1 3 Cancer Chemother Pharmacol DOI 10.1007/s00280-014-2557-y ORIGINAL ARTICLE The combination of Cl-IB-MECA with paclitaxel: a new anti-metastatic therapeutic strategy for melanoma Ana S. Soares · Vera M. Costa · Carmen Diniz · Paula Fresco Received: 19 May 2014 / Accepted: 26 July 2014 © Springer-Verlag Berlin Heidelberg 2014 although with dissimilar importance on the two melanoma cell lines. Inosine also increased colony formation on A375 cells. Levels of ERK1/2 were increased after inosine expo- sure and that increase was dependent on A 3 adenosine receptor activation in both cell lines. Moreover, microves- sel sprouting stimulated by inosine was decreased by the combination of Cl-IB-MECA with paclitaxel. Conclusions Cl-IB-MECA combined with paclitaxel was able to impair almost all of the referred metastatic related mechanisms induced by inosine, making this approach a valuable tool for combinatory therapy against metastatic melanoma. Keywords Melanoma · Cl-IB-MECA · Paclitaxel · Inosine · Metastatic potential Abbreviations AR Adenosine receptor BME Basement membrane extract BSA Bovine serum albumin Cl-IB-MECA 2-Chloro-N(6)-(3-iodobenzyl)-adenosine- 5′-N-methyl-uronamide CTR Control DMSO Dimethyl sulfoxide DMEM-HG Dulbecco’s modified Eagle’s medium– high glucose ECM Extracellular matrix ERK Extracellular signal-regulated kinase FBS Foetal bovine serum INO Inosine MAPK Mitogen-activated protein kinase MRE3008F20 N-[2-(2-furanyl)-8-propyl-8H- pyrazolo[4,3-e][1,2,4]triazolo[1,5-c] pyrimidine-5-yl]-N′-(4-methoxyphenyl) urea Abstract Purpose Metastatic melanoma is considered one of the most aggressive malignant tumours, representing the dead- liest form of skin cancer. Melanoma progression is associ- ated with the abrogation of normal controls that limit cell proliferation, migration, and invasion, eventually leading to metastasis. Based on the variety of cellular mechanisms involved in metastatic progression, we aimed to evaluate the effect of inosine (50 μM) and of the combination of Cl- IB-MECA (10 μM) with paclitaxel (10 ng/mL) on several stages of melanoma progression. Methods Proliferation, migration, adhesion, invasion, and colony formation assays were performed on human C32 and A375 metastatic melanoma cells. Levels of ERK1/2 were also determined using an ELISA kit. Moreover, mouse aortic rings were treated with vascular endothelial growth factor in order to assess the microvessel sprout- ing (an indicator of angiogenesis) in the presence of the referred compounds. Results We demonstrate that inosine induced, through A 3 adenosine receptor activation, proliferation, migration, adhesion, and invasion on C32 and A375 melanoma cells, A. S. Soares · C. Diniz · P. Fresco (*) REQUIMTE/Laboratório de Farmacologia, Departamento de Ciências do Medicamento, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal e-mail: pfresco@ff.up.pt A. S. Soares · C. Diniz · P. Fresco MedInUP – Centro de Investigação Farmacológica e Inovação Medicamentosa, Universidade do Porto, Porto, Portugal V. M. Costa REQUIMTE/Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal