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Interactions between Endoplasmic Reticulum Stress and Autophagy: Implications for Apoptosis and Neuroplasticity-Related Proteins in Palmitic Acid-Treated Prefrontal Cells.

ABSTRACT: Lipotoxicity of palmitic acid (PA) or high-fat diets has been reported to increase endoplasmic reticulum (ER) stress and autophagy in peripheral tissue as well as apoptotic cell death. It also can lead to an AD-like pathological pattern. However, it has been unknown that PA-induced ER stress and autophagy are involved in the regulation of neuroplastic abnormalities. Here, we investigated the roles of ER stress and autophagy in apoptosis and neuroplasticity-related protein expression in PA-treated prefrontal cells. Prefrontal cells dissected from newborn Sprague-Dawley rats were treated with PA compound with ER stress inhibitor 4-phenylbutyric acid (4-PBA) and autophagy inhibitor 3-methyladenine (3-MA) or PA alone. PA promoted ER stress and autophagy and also cause apoptosis as well as a decline in the expression of neuroplasticity-related proteins. Inhibition of ER stress decreased the expressions of neuroplasticity-related proteins and reduced autophagy activation and apoptosis in PA-treated prefrontal cells. Inhibition of autophagy exacerbated apoptosis and enhanced ER stress in PA-treated prefrontal cells. The present study illustrated that both ER stress and autophagy could be involved in apoptosis and decreased neuroplasticity-related proteins, and the interaction between ER stress and autophagy may play a critical role in apoptosis in PA-treated prefrontal cells. Our results provide new insights into the molecular mechanisms in vitro of lipotoxicity in obesity-related cognitive dysfunction.

SUBMITTER: Xue X

PROVIDER: S-EPMC8505096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PURPOSE:Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. METHODS:Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. RESULTS:Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced. DISCUSSION:Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.

Project description:BackgroundPalmitic acid (PA), the main component of dietary saturated fat, causes apoptosis in many cell types, including mouse granulosa cell. Melatonin, an important endogenous hormone, has beneficial effects on female reproductive processes. Since elevated PA levels are present in follicular fluid (FF) of patients with infertility and are shown to be toxic for granulosa cells, we investigated the molecular mechanisms of PA toxicity in mouse granulosa cells and explored the effects of melatonin on PA-induced apoptosis.MethodsGranulosa cells from immature female mice were cultured for 24 h in medium containing PA and/or melatonin. Then, the effects of PA alone or combined with melatonin on viability, apoptosis and endoplasmic reticulum (ER) stress in granulosa cells were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry assay and western blot. After 48 h of PA and/or melatonin treatment, the concentrations of estradiol (E2) and progesterone (P4) in the culture supernatants were measured with ELISA kits.ResultsIn this study, we explored the effects of melatonin on cell viability and apoptosis in PA-treated mouse granulosa cells and uncovered the signaling pathways involved in these processes. Our results showed that 200-800 μM PA treatment reduces cell viability, induces cell apoptosis, enhances the expression of apoptosis-related genes (Caspase 3 and B-cell lymphoma-2 (BCL-2) associated X protein (BAX)), and activates the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Melatonin treatment (1-10 μM) suppresses 400 μM PA-induced cell viability decrease, cell apoptosis, Caspase 3 activation, and BAX, CHOP, and GRP78 expression. In addition, we found that 10 μM melatonin successfully attenuated the 400 μM PA-induced estrogen (E2) and progesterone (P4) decreases.ConclusionsThis study suggests that PA triggers cell apoptosis via ER stress and that melatonin protects cells against apoptosis by inhibiting ER stress in mouse granulosa cells.

Project description:Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2? as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2? or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2?/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.

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Interactions between Endoplasmic Reticulum Stress and Autophagy: Implications for Apoptosis and Neuroplasticity-Related Proteins in Palmitic Acid-Treated Prefrontal Cells.

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