Abstract 2663: Regulation of ER stress-mediated cell death by calcium mobilization: A potential mitochondrial pathway for 15-deoxy-Δ12,14prostamide J2cytotoxicity in melanoma

Abstract

Abstract Melanoma is the most aggressive and deadly form of cutaneous cancer in the United States, representing a major clinical challenge. Our lab previously demonstrated that the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15d-PMJ2), was an effective and selective inducer of apoptotic cell death in B16F10 murine melanoma cells. Furthermore, we found that 15d-PMJ2 mediated apoptosis was regulated by endoplasmic reticulum (ER) stress. Numerous studies have determined that ER stress causes Ca+2 flux, and that this action initiates cellular apoptosis. As such, we investigated Ca+2 mobilization in the context of 15d-PMJ2-mediated ER stress. In B16F10 melanoma cells, 15d-PMJ2 significantly increased cytosolic Ca+2 levels. Blockade of Ca+2 channels using ruthenium red (a pan Ca+2 channel inhibitor) or 2-APB (an IP3 selective inhibitor) decreased the cytotoxicity of 15d-PMJ2. To investigate the role of ER stress in Ca+2 mobilization, cells were pretreated with the ER stress inhibitors, phenylbutyric acid (PBA) and GSK2602 (a PERK-selective inhibitor). Both inhibitors significantly lowered cytosolic Ca+2 levels suggesting Ca+2 flux is mediated by ER stress. Moreover, BAP31, an important regulator of endoplasmic Ca+2 release and ER stress was activated following exposure to 15d-PMJ2. To understand the impact of elevated Ca+2 levels, we investigated cysteine proteases (calpains) and mitochondrial Ca+2, both of which induce apoptosis in a Ca+2-dependent manner. Our results show that mitochondrial Ca+2 levels were significantly increased by 15d-PMJ2 and that this effect was prevented by blocking ER stress. In contrast, 15d-PMJ2 increased calpain-2 activity however, the inhibition of calpain had no effect on the cytotoxicity of 15d-PMJ2. These findings indicate that mitochondrial Ca2+ flux, but not calpains regulate ER stress apoptosis induced by 15d-PMJ2. It has been reported that the activity of J-series prostaglandins is conferred by an electrophilic double bond contained within its cyclopentenone ring. To evaluate the significance of this moiety in 15d-PMJ2, a neutral analog (neutral-15d-PMJ2) lacking the double bond was synthesized. We determined that that the accumulation of intracellular and mitochondrial Ca2+ was dependent on the electrophilic double bond, suggesting it is a critical structural component required for activity. Taken together, these data indicate that the mobilization of calcium to the cytoplasm and mitochondria may critically regulate ER stress apoptosis that is mediated by 15d-PMJ2. Citation Format: Daniel Alexander Ladin, Estefani Cota, Rukiyah Van Dross. Regulation of ER stress-mediated cell death by calcium mobilization: A potential mitochondrial pathway for 15-deoxy-Δ12,14prostamide J2cytotoxicity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2663.