Abstract 3809: Synthesis of the novel prostamide, 15-deoxy-Δ12,14 prostaglandin J2-ethanolamide, and characterization of its anti-tumor activity

Non-melanoma skin cancer (NMSC), is the most prevalent form of cancer in the United States. Many epithelial cancers including NMSC over-express the enzyme cyclooxygenase-2 (COX-2). COX-2 converts arachidonic acid (AA) to prostaglandins including prostaglandin E2 (PGE2). The interaction of PGE2 with the EP receptor has been shown to promote cancer by activating anti-apoptotic signals and angiogenesis. AA is also metabolized by COX-2 to 15deoxy, Δ12,14 prostaglandin J2 (15d-PGJ2) which promotes apoptosis via mechanisms including oxidative and endoplasmic reticulum (ER) stress. Arachidonoylethanolamide (AEA) is an endogenous cannabinoid that causes death in numerous cancer cell lines. Our lab previously demonstrated that AEA-induced apoptosis in NMSC cells occurs in a COX-2-dependent manner. Furthermore, mass spectral analysis of the products of AEA metabolism by COX-2 reveal the presence of a novel ethanolamide-conjugated J-series prostaglandin, (15d-PGJ2-EA). We hypothesize that the apoptotic effects of AEA are mediated by its primary cytotoxic metabolite, 15d-PGJ2-EA. To determine if 15d-PGJ2-EA mediates the effects of AEA we synthesized 15d-PGJ2-EA using 15d-PGJ2 as a substrate. The synthesis was accomplished using an uronium coupling reagent in the presence of ethanolamine with verification performed by H1-NMR. Tumorigenic JWF2 and non-tumorigenic HaCaT cell lines were treated with various concentrations of 15d-PGJ2-EA or 15d-PGJ2 and viability was measured using MTS assays. At 5 μM 15d-PGJ2-EA there was a 4.1-fold difference while at 10 μM there was a 217- fold difference in JWF-2 cell compared with HaCaT cell survival. In cells treated the AA metabolite, 15d-PGJ2, a 1.4-fold difference at 5 μM and a 1.7 fold difference at 10 μM in JWF2 compared to HaCaT cell survival was observed. To verify that cell death was due to apoptosis, the cleavage of caspase-3 and PARP was examined by conducting Western blot analysis. 15d-PGJ2-EA and 15d-PGJ2 increased caspase-3 and PARP cleavage in the tumorigenic but not in the non-tumorigenic cell line. To evaluate the mechanism of apoptosis, we examined oxidative- and ER-stress production in tumorigenic JWF2 cells. 15d-PGJ2-EA treated cells showed a 300% increase in DCF fluorescence (oxidative stress) and an 8.5-fold increase in CHOP-10 expression (ER stress) while 15d-PGJ2 treated cells showed 200% increase in DCF and a 3.5 fold increase in CHOP-10. Collectively, these findings suggest that AEA-induced apoptosis is mediated by 15d-PGJ2-EA since 15d-PGJ2-EA and AEA exhibit the same molecular effects. In addition, 15d-PGJ2-EA selectively induced cell death in tumorigenic but not non-tumorigenic keratinocytes. Furthermore, 15d-PGJ2-EA was more potent than 15d-PGJ2. Hence, the endocannabinoid metabolite 15d-PGJ2-EA may be an effective agent for eliminating cancer. Citation Format: Daniel Ladin, Colin Burns, Rukiyah T. Van Dross. Synthesis of the novel prostamide, 15-deoxy-Δ12,14 prostaglandin J2-ethanolamide, and characterization of its anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3809. doi:10.1158/1538-7445.AM2015-3809