Induction Of Endoplasmic Reticulum Stress Research Articles

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-β-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-β-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Angiopoietin-like protein 4 induces growth hormone variant secretion and aggravates insulin resistance during pregnancy, linking obesity to gestational diabetes mellitus.

Angiopoietin-like protein 4 (ANGPTL4) is a secretory glycoprotein involved in regulating glucose homeostasis in non-pregnant subjects. However, its role in glucose metabolism during pregnancy and the pathophysiology of gestational diabetes mellitus (GDM) remains elusive. Thus, this study aimed to clarify the relationship between ANGPTL4 and GDM and investigate the pathophysiology of placental ANGPTL4 in glucose metabolism. We investigated this issue using blood and placenta samples in 957 pregnant women, the human 3A-sub-E trophoblast cell line, and the L6 skeletal muscle cell line. We found that ANGPTL4 expression in the placenta was higher in obese pregnant women than in lean controls. Palmitic acid significantly induced ANGPTL4 expression in trophoblast cells in a dose-response manner. ANGPTL4 overexpression in trophoblast cells resulted in endoplasmic reticulum (ER) stress, which stimulated the expression and secretion of growth hormone-variant (GH2) but not human placental lactogen. In L6 skeletal muscle cells, soluble ANGPTL4 suppressed insulin-mediated glucose uptake through the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinases 1/2 (ERK 1/2) pathways. In pregnant women, plasma ANGPTL4 concentrations in the first trimester predicted the incidence of GDM and were positively associated with BMI, plasma triglyceride, and plasma GH2 in the first trimester. However, they were negatively associated with insulin sensitivity index ISI0,120 in the second trimester. Overall, placental ANGPTL4 is induced by obesity and is involved in the pathophysiology of GDM via the induction of ER stress and GH2 secretion. Soluble ANGPTL4 can lead to insulin resistance in skeletal muscle cells and is an early biomarker for predicting GDM.

Copper exposure induces mitochondrial dysfunction and hepatotoxicity via the induction of oxidative stress and PERK/ATF4 -mediated endoplasmic reticulum stress

Copper is an essential trace element, and excessive exposure could result in hepatoxicity, however, the underlying molecular mechanisms remain incompletely understood. The present study is aimed to investigate the molecular mechanisms of copper sulfate (CuSO4) exposure-induced hepatoxicity both in vivo and in vitro. In vitro, HepG2 and L02 cells were exposed to various doses of CuSO4 for 24 h. Cell viability, ROS production, oxidative stress biomarkers, mitochondrial functions, ultrastructure, intracellular calcium (Ca2+) concentration, and the expression of proteins related to mitochondrial apoptosis and endoplasmic reticulum (ER) stress were assessed. In vivo, C57BL/6 mice were treated with CuSO4 at doses of 10 and 30 mg/kg BW/day and co-treated with 4-PBA at 100 mg/kg BW/day for 35 days. Subsequently, liver function, histopathological features, and protein expression were evaluated. Results found that exposure to CuSO4 at concentrations of 100–400 μM for 24 h significantly decreased the viabilities of HepG2 and L02 cells and it was in a dose-dependent manner. Additionally, CuSO4 exposure induced significant oxidative stress and mitochondrial dysfunction in HepG2 cells, which were partially ameliorated by the antioxidant N-acetylcysteine (NAC). Furthermore, CuSO4 exposure prominently triggered ER stress, as evidenced by the upregulation of GRP94, GRP78, phosphorylated forms of PERK and eIF2α, and CHOP proteins in livers of mice and HepG2 cells. NAC treatment significantly inhibited CuSO4 exposure -induced ER stress in HepG2 cells. Pharmacological inhibition of ER stress through co-treatment with 4-PBA and the PERK inhibitor GSK2606414, as well as genetic knockdown of ATF4, partially mitigated CuSO4-induced cytotoxicity in HepG2 cells by reducing mitochondrial dysfunction and inhibiting the mitochondrial apoptotic pathway. Moreover, 4-PBA treatment significantly attenuated CuSO4-induced caspase activation and hepatoxicity in mice. In conclusion, these results reveal that CuSO4-induced hepatotoxicity involves mitochondrial dysfunction and ER stress by activating oxidative stress induction and PERK/ATF4 pathway.

MitoTempo protects against nε-carboxymethyl lysine-induced mitochondrial dyshomeostasis and neuronal cells injury

Enhanced formation of advanced glycation end products (AGEs) is a pivotal factor in diabetes pathophysiology, increasing the risk of diabetic complications. Nε-carboxy-methyl-lysine (CML) is one of the most relevant AGEs found in several tissues including the peripheral blood of diabetic subjects. Despite recognizing diabetes as a risk factor for neurodegenerative diseases and the documented role of mitochondrial abnormalities in this connection, the impact of CML on neuronal mitochondria and its contribution to diabetes-related neurodegeneration remain uncertain. Here, we evaluated the effects of CML in differentiated SH-SY5Y human neuroblastoma cells. Due to the association between mitochondrial dysfunction and increased production of reactive oxygen species (ROS), the possible protective effects of MitoTempo, a mitochondria-targeted antioxidant, were also evaluated. Several parameters were assessed namely cells viability, mitochondrial respiration and membrane potential, ATP and ROS production, Ca2+ levels, mitochondrial biogenesis and dynamics, mito/autophagy, endoplasmic reticulum (ER) stress and amyloidogenic and synaptic integrity markers. CML caused pronounced mitochondrial defects characterized by a significant decrease in mitochondrial respiration, membrane potential, and ATP production and an increase in ROS production. An accumulation of individual mitochondria associated with disrupted mitochondrial networks was also observed. Furthermore, CML caused mitochondrial fusion and a decrease in mitochondrial mass and induced ER stress associated with altered unfolded protein response and Ca2+ dyshomeostasis. Moreover, CML increased the protein levels of β-secretase-1 and amyloid precursor protein, key proteins involved in Alzheimer's Disease pathophysiology. All these effects contributed to the decline in neuronal cells viability. Notable, MitoTempo was able to counteract most of CML-mediated mitochondrial defects and neuronal cells injury and death. Overall, these findings suggest that CML induces pronounced defects in neuronal mitochondria and ER stress, predisposing to neurodegenerative events. More, our observations suggest that MitoTempo holds therapeutic promise in mitigating CML-induced mitochondrial imbalance and neuronal damage and death.

Biomaterials for Targeting Endoplasmic Reticulum in Cancer.

Endoplasmic reticulum (ER) is one of the most important sub-cellular organelles which controls myriads of biological functions including protein biosynthesis with proper functional folded form, protein misfolding, protein transport into Golgi body for secretion, Ca2+ homeostasis and so on. Subsequently, dysregulation in ER function leads to ER stress followed by disease pathology like cancer. Hence, targeting ER in the cancer cells emerged as one of the futuristic strategies for cancer treatment. However, the major challenge is to selectively and specifically target ER in the sub-cellular milieu in the cancer tissues, due to the lack of ER targeting chemical moieties to recognize the ER markers. To address this, in the last decade, numerous biomaterials were explored to selectively impair and image ER in cancer cells to induce ER stress. This review outlines those biomaterials which consists of carbon and silicon materials, lipid nanoparticles (liposomes and micelles), supramolecular self-assembled nanostructures, cell membrane-coated nanoparticles and metallic nanoparticles. Moreover, we also discuss the challenges and possible solutions of this promising field to usher the readers towards next-generation ER targeted cancer therapy.

P20BAP31 Induces Autophagy in Colorectal Cancer Cells by Promoting PERK-Mediated ER Stress.

B-cell receptor-associated protein 31 (BAP31) is an endoplasmic reticulum (ER) membrane protein involved in apoptosis and autophagy by communication with ER and mitochondria. BAP31 is cleaved by caspase-8 and generates a proapoptotic fragment, p20BAP31, which has shown to induce ER stress and apoptosis through multiple pathways. In this study, we found that p20BAP31 significantly increased the agglomeration of LC3 puncta, suggesting the occurrence of autophagy. Therefore, it is meaningful to explore the mechanism of p20BAP31-induced autophagy, and further analyze the relationships among p20BAP31-induced autophagy, ER stress and apoptosis. The data showed that p20BAP31 induced autophagy by inhibition of the PI3K/AKT/mTOR signaling in colorectal cells. ER stress inhibitor 4-PBA and PERK siRNA alleviated p20BAP31-induced autophagy; in turn, autophagy inhibitors 3-MA and CQ did not affect p20BAP31-induced ER stress, suggesting that p20BAP31-induced ER stress is the upstream of autophagy. We also discovered that ROS inhibitor NAC inhibited p20BAP31-induced autophagy. Furthermore, inhibition of autophagy by CQ suppressed p20BAP31-induced apoptosis and ameliorated cell proliferation. Importantly, p20BAP31 markedly reduced the tumor size in vivo, and significantly enhanced the autophagy levels in the tumor tissues. Collectively, p20BAP31 initiates autophagy by inhibiting the PI3K/AKT/mTOR signaling and activating the PERK-mediated ROS accumulation, further promotes p20BAP31-induced apoptosis and ultimately results in cell death. This study comprehensively reveals the potential mechanism of p20BAP31-induced cell death, which may provide new strategies for antitumor therapy.

Urolithin A, induces apoptosis and autophagy crosstalk in Oral Squamous Cell Carcinoma via mTOR /AKT/ERK1/2 pathway

BackgroundOral squamous cell carcinoma (OSCC) is the most prevalent malignancy in the world with an alarming rate of mortality. Despite the advancement in treatment strategies and drug developments, the overall survival rate remains poor. Therefore, it is imperative to develop alternative or complimentary anti cancer drugs with minimum off target effects. Urolithin A, a microbial metabolite of ellagic acid and ellagitannins produced endogenously by human gut micro biome is considered to have anti-cancerous activity. However anti tumorigenic effect of urolithin A in OSCC is yet to be elucidated. In this study, we examined whether urolithin A inhibits cell growth and induces both apoptosis and autophagy dependent cell death in OSCC cell lines. PurposeThe present study aims to evaluate the potential of urolithin A to inhibit OSCC and its regulatory effect on OSCC proliferation and invasion in vitro and in vivo mouse models. MethodsWe evaluated whether urolithin A could induce cell death in OSCC in vitro and in vivo mouse models. ResultsFlow cytometric and immunoblot analysis on Urolithin A treated OSCC cell lines revealed that urolithin A markedly induced cell death of OSCC via the induction of endoplasmic reticulum stress and subsequent inhibition of AKT and mTOR signaling as evidenced by decreased levels of phosphorylated mTOR and 4EBP1. This further revealed a possible cross talk between apoptotic and autophagic signaling pathways. In vivo study demonstrated that urolithin A treatment reduced tumor size and showed a decrease in mTOR, ERK1/2 and Akt levels along with a decrease in proliferation marker, Ki67. Taken together, in vitro as well as our in vivo data indicates that urolithin A is a potential anticancer agent and the inhibition of AKT/mTOR/ERK signalling is crucial in Urolithin A induced growth suppression in oral cancer. ConclusionUrolithin A exerts its anti tumorigenic activity through the induction of apoptotic and autophagy pathways in OSCC. Our findings suggest that urolithin A markedly induced cell death of oral squamous cell carcinoma via the induction of endoplasmic reticulum stress and subsequent inhibition of AKT and mTOR signaling as evidenced by decreased levels of phosphorylated mTOR and 4EBP1. Urolithin A remarkably suppressed tumor growth in both in vitro and in vivo mouse models signifying its potential as an anticancer agent in the prevention and treatment of OSCC. Henceforth, our findings provide a new insight into the therapeutic potential of urolithin A in the prevention and treatment of OSCC.

Endoplasmic reticulum stress is attenuated by glycolysis in lymphatic malformations.

This study aims to investigate the role of endoplasmic reticulum stress (ER stress) in human dermal lymphatic endothelial cells (HDLECs) and lymphatic malformations (LMs) and its relationship with aerobic glycolysis and inflammation. The proliferation and apoptosis of HDLECs were examined with lipopolysaccharide (LPS) treatment. ER stress-associated proteins and glycolysis-related markers were detected by western blot. Glycolysis indexes were detected by seahorse analysis and lactic acid production assay kits. Immunohistochemistry was used to reveal the ER stress state of lymphatic endothelial cells (LECs) in LMs. LPS induced ER stress in HDLECs but did not trigger detectable apoptosis. Intriguingly, LPS-treated HDLECs also showed increased glycolysis flux. Knockdown of Hexokinase 2, a key enzyme for aerobic glycolysis, significantly inhibited the ability of HDLECs to resist ER stress-induced apoptosis. Moreover, compared to normal skin, glucose-regulated protein 78 (GRP78/BIP), and phosphorylation protein kinase R-like kinase (p-PERK), two key ER stress-associated markers, were upregulated in LECs of LMs, which was correlated with the inflected state. In addition, excessively activated ER stress inhibited the progression of LMs in rat models. These data indicate that glycolysis could rescue activated ER stress in HDLECs, which is required for the accelerated development of LMs. Inflammation enhances both ER stress and glycolysis in LECs while glycolysis is required to attenuate the pro-apoptotic effect of ER stress. Endoplasmic reticulum (ER) stress is activated in lymphatic endothelial cells (LECs) of LMs, especially in inflammatory condition. The expression of ER stress-related proteins is increased in LMs and correlated with Hexokinase 2 expression. Pharmacological activation of ER stress suppresses the formation of LM lesions in the rat model. ER stress may be a promising and effective therapeutic target for the treatment of LMs.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

Abstract PO4-08-01: Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer

Abstract Background Endoplasmic reticulum stress (ER stress) is a process in which cells activate unfolded protein response and other signaling pathways in response to misfolded and unfolded protein aggregation and calcium ion balance disturbance in the endoplasmic reticulum cavity. Depending on the intensity of endoplasmic reticulum stress, it can not only induce chaperone expression to play a protective effect, but also induce apoptosis independently. Metabolic reprogramming is one of the characteristics of tumor, which refers to the metabolic changes that cells make in response to various stimuli. Here, we demonstrate that methylsterol monooxygenase 1 (MSMO1) regulates cellular cholesterol metabolism and influences breast cancer susceptibility to chemotherapy by regulating the relative intracellular T-MAS content. Methods RNA-seq was performed on core needle biopsy samples from 26 patients receiving neoadjuvant chemotherapy for breast cancer to screen for genes that may influence neoadjuvant chemotherapy sensitivity. Cell proliferation, CCK8 assay and apoptosis assay were performed for MSMO1 phenotype study. Transmission electron microscopy (TEM) was conducted to visualize the structure of endoplasmic reticulum. Western Blot was used to detect the activation level of the unfolded protein response pathway. The relative content of sterols in MSMO1 knockdown cells was detected by targeted lipid mass spectrometry. AO/PI staining of breast cancer organoids was performed to verify the effect of MSMO1 metabolic substrate T-MAS on chemotherapy drug sensitivity. Results RNA-seq of core needle biopsy samples from breast cancer patients receiving neoadjuvant chemotherapy suggested that MSMO1 was highly expressed in non-pCR patients, and clinical survival data suggested that MSMO1 was associated with poor prognosis in patients with all types of breast cancer, suggesting that MSMO1 might be related to chemotherapy sensitivity of breast cancer. Phenotypic experiments revealed that MSMO1 regulates chemotherapy sensitivity of breast cancer both in vivo and in vitro. Ectopic overexpression of MSMO1 promoted cancer cell resistance to chemotherapy. To uncover the underlying mechanisms, RNA-seq of MSMO1 knock-down cells were carried out and identified that MSMO1 was associated with ER stress. TEM and western blot assay indicated that downregulation of MSMO1 expression leads to endoplasmic reticulum swelling and activation of the unfolded protein response. Considering that MSMO1 is a cholesterol metabolizing enzyme, targeted lipids mass spectrometry was performed to detect the changes in the contents of various intermediate metabolites in the cholesterol pathway, which indicated that the relative content of T-MAS, a metabolic substrate of MSMO1, was significantly up-regulated in MSMO1 knockdown cells. Then, in order to verify whether this metabolite can independently regulate ER stress, breast cancer cells were given T-MAS externally, and TEM and Western Blot analysis indicated that T-MAS could induce ER stress and activate the unfolded protein response independently. Apoptosis assay and AO/PI staining of breast cancer organoids revealed that T-MAS can improve the sensitivity of breast cancer to chemotherapy drugs. Conclusion In summary, these results shed light on the role of MSMO1 in cholesterol metabolism reprogramming, which adjust the endoplasmic reticulum stress state of cells by changing the relative content of intracellular T-MAS, and thereby influencing the sensitivity of breast cancer chemotherapy. As an intermediate metabolite of cholesterol metabolism, T-MAS has potential clinical application value of chemotherapy sensitization. Citation Format: Hengyu Ren, Liren Wangxu, Yayun Chi, Jiong Wu. Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-01.

Cytotoxic sigma-2 ligands trigger cancer cell death via cholesterol-induced-ER-stress

Sigma-2-ligands (S2L) are characterized by high binding affinities to 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 (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 cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. 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.

Borax affects cellular viability by inducing ER stress in hepatocellular carcinoma cells by targeting SLC12A5.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

Abstract 1040: Investigating The Role Of Lipoproteins In Macrophage Er Stress

Atherosclerosis, a major cause of cardiovascular disease (CVD), is an inflammatory disease defined by the progressive buildup of lipid-laden macrophages and cholesterol-filled plaques. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) play critical roles in the pathogenesis of atherosclerosis due to their roles in transporting cholesterol between tissues and cell types, including macrophages. Epidemiological studies have shown that high levels of HDL-cholesterol (HDL-C) are correlated with decreased CVD. However, HDL-C raising therapeutics failed to show clinical benefits, suggesting that HDL function may be a better indicator of CVD risk. Recent studies from our lab demonstrated that HDL modified by reactive aldehydes has impaired cardioprotective functions in macrophages. Other studies have also shown that oxidized LDL triggers ER stress in vascular cells and macrophages, a process that can be blunted by the presence of HDL. Therefore, we tested the hypothesis that HDL modified by reactive aldehydes triggers ER stress in macrophages, with the goal of identifying mechanisms by which dysfunctional HDL contributes to atherosclerosis progression. Primary murine macrophages were incubated with native or modified forms of lipoproteins and ER stress markers (XBP1, BiP, p-eIF2a, ATF6) were assessed by immunoblot analysis or semi-quantitative PCR. Through a comprehensive and extensive analysis, our findings led us to discover three novel findings in primary murine macrophages: (i) oxLDL alone was unable to induce ER stress; (ii) co-incubation with oxLDL or HDL in the presence of thapsigargin (an inducer of ER stress) resulted in increased and prolonged expression of ER stress markers as compared to cells treated with thapsigargin alone; and (iii) neither HDL nor HDL modified by reactive aldehydes was able blunt the ER stress induced by thapsigargin in the presence or absence of oxLDL. In summary, our highly systematic approach to assess the role of lipoproteins in mediating ER stress in primary macrophages generated convincing data that refutes previously-published findings, thus resulting in an inability to make definitive conclusions on the role of HDL modification in ER stress.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

A 700nm LED Light Activated Ru(II) Complex Destroys Tumor Cytoskeleton via Photosensitization and Photocatalysis.

Photoactivable chemotherapy (PACT) using metallic complexes provides spatiotemporal selectivity over drug activation for targeted anticancer therapy. However, the poor absorption in near-infrared (NIR) light region of most metallic complexes renders tissue penetration challenging. Herein, an NIR light triggered dinuclear photoactivable Ru(II) complex (Ru2) is presented and the antitumor mechanism is comprehensively investigated. The introduction of a donor-acceptor-donor (D-A-D) linker greatly enhances the intramolecular charge transition, resulting in a high molar extinction coefficient in the NIR region with an extended triplet excited state lifetime. Most importantly, when activated by 700nm NIR light, Ru2 exhibits unique slow photodissociation kinetics that facilitates synergistic photosensitization and photocatalytic activity to destroy diverse intracellular biomolecules. In vitro and in vivo experiments show that when activated by 700nm NIR light, Ru2 exhibits nanomolar photocytotoxicity toward 4T1 cancer cells via the induction of calcium overload and endoplasmic reticulum (ER) stress. These findings provide a robust foundation for the development of NIR-activated Ru(II) PACT complexes for phototherapeutic application.

Dysregulation of miR-146a is associated with exacerbated inflammation, oxidative and endoplasmic reticulum stress in the progression of diabetic foot ulcer.

Recent evidence has implicated the role of microRNA-146a (miR-146a) in regulating inflammatory responses. In the present study, we investigated the role of miRNA-146a in the progression of diabetic foot ulcer (DFU) in type 2 diabetes mellitus patients (T2DM) and studied its correlation with stress mediators such as Endoplasmic Reticulum (ER) and oxidative stress. Ninety subjects were enrolled and evenly distributed among three groups: Controls (n = 30), T2DM without complications (n = 30) and T2DM with foot ulcers (n = 30). Subsequently, each group was further subdivided based on the University of Texas classification. Peripheral blood was collected from all the study subjects, while tissue biopsies were taken only from DFU patients. Total RNA from both PBMCs and wound tissues were isolated using miRNA isolation kit and qPCR was performed to check the expression of miR-146a, ER stress and oxidative stress markers. Our findings revealed a significant decrease in miR-146a expression among T2DM patients with Grade 2 and Grade 3 DFUs compared with those with Grade 0 and Grade 1 DFUs. Notably, inflammatory genes regulated by miR-146a, including TRAF6, IRAK-1 and ADAM, were all upregulated in T2DM patients with Grade 2 and Grade 3 DFUs. Moreover, reduced miR-146a levels were correlated with increased markers of ER stress and oxidative stress in Grade 2 and Grade 3 DFU patients. Furthermore, our in vitro experiment using mouse 3T3 fibroblasts demonstrated a downregulation of miR-146a following induction of hyperglycaemia, ER stress and oxidative stress in these cells. These findings suggest a potential link between diminished miR-146a expression and heightened oxidative and ER stress in T2DM patients with more severe grades of DFUs. Our results imply that targeting miR-146a may hold therapeutic promise for managing disease progression in DFU patients, as it could help alleviate oxidative and ER stress associated with diabetic complications.

Analysis of Toxic Effects of Fluoride on Ovine Follicular Granulosa Cells Using RNA-Seq.

Fluoride is abundant in the environment and is an essential trace element in living organisms. However, prolonged excessive fluoride intake can lead to fluorosis, which poses a threat to the reproductive health of animals and humans. Although previous research has mainly focused on animal models, the impact of fluoride on ovine follicular granulosa cells (GCs) has not been comprehensively elucidated. This study employed RNA-Seq technology to elucidate the toxic effects of fluoride on ovine follicular GCs and its mechanism of action. Culturing primary ovine follicular GCs in vitro and subjecting them to fluoride treatment revealed 3218 differentially expressed genes (DEGs), with 2278 upregulated and 940 downregulated. Significantly, this study unveiled fluoride's induction of endoplasmic reticulum (ER) stress in cells, triggering a cascade involving the PERK pathway factor ATF4, leading to cell death via DDIT3/CHOP activation and the subsequent upregulation of CHAC1, ATF3, ERO1α, and TRIB3. These findings provide crucial insights into the toxicity of fluoride in ovine, offering a foundation for mitigating fluoride-related losses in the farming industry.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

The Interplay Between Endoplasmic Reticulum Stress and Oxidative Stress in Chondrocyte Catabolism.

Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation. Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM). Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA in vivo. Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.