Gessi Stefania, Bencivenni Serena, Battistello Enrica, Vincenzi Fabrizio, Colotta Vittoria, Catarzi Daniela, Varano Flavia, Merighi Stefania, Borea Pier Andrea, Varani Katia
Department of Medical Sciences, Pharmacology Section, University of Ferrara, Ferrara, Italy.
Department of Neuroscience, Psychology, Drug Research and Child Health, Pharmaceutical and Nutraceutical Section, University of Florence, Florence, Italy.
Front Pharmacol. 2017 Dec 1;8:888. doi: 10.3389/fphar.2017.00888. eCollection 2017.
Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A, A, A, and A receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective AAR agonist 2--(2-carboxyethyl)phenethylamino-5'--ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the AAR antagonist TP455, as well as by the reference AAR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of AAR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the AAR. In conclusion, the AAR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective AAR antagonists as potential new therapeutics.
多项证据表明，普遍存在的核苷腺苷通过A1、A2A、A2B和A3受体（AR）亚型发挥作用，在肿瘤发展中起关键作用。根据不同受体亚型在各种肿瘤中的参与情况，腺苷对细胞增殖具有相反的作用。鉴于新型A3AR拮抗剂2-(2-呋喃基)-N-(2-甲氧基苄基)[1,3]噻唑并[5,4-d]嘧啶-5,7-二胺（TP455）的可用性，已评估了A3ARs在人A375黑色素瘤以及人A549肺癌和大鼠MRMT1乳腺癌增殖中的作用。具体而言，研究了在不同来源癌细胞中触发的信号通路以及TP455的拮抗作用。通过受体结合试验评估A3AR蛋白表达。此外，研究了A3AR激活在24、48和72小时对细胞增殖的影响。选择性A3AR激动剂2-（2-羧乙基）苯乙氨基-5'-乙基羧酰胺腺苷盐酸盐（CGS21680）浓度依赖性地诱导A375、A549和MRMT1癌细胞的增殖，A3AR拮抗剂TP455以及参考A3AR阻滞剂4-(2-[7-氨基-2-(2-呋喃基)[1,2,4]三唑并[2,3-a][1,3,5]三嗪-5-基氨基]乙基)苯酚（ZM241385）可有效拮抗该作用。至于在此反应中募集的信号通路，我们证明，通过使用信号转导通路的特异性抑制剂，A3AR刺激的作用是通过磷脂酶C（PLC）和蛋白激酶C-δ（PKC-δ）诱导的。此外，我们通过AlphaScreen SureFire磷酸化（p）蛋白测定法评估了A3AR募集以刺激细胞增殖的激酶，发现蛋白激酶B（AKT）、细胞外调节激酶（ERK1/2）和c-Jun N端激酶（JNKs）的参与。实际上，我们证明，在新的强效化合物TP455以及ZM241385存在下，CGS21680对激酶的刺激作用大大降低，证实了A3AR的作用。总之，A3AR激活刺激A375、A549和MRMT1癌细胞的增殖，重要的是TP455显示出其抵消这种作用的能力，表明选择性A3AR拮抗剂作为潜在的新疗法。
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