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Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis.

ABSTRACT: Unfolded protein response (UPR) is an adaptive reaction for cells to reduce endoplasmic reticulum (ER) stress. In many types of cancers, such as lung cancer and pancreatic cancer, cancer cells may harness ER stress to facilitate their survival and growth. Prion protein (PrP) is a glycosylated cell surface protein that has been shown to be up-regulated in many cancer cells. Since PrP is a protein prone to misfolding, ER stress can result in under-glycosylated PrP, which in turn may activate ER stress. To assess whether ER stress leads to the production of under-glycosylated PrP and whether under-glycosylated PrP may contribute to ER stress thus leading to cancer cell apoptosis, we treated different cancer cells with brefeldin A (BFA), thapsigargin (Thps), and tunicamycin (TM). We found that although BFA, Thps, and TM treatment activated UPR, only ATF4 was consistently activated by these reagents, but not other branches of ER stress. However, the canonical PERK-eIF2α-ATF4 did not account for the observed activation of ATF4 in lung cancer cells. In addition, BFA, but neither Thps nor TM, significantly stimulated the expression of cytosolic PrP. Finally, we found that the levels of PrP contributed to anti-apoptosis activity of BFA-induced cancer cell death. Thus, the pathway of BFA-induced persistent ER stress may be targeted for lung and pancreatic cancer treatment.

SUBMITTER: Gao Z

PROVIDER: S-EPMC6513834 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1-20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

Project description:BackgroundCisplatin (CDDP), a widely used chemotherapeutic agent, can induce excessive granulosa cell apoptosis, follicle loss and even premature ovarian insufficiency (POI). However, the mechanism remains elusive, although some studies have indicated the involvement of endoplasmic reticulum stress (ERS). The aim of our study was to investigate the possible mechanism ERS in CDDP-induced granulosa cell apoptosis and follicle loss.MethodsA POI mouse model was generated by CDDP. The ovaries samples were collected and processed for isobaric tags for relative and absolute quantification analysis (iTRAQ) to screen out our interested proteins of HSPA5 and HSP90AB1, and the decline in their expression were verified by a real-time quantitative PCR and a western blotting assay. In vitro, human granulosa cells, KGN and COV434 cells were transfected with siRNA targeting HSPA5 and HSP90AB1 and then treated with CDDP, or treated with CDDP with/without CDDP+?4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA). The levels of ERS, autophagy and apoptosis were evaluated by western blotting, DALGreen staining and flow cytometry. In vivo, ovaries from mice that received intraperitoneal injections of saline, CDDP, CDDP+?4-PBA and CDDP+?3-MA were assayed by immunofluorescence, hematoxylin and eosin (H&E) staining for follicle counting, and terminal-deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining for cell apoptosis assay. The plasma hormone levels were measured by an enzyme-linked immunosorbent assay (ELISA) kit.ResultsWe have clarified the relationships between ERS, autophagy, and apoptosis in CDDP-induced granulosa cell apoptosis, both in vitro and in vivo. Alleviating ERS by inhibiting HSPA5 and HSP90AB1 attenuated CDDP-induced autophagy and apoptosis. 4-PBA treatment significantly attenuated CDDP-induced cell autophagy and apoptosis in cultured KGN and COV434 cells. However, inhibiting cell autophagy with 3-MA negligibly restored the CDDP-induced changes in ERS and apoptosis. In vivo experiments also demonstrated that treatment with 4-PBA, but not 3-MA, prevented CDDP-induced ovarian damage and hormone dysregulation.ConclusionsCDDP-induced ERS could promote autophagy and apoptosis in granulosa cells, causing excessive follicle loss and endocrine disorders. Alleviation of ERS with 4-PBA, but not of autophagy with 3-MA, protect against CDDP-induced granulosa cell apoptosis and ovarian damage. Thus, 4-PBA can be used to protect the ovary during chemotherapy in women.

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Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis.

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