title: AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.
abstract: Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.
authors.affiliation: Departments of aPharmacy bTranslational Research and New Technologies in Medicine and Surgery cVeterinary Sciences, University of Pisa dMedical Oncology Unit, University Hospital of Pisa, Pisa, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Carpi
authors.forename: Stefano
authors.initials: S
authors.lastname: Fogli
authors.forename: Antonella
authors.initials: A
authors.lastname: Romanini
authors.forename: Mario
authors.initials: M
authors.lastname: Pellegrino
authors.forename: Barbara
authors.initials: B
authors.lastname: Adinolfi
authors.forename: Adriano
authors.initials: A
authors.lastname: Podestà
authors.forename: Barbara
authors.initials: B
authors.lastname: Costa
authors.forename: Eleonora
authors.initials: E
authors.lastname: Da Pozzo
authors.forename: Claudia
authors.initials: C
authors.lastname: Martini
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Breschi
authors.forename: Paola
authors.initials: P
authors.lastname: Nieri
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015220:Calcium Channels / Q000378:metabolism; D000068579:Celecoxib; D045744:Cell Line, Tumor / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D054314:Drug Inverse Agonism; D004357:Drug Synergism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D008545:Melanoma / Q000473:pathology*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology*; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors*; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012878:Skin Neoplasms / Q000473:pathology*; D013449:Sulfonamides / Q000494:pharmacology; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D015220:Calcium Channels; C524885:GPR55 protein, human; D009419:Nerve Tissue Proteins; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled; D013449:Sulfonamides; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; C103505:AM 251; D000242:Cyclic AMP; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D000068579:Celecoxib