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

Metastatic melanoma is the deadliest form of skin cancer. It is highly resistant to conventional therapies,particularly to drugs that cause apoptosis as the main anticancer mechanism. Recently, induction of autophagic cell death is emerging as a novel therapeutic target for apoptotic-resistant cancers. We aimed to investigate the underlying mechanisms elicited by the cytotoxic combination of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide(Cl-IB-MECA, a selective A(3) adenosine receptor agonist; 10 μM) and paclitaxel (10 ng/mL) on human C32 and A375 melanoma cell lines.. Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase leakage assays, after 48-h incubation. Autophagosome and autolysosome formation was detected by fluorescence through monodansylcadaverine-staining and CellLight(®) Lysosomes-RFP-labelling, respectively. Cell nuclei were visualized by Hoechst staining, while levels of p62 were determined by an ELISA kit. Levels of mammalian target of rapamycin (mTOR) and the alterations of microtubule networks were evaluated by immunofluorescence.. We demonstrated, for the first time, that the combination of Cl-IB-MECA with paclitaxel significantly increases cytotoxicity, with apoptosis and autophagy the major mechanisms involved in cell death. Induction of autophagy, using clinically relevant doses,was confirmed by visualization of autophagosome and autolysosome formation, and downregulation of mTOR and p62 levels. Caspase-dependent and caspase-independent mitotic catastrophe evidencing micro- and multinucleation was also observed in cells exposed to our combination.. The combination of Cl-IB-MECA and paclitaxel causes significant cytotoxicity on two melanoma cell lines through multiple mechanisms of cell death. This multifactorial hit makes this therapy very promising as it will help to avoid melanoma multiresistance to chemotherapy and therefore potentially improve its treatment.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Blotting, Western; Cell Proliferation; Drug Synergism; Drug Therapy, Combination; Fluorescent Antibody Technique; Humans; Melanoma; Mitosis; Paclitaxel; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

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

Cl-IB-MECA is a selective A3 adenosine receptor agonist, which plays a crucial role in limiting tumor progression. In mice, Cl-IB-MECA administration enhances the anti-tumor T cell-mediated response. However, little is known about the activity of Cl-IB-MECA on CD8+ T cells. The aim of this study was to investigate the effect of ex vivo Cl-IB-MECA treatment of CD8+ T cells, adoptively transferred in melanoma-bearing mice. Adoptive transfer of Cl-IB-MECA-treated CD8+ T cells or a single administration of Cl-IB-MECA (20 ng/mouse) inhibited tumor growth compared with the control group and significantly improved mouse survival. This was associated with the release of Th1-type cytokines and a greater influx of mature Langerin+ dendritic cells (LCs) into the tumor microenvironment. CD8+ T cells treated with Cl-IB-MECA released TNF-α which plays a critical role in the therapeutic efficacy of these cells when injected to mice. Indeed, neutralization of TNF-α by a specific monoclonal Ab significantly blocked the anti-tumor activity of Cl-IB-MECA-treated T cells. This was due to the reduction in levels of cytotoxic cytokines and the presence of fewer LCs. In conclusion, these studies reveal that ex vivo treatment with Cl-IB-MECA improves CD8+ T cell adoptive immunotherapy for melanoma in a TNF-α-dependent manner.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Animals; CD8-Positive T-Lymphocytes; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Immunohistochemistry; Immunotherapy, Adoptive; Melanoma; Mice; Mice, Inbred C57BL

Adenosine exerts its effects through four subtypes of G-protein-coupled receptors: A(1), A(2A), A(2B), and A(3). Stimulation of the human A(3) receptor has been suggested to influence cell death and proliferation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Due to their importance, the cross-talk between these two pathways has been investigated. Here, we show that the A(3) adenosine receptor agonist Cl-IB-MECA stimulates PI3K-dependent phosphorylation of Akt leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation. The response to Cl-IB-MECA was not blocked by A(1), A(2A), or A(2B) receptor antagonists, although it was abolished by A(3) receptor antagonists. Furthermore, the response to Cl-IB-MECA was generated at the cell surface, since the inhibition of A(3) receptor expression, by using small interfering RNA, abolished agonist effects. Using A375 cells, we show that A(3) adenosine receptor stimulation results in PI3K-dependent phosphorylation of Akt, leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Base Sequence; Cell Division; Cell Line, Tumor; Humans; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, Adenosine A3; RNA, Small Interfering