AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells

Year: 2015

Authors: Carpi, S.; Fogli, S.; Romanini, A.; Pellegrino, M.; Adinolfi, B.; Podesta, A.; Costa, B.; Da Pozzo, E.; Martini, C.; Breschi, M.C.; Nieri, P.

Autors Affiliation: University of Pisa, Deptments of Pharmacy, I-56126 Pisa, Italy; University of Pisa, Dept. Translational Research and New Technologies in Medicine and Surgery, I-56126 Pisa, Italy; University of Pisa, Dept. Veterinary Sciences, I-56126 Pisa, Italy; University Hospital of Pisa, Medical Oncology Unit, Pisa, Italy

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.


Volume: 26 (7)      Pages from: 754  to: 762

More Information: This work was supported by the Association against Melanoma Onlus (Italy).
KeyWords: A375 cells; AM251; apoptosis; Bax; Bcl-2
DOI: 10.1097/CAD.0000000000000246

Citations: 18
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