Carpi Sara, Fogli Stefano, Romanini Antonella, Pellegrino Mario, Adinolfi Barbara, Podestà Adriano, Costa Barbara, Da Pozzo Eleonora, Martini Claudia, Breschi Maria Cristina, Nieri Paola
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.
Anticancer Drugs. 2015 Aug;26(7):754-62. doi: 10.1097/CAD.0000000000000246.
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.
人类皮肤黑色素瘤是一种侵袭性且对化疗耐药的癌症类型。AM251是一种1型大麻素（CB1）受体拮抗剂/反向激动剂，对胰腺和结肠癌细胞具有脱靶抗肿瘤活性。本研究旨在表征AM251的体外抗黑色素瘤活性。BRAF V600E突变黑色素瘤细胞系A375被用作体外模型系统。表征工具包括细胞活力测定、核形态评估、基因表达、蛋白质印迹、Annexin V-FITC/7-AAD双重染色流式细胞术、细胞周期分析以及细胞内cAMP和钙浓度变化的测量。AM251对A375人黑色素瘤细胞具有显著的细胞毒性作用，其效力与顺铂相当，而对人皮肤成纤维细胞的活力无显著影响。AM251在接近IC50的浓度下，下调了编码抗凋亡蛋白（BCL2和存活素）的基因，并增加了促凋亡BAX的转录水平，诱导了Annexin V反应性改变、DNA片段化、细胞核内染色质浓缩以及G2/M期阻滞。AM251还使基础cAMP水平升高了40%，但不影响细胞内钙浓度。排除了GPR55、TRPA1和COX-2参与AM251的作用机制。AM251与塞来昔布联合产生了协同抗肿瘤活性，尽管这种作用的潜在机制仍有待阐明。本研究首次提供了AM251对A375细胞具有促凋亡作用和G2/M细胞周期阻滞的证据。该化合物可能是开发有前景的二芳基吡唑衍生物以用于人类皮肤黑色素瘤评估的潜在原型。
