Investigations of the mechanisms of cell death induced by 15-deoxy delta(12,14) prostaglandin J2

15-deoxy-Delta12,14-Prostaglandin J 2 (15d-PGJ2), an arachidonic acid metabolite induced non-apoptotic, non-autophagic cell death in malignant cancer cell lines HCT116, MDAMB231 and DU145 derived from colon, breast and prostate, respectively. Non-apoptotic cell death induced by 15d-PGJ2 in these cancer cells was characterized by formation of large number of cytoplasmic vacuoles, which were identified as dilated cisternae of endoplamic reticulum. The failure of inhibitors of apoptosis (zVAD) to block cytoplasmic vacuolation induced cell death and the lack of autophagic vacuoles suggested that this form of cell death is non-apoptotic and non-autophagic. Notably, cell death induced by 15d-PGJ2 in these cancer cells was associated with upregulation and processing of an autophagy marker, MAP1LC3 (LC3). Moreover, knockdown of LC3 expression conferred significant protection from 15d-PGJ2 induced cell death suggesting a novel role of LC3 in cytoplasmic vacuolation and cell death. Cytoplasmic vacuolation induced cell death was also accompanied by activation of ERK pathway and ERK inhibitors significantly blocked not only the induction and processing LC3, but also reduced 15d-PGJ2 induced cytoplasmic vacuolation. In contrast to malignant cells, 15d-PGJ2 induced apoptotic cell death in non-malignant immortalized rat kidney proximal tubular cell line, RPTC, by activating Bax translocation to the mitochondrial outer membrane and the release of cytochrome c into the cytosol. 15d-PGJ2 induced apoptosis in RPTC was associated with increased expression and phosphorylation of BH3 only protein, Bim through activation of the ERK pathway. Inhibition of the ERK activity by U0126, prevented phosphorylation of Bim, and significantly blocked Bax oligomerization, and apoptosis suggesting a critical role of ERK mediated Bim phosphorylation in 15d-PGJ2 induced apoptosis of RPTC. Interestingly, 15d-PGJ2 induced apoptotic cell death in non-transformed cells as well as non-apoptotic cell death in cancer cells were mediated through disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and eventual cell death. Recently, changes in mitochondrial shape has been shown to regulate apoptotic sensitivity of a variety of cells, with mitochondrial fusion or elongation decreasing the sensitivity and mitochondria fission or division increasing the sensitivity to death inducing agents. As opposed to previous reports, where mitochondrial fragmentation precedes apoptosis, 15d-PGJ2 induced apoptosis in RPTC was preceded by extensive mitochondrial fusion. The mechanism for 15d-PGJ2 induced mitochondrial elongation was most likely due to the failure of mitochondrial fission protein, Drp1, to disassemble from an oligomeric complex. Nevertheless, activity of the fusion proteins was still required for mitochondrial elongation. Initial mitochondrial fusion by 15d-PGJ2 was followed by loss of mitochondrial tubular rigidity resulting in the formation of large spherical mitochondria with disorganized cristae. The mechanism that resulted in such transformation of mitochondrial morphology were the ubiquitination of Opa 1 and the loss of mitochondrial fusion proteins, mitofusins and Opa1. This dissertation points to a novel role of autophagy marker LC3, in a non-autophagic cell death process. Furthermore, this study provides a new role for ERK in potentiating Bax-induced apoptosis through phosphorylation of BH3 only protein Bim. This work also provides important insights into the mechanisms involved in changes of mitochondrial morphology and provides a unique model with the precedence of mitochondrial fusion in the apoptotic death.