2-chloro-n(6)-(3-iodobenzyl)adenosine-5--n-methyluronamide and Neoplasm-Metastasis

Metastatic melanoma is considered one of the most aggressive malignant tumours, representing the deadliest form of skin cancer. Melanoma progression is associated 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.. 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 sprouting (an indicator of angiogenesis) in the presence of the referred compounds.. We demonstrate that inosine induced, through A3 adenosine receptor activation, proliferation, migration, adhesion, and invasion on C32 and A375 melanoma cells, 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 exposure and that increase was dependent on A3 adenosine receptor activation in both cell lines. Moreover, microvessel sprouting stimulated by inosine was decreased by the combination of Cl-IB-MECA with paclitaxel.. 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.

Topics: Adenosine; Animals; Antineoplastic Agents; Cell Physiological Phenomena; Disease Progression; Drug Screening Assays, Antitumor; Humans; Inosine; Melanoma; Mice; Neoplasm Metastasis; Neoplasm Staging; Neovascularization, Pathologic; Paclitaxel; Receptor, Adenosine A3; Tumor Cells, Cultured

Metastatic melanoma monotherapies with drugs such as dacarbazine, cisplatin or paclitaxel (PXT) are associated with significant toxicity and low efficacy rates. These facts reinforce the need for development of novel agents or combinatory strategies. Cl-IB-MECA is a small molecule, orally bioavailable, well tolerated and currently under clinical trials as an anticancer agent. Our aim was to investigate a possible combinatory therapeutic strategy using PXT and Cl-IB-MECA on human C32 melanoma cells and its underlying mechanisms. Cytotoxicity was evaluated using MTT reduction, lactate dehydrogenase leakage and neutral red uptake assays, for different concentrations and combinations of both agents, at 24 and 48 h. Apoptosis was also assessed using fluorescence microscopy and through the evaluation of caspases 8, 9, and 3 activities. We demonstrated, for the first time, that combination of PXT and Cl-IB-MECA significantly increases cytotoxicity for clinically relevant concentrations. This combination seems to act synergistically in disrupting membrane integrity, but also causing lysosomal and mitochondrial dysfunction. When using the lowest PTX concentration (10 ng/mL), co-incubation with CI-IB-MECA (micromolar concentrations) potentiated overall cytotoxic effects and morphological signs of apoptosis. All combinations studied enhanced caspase 8, 9, and 3 activities, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. The possibility that cytotoxicity elicited by Cl-IB-MECA, alone or in combination with PXT, involves adenosine receptor activation was discarded and results confirmed that oxidative stress is only involved in cytotoxicity after treatment with PXT, alone. Being melanoma a very apoptosis-resistance cancer, this combination seems to hold promise as a new therapeutic strategy for melanoma.

Topics: Adenosine; Antineoplastic Combined Chemotherapy Protocols; Caspases; Cell Culture Techniques; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Humans; Melanoma; Neoplasm Metastasis; Paclitaxel