title: Potentiation of cytotoxicity of paclitaxel in combination with Cl-IB-MECA in human C32 metastatic melanoma cells: A new possible therapeutic strategy for melanoma.
abstract: 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.
authors.affiliation: REQUIMTE/Laboratório de Farmacologia, Departamento de Ciências do Medicamento, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal.
authors.forename: Ana S
authors.initials: AS
authors.lastname: Soares
authors.forename: Vera M
authors.initials: VM
authors.lastname: Costa
authors.forename: Carmen
authors.initials: C
authors.lastname: Diniz
authors.forename: Paula
authors.initials: P
authors.lastname: Fresco
mesh_terms: D000241:Adenosine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004789:Enzyme Activation; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000473:pathology; D009362:Neoplasm Metastasis; D017239:Paclitaxel / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D020169:Caspases; D000241:Adenosine; D017239:Paclitaxel; C090034:2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide
keywords: 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; AR; Ac-DEVD-CHO; Adenosine receptor; Cl-IB-MECA; DMEM-HG; DMSO; Dimethyl sulfoxide; Dulbecco's Modified Eagle's Medium–High Glucose; FBS; Fetal bovine serum; IAP; Inhibitors of apoptosis; LDH; Lactate dehydrogenase; MTT; Melanoma; N-Ac-Asp-Glu-Val-Asp-CHO; N-acetyl-cysteine; NAC; NR; Neutral red; PXT; Paclitaxel; ROS; Reactive oxygen species; TNF; Tumour necrosis factor