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Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways.

ABSTRACT: We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca(2+) homeostasis manifested by Ca(2+) rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

SUBMITTER: Moserova I

PROVIDER: S-EPMC3293927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sigma-2-ligands (S2L) are characterized by high-affinity interaction with their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes following chemical conjugation (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across pancreatic, ovarian, and synovial cancer cell lines. While C6 and C10 displayed distinct cytotoxicities (extended aliphatic side chains), C0 (no aliphatic side chain) and ISO1 (structurally unrelated) were essentially non-toxic, enabling us to investigate the unique mechanism of S2L cytotoxicity. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced an increase in intralysosomal free cholesterol, a corresponding increase in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking and processing, and cytotoxicity due to cholesterol excess, a phenomenon that contrasts the effects of inhibiting NPC1 function. RNA-sequencing revealed that gene clusters involved in cholesterol homeostasis and ER stress response were activated exclusively by cytotoxic S2L. ER stress markers were confirmed on qPCR, and ER stress modulators inhibited or enhanced cytotoxicity of C10 confirming the involvement of ER stress response in the cytotoxicity of S2L. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) which were also confirmed with qPCR and at the protein level and were found to be a pro-survival mechanism activated by ER stress response. Moreover, inhibiting the processes downstream this adaptive response with simvastatin resulted in synergistic treatment outcomes when combined with C10 in vitro and in vivo. Of note, the conjugates retained the S2L-mediated cholesterol phenotype of their parental ligands, which suggests that disrupting cholesterol homeostasis might contribute to the overall cytotoxicity of the drug conjugates. Furthermore, these phenomena were seen exclusively with cytotoxic S2L. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

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Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways.

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