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    • The AR signalling pathways play important roles in several p

    2023-02-01

    The AR signalling pathways play important roles in several physiopathological processes associated with ischaemia, inflammation, and tumourigenesis [35]. Moreover, A3AR is overexpressed in different tumour 4-aminopyridine australia and seems implicated in pro- or anti-apoptotic effects [36]. In particular, activation of A3AR subtype, by agonists, is believed to induce tumour growth inhibition in vitro and in vivo, via the modulation of Wnt and the NF-kappaβ signalling pathways [37]. The A3AR agonist Cl-IB-MECA was deeply studied in vitro and in vivo on tumour cells proliferation. The rationale for using low concentrations of Cl-IB-MECA was based on its high A3AR affinity and selectivity. Notably, at low concentration range Cl-IB-MECA induced a differential effect on tumour and normal cell proliferation. In previous papers, it was reported the synthesis and in vitro biological evaluation of a series of substituted Ado derivatives endowed with low or subnanomolar affinity for the human A3AR and very high selectivity versus the other AR subtypes [17]. Among them, compound 1 (Fig. 1) was found very potent and selective for A3AR. Hence, this ligand and other two of its simplified analogues, compounds 2 and 3 (Fig. 1), which behaved with good affinity and selectivity at the A3AR subtype (Table 1), have been chosen to be tested in different tumour cell lines in comparison with Cl-IB-MECA. In particular, compound 2 is substituted only at 2 and N6 position, while compound 3 is substituted only in 2 and 4′ positions. Furthermore, the newly synthesized derivatives 4–6 (Fig. 1), characterized by the presence of sterically hindered groups in N6 position, have been included in the present study. Compound 6 is characterized by a phenylethyl group at N6 position, similar to that present in the same position in the molecule of Cl-IB-MECA, while, in compounds 4 and 5 a more steric hindered methylbenzhydryl group is present. In order to study the activity of the known and newly synthesized A3AR agonists 1–6 as anticancer drugs, human prostatic cancer cell line PC3 [38] was used. After the preliminary screening, to generalize the activity of A3AR mediated cell death process, we also used human hepatocellular carcinoma cell line Hep G2 [39] and human epithelial colorectal carcinoma Caco-2 [40]. All these three cell lines are very well studied and they are known to express A3AR [[41], [42], [43]]. Only compounds 5 and 6 caused proliferation inhibition in time- and dose- dependent manners. Compounds 5 and 6 showed to induce 100% and 56% mortality at 100 μM, and the reference A3AR full agonist Cl-IB-MECA induced a 100% mortality when tested in the same conditions. On the contrary, the other compounds, which show a very good affinity for A3AR, did not induce mortality higher than 10% in the same cell line, hence they are not able to affect cell survival on these tumour cells. It is worthwhile to note that compound 1 is a very potent and a selective full agonist of A3AR. It is possible that the effects induced by the Cl-IBMECA and compounds 5, 6 is not mediated by the A3AR but are dependent intracellular mechanisms, as shown from Mlejnek and collaborators [44]. These authors observed that the cytotoxic effects of Cl-IB-MECA are related to high intracellular levels of the molecule that interacts with the P-gp protein [44]. Results obtained from the present study suggest that by the Cl-IBMECA and compounds 5, 6 interferes with tumour growth of PC3 by a molecule mechanism independent of A3AR activation. In support of these results, other authors have already previously described a mechanism independent of receptor activation on antitumor effects induced by the treatment of selective A3AR agonists [[45], [46], [47]]. In order to verify if the reduction of viability was due to other mechanisms a specific experiment was performed. In this experiment was compared CHO cells transfected with human recombinant A3AR and their counter partner wild-type CHO cells, both cells were incubated with compounds 5 and 6. Results showed that, at 100 μM concentration, the percentage of mortality in both cells was almost the same. In fact, compounds 5 and 6 induce mortality of 100% and 48% in CHO cells transfected with human recombinant A3AR and of 100%, 45% in CHO wild type, respectively. These results confirm that the antitumor activity is not due to the contribution of A3AR.