Abstract 3217: Novel prostamide, 15-deoxy-delta12,14 prostamide J2, displays activity against melanoma in vitro and in vivo: potential role of endoplasmic reticulum stress

Abstract

Abstract Melanoma is the most aggressive and deadly form of cutaneous neoplasm in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid, arachidonoyl ethanolamide (AEA), induced cell death in non-melanoma skin cancer (NMSC) cells through the cyclooxygenase-2 (COX-2) mediated formation of novel J-series prostamides (PMJs). We were the first to chemically synthesize the primary metabolite, 15-deoxy-Δ12,14 prostamide J2 (15d-PMJ2), which displayed potent and selective cytotoxicity in NMSC cells. As such, we hypothesize that the selective cytotoxicity of 15d-PMJ2 would be observed in other forms of skin cancer, including melanoma. B16F10 murine melanoma cells and nontumorigenic Melan-A cells were treated with different concentrations of 15d-PMJ2 for 24 hours and cell viability was measured using MTS assays. At 5µM, 15d-PMJ2 decreased viability by 63% in B16F10 cells, while Melan-A viability was not affected. To verify that cell death was due to apoptosis, the cleavage of apoptotic markers caspase-3 and PARP was examined by conducting Western blot analysis. 15d-PMJ2 markedly increased caspase-3 and PARP cleavage only in B16F10 melanoma cells. Previous studies in NMSC indicated that 15d-PMJ2 induced ER-stress and apoptosis. To investigate the mechanism of 15d-PMJ2-mediated death in melanoma, we examined ER-stress responses. Melan-A and B16F10 melanoma cells were treated with 5µM 15d-PMJ2 and evaluated for CHOP10 and p-PERK expression by Western blot analysis. B16F10, but not Melan-A cells exhibited a notable increase in CHOP10 and p-PERK expression when treated with 15d-PMJ2. To further examine the role of ER-stress on 15d-PMJ2 mediated apoptosis, B16F10 cells were pretreated with the ER-stress inhibitors salubrinal and 4-phenylbutyric acid (PBA). Both salubrinal and PBA decreased activation of caspase-3/7, suggesting that ER-stress plays an important role in 15d-PMJ2 mediated tumor cell death. To determine the anti-melanoma activity of 15d-PMJ2 in vivo, B16F10 allograft tumors grown in C57BL/6 mice were dosed subcutaneously with 0.5 or 5.0 mg/kg 15d-PMJ2 for 5 days. Tumors treated with 15d-PMJ2 exhibited significantly reduced growth and mean weights compared to vehicle and untreated animals. TUNEL analysis of tumor tissues indicated a large presence of necrotic and apoptotic cells in 15d-PMJ2-treated tumors compared to vehicle and untreated tumors. To determine whether 15d-PMJ2 induced ER-stress in vivo, tumors were assayed for p-PERK and CHOP10 levels by immunohistochemistry (IHC). These markers were significantly elevated in 15d-PMJ2-treated tumors. Similarly, the viability of primary patient-derived melanoma cells was significantly decreased by 15d-PMJ2. These findings suggest that the novel prostamide, 15d-PMJ2, possesses potent and selective anti-melanoma activity in vitro and in vivo. Citation Format: Daniel A. Ladin, Li V. Yang, Timothy L. Fitzgerald, Rukiyah Van Dross. Novel prostamide, 15-deoxy-delta12,14 prostamide J2, displays activity against melanoma in vitro and in vivo: potential role of endoplasmic reticulum stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3217. doi:10.1158/1538-7445.AM2017-3217