Pharmacological study of A3 Adenosine receptor agonist (AB Meca) in xenograft lung cancer model in mice through in silico and in vivo approach: Targeting TNF-α

Background: Lung cancer is the leading cause of mortality in India. Adenosine receptor (AR) has emerged as a novel cancer-specific target. A3AR levels are upregulated in various tumor cells, which may mean that the specific AR may act as a biological marker and target specific ligands leading to cell growth inhibition. Aim: Our aim was to study the utility of the TNF-α agonist, AB MECA, by in silico (molecular docking) and in vitro (human cancer cells in xenografted mice) studies. Method: Molecular docking on the AB-meca and TNF-α was performed using AutoDock. A549 Human lung cancer 2 ×106 cells per microliter per mouse injected via intrabronchial route. Rat TNF-α level was assessed by ELISA method. Results: AB Meca's predicted binding energy (beng) with TNF-α was 97.13 kcal/mol, and the compatible docking result of a small molecular inhibitor with TNF-α native ligand beng was 85.76 kcal/mol. In vivo, a single dose of lung cancer cell A549 is being researched to potentiate tumor development. Doxorubicin and A3AR agonist therapies have lowered TNF-alpha levels that were associated with in silico function. The A3AR Agonist myeloprotective effect was also found in groups treated with doxorubicin. Conclusion: AB MECA’s higher binding energy (beng) with TNF-α mediated reduction of tumor growth in our lung cancer in vivo model suggests that it may be an effective therapy for lung cancer.

Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.