition, we evaluate the structure- and context-related limitations of targeting this pathway and highlight avenues that could possibly be exploited in future adenosinergic therapies.

• Keywords
• adenosine, adenosine receptors, adenosinergic therapy, adverse effects, cancer, immuno-oncology, immunosurveillance, tumour microenvironment,
• MeSH
• Adenosine biosynthesis   genetics   immunology   therapeutic use   MeSH
• Molecular Targeted Therapy * MeSH
• Immunotherapy trends   MeSH
• Carcinogenesis drug effects   immunology   MeSH
• Humans MeSH
• Tumor Microenvironment drug effects   immunology   MeSH
• Neoplasms genetics   immunology   therapy   MeSH
• Receptors, Purinergic P1 immunology   therapeutic use   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Adenosine MeSH
• Receptors, Purinergic P1 MeSH

The synthetic curcumin analogue, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), suppresses NF-κB activity and exhibits antiproliferative effects against a variety of cancer cells in vitro. Recently, it was reported that EF-24-induced apoptosis was mediated by a redox-dependent mechanism. Here, we studied the effects of N-acetylcysteine (NAC) on EF-24-induced cell death. We also addressed the question of whether the main drug transporters, ABCB1 and ABCG2, affect the cytotoxic of EF-24. We observed that EF-24 induced cell death with apoptotic hallmarks in human leukemia K562 cells. Importantly, the loss of cell viability was preceded by production of reactive oxygen species (ROS), and by a decrease of reduced glutathione (GSH). However, neither ROS production nor the decrease in GSH predominantly contributed to the EF-24-induced cell death. We found that EF-24 formed an adduct with GSH, which is likely the mechanism contributing to the decrease of GSH. Although NAC abrogated ROS production, decreased GSH and prevented cell death, its protective effect was mainly due to a rapid conversion of intra- and extra-cellular EF-24 into the EF-24-NAC adduct without cytotoxic effects. Furthermore, we found that neither overexpression of ABCB1 nor ABCG2 reduced the antiproliferative effects of EF-24. In conclusion, a redox-dependent-mediated mechanism only marginally contributes to the EF-24-induced apoptosis in K562 cells. The main mechanism of NAC protection against EF-24-induced apoptosis is conversion of cytotoxic EF-24 into the noncytotoxic EF-24-NAC adduct. Neither ABCB1 nor ABCG2 mediated resistance to EF-24.

• Keywords
• EF-24-GSH adduct, EF-24-NAC adduct, K562 cells, NF-κB, Nrf2,
• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 genetics   metabolism   MeSH
• Acetylcysteine metabolism   MeSH
• Apoptosis drug effects   MeSH
• Benzylidene Compounds pharmacology   MeSH
• Glutathione metabolism   MeSH
• Leukemia metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins genetics   metabolism   MeSH
• Oxidative Stress * MeSH
• ATP Binding Cassette Transporter, Subfamily B genetics   metabolism   MeSH
• Piperidones pharmacology   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,5-bis(2-fluorobenzylidene)piperidin-4-one MeSH Browser
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ABCB1 protein, human MeSH Browser
• ABCG2 protein, human MeSH Browser
• Acetylcysteine MeSH
• Benzylidene Compounds MeSH
• Glutathione MeSH
• Neoplasm Proteins MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Piperidones MeSH
• Antineoplastic Agents MeSH
• Reactive Oxygen Species MeSH

The question as to whether A3 adenosine receptor (A3AR) agonists, N (6)-(3-iodobenzyl)-adenosine-5'-N- methyluronamide (IB-MECA) and 2-chloro-N (6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), could exert cytotoxic effects at high concentrations with or without the involvement of A3AR has been a controversial issue for a long time. The initial findings suggesting that A3AR plays a crucial role in the induction of cell death upon treatment with micromolar concentrations of IB-MECA or Cl-IB-MECA were revised, however, the direct and unequivocal evidence is still missing. Therefore, the sensitivity of Chinese hamster ovary (CHO) cells transfected with human recombinant A3AR (A3-CHO) and their counter partner wild-type CHO cells, which do not express any of adenosine receptors, to micromolar concentrations of IB-MECA and Cl-IB-MECA was studied. We observed that IB-MECA and Cl-IB-MECA exhibited a strong inhibitory effect on cell proliferation due to the blockage of cell cycle progression at G1/S and G2/M transitions in both A3-CHO and CHO cells. Further analysis revealed that IB-MECA and Cl-IB-MECA attenuated the Erk1/2 signalling irrespectively to A3AR expression. In addition, Cl-IB-MECA induced massive cell death mainly with hallmarks of a necrosis in both cell lines. In contrast, IB-MECA affected cell viability only slightly independently of A3AR expression. IB-MECA induced cell death that exhibited apoptotic hallmarks. In general, the sensitivity of A3-CHO cells to micromolar concentrations of IB-MECA and Cl-IB-MECA was somewhat, but not significantly, higher than that observed in the CHO cells. These results strongly suggest that IB-MECA and Cl-IB-MECA exert cytotoxic effects at micromolar concentrations independently of A3AR expression.

• MeSH
• Adenosine analogs & derivatives   pharmacology   MeSH
• Adenosine A3 Receptor Agonists pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Cytotoxins pharmacology   MeSH
• Cell Cycle Checkpoints drug effects   MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinase 1 antagonists & inhibitors   genetics   metabolism   MeSH
• Mitogen-Activated Protein Kinase 3 antagonists & inhibitors   genetics   metabolism   MeSH
• Cell Proliferation drug effects   MeSH
• Proto-Oncogene Proteins c-akt genetics   metabolism   MeSH
• Receptor, Adenosine A3 genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction drug effects   MeSH
• Transfection MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide MeSH Browser
• Adenosine MeSH
• Adenosine A3 Receptor Agonists MeSH
• Cytotoxins MeSH
• MAPK1 protein, human MeSH Browser
• Mitogen-Activated Protein Kinase 1 MeSH
• Mitogen-Activated Protein Kinase 3 MeSH
• N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine MeSH Browser
• Proto-Oncogene Proteins c-akt MeSH
• Receptor, Adenosine A3 MeSH