TL;DR
The involvement of ER stress-associated MAPK/JNK signaling in esculetin-induced apoptosis and functional impairment in INS-1 pancreatic β-cells is suggested, providing mechanistic insight into stress-associated β-cell dysfunction.
Cancer cells are able to survive under conditions of high endoplasmic reticulum (ER) stress by activating the adaptive unfolded protein response (UPR), which is closely linked with autophagy. On the other hand, excessive and prolonged ER stress leads to apoptosis. However, the relationships between different UPR branches and apoptosis or autophagy vary in cancer cells of different origins and depend on the extent and nature of the stress signal. This study was designed to establish the role of ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 (IRE1α) in apoptosis or autophagy signaling in T-lymphoblastic leukemia MOLT-3 cells via the RNA interference method. The cells were transfected with small interfering RNAs (si-PERK, si-IRE1α, or si-Cont) for 6 h and further cultured under normal conditions for 72 h to provide an insight into chronic effects of the gene silencing. The expression of apoptosis and autophagy effectors at the mRNA and protein levels was compared using RT-PCR and Western blot assays, respectively. Transfection of the cells with PERK siRNA led to a significant decrease in PERK protein and gene expression, and decreased phosphorylation of its downstream effector eukaryotic initiation factor 2α (eIF2α). PERK silencing was accompanied by activation of apoptosis-related genes and proteins—BCL2-associated X (Bax), caspase-3, C/EBP homologous protein (CHOP), while the levels of autophagy markers (Unc-51 like autophagy activating kinase 1 (ULK1), Beclin-1, and microtubule-associated proteins 1A/1B light chain 3 (LC3A/B)) remained stable. In contrast, treatment of the cells with si-IRE1α reduced the content of IRE1α, X-box-binding protein 1 (sXBP1), and glucose-regulated protein 78 (GRP78) proteins, but increased ERN1 gene expression. IRE1α RNA interference did not affect the levels of the pro-apoptotic marker Bax, but suppressed caspase-3, CHOP, c-Jun N-terminal kinase (JNK), and autophagy signaling molecules (ULK1, Beclin-1, LC3A/B) at both the transcriptional and translational levels. These results indicate that the PERK pathway is an important contributor to the survival of MOLT-3 cells under basal ER stress, while PERK depletion compromises the resistance of cells to UPR-mediated apoptosis. The IRE1α UPR branch is directly linked with autophagy-dependent signaling, although IRE1α knockdown exerted a more complicated influence on the cells, probably via activation of multiple pro-death and compensatory pro-survival regulatory mechanisms.
Endoplasmic reticulum (ER) stress contributes to β cell death in both Type 1 and Type 2 diabetes (T1D and T2D). However, the molecular mechanisms driving β cell death during ER stress remain insufficiently defined, limiting development of protective therapies. GRP78, an ER chaperone, is the master regulator of unfolded protein response (UPR), suppressing UPR initiators during the unstressed state and releasing them to allow UPR activation during stress. To dissect the pathways leading to ER-stress response related β cell decompensation, we engineered mice genetically lacking GRP78 in pancreatic β cells. GRP78 deletion caused acute insulin-deficient diabetes in pups before weaning, with reduced β cell mass due to increased apoptosis. Molecular studies identified deregulated UPR, specifically IRE1 activity, as driving cell death. Unbiased and targeted analyses identified a JNK-p53 axis downstream of IRE1 kinase as a key mediator of β cell death during UPR activation. In vivo JNK inhibition protected against β cell death in 2 distinct ER stress diabetes models. In human β cells, pharmacological inhibition of both JNK and p53 improved β cell survival during GRP78 knockdown–induced UPR. These findings provide insight into mechanisms causing β cell death during ER stress and outline possible therapeutic targets to preserve insulin secretory capacity in diabetes.
Glioblastoma (GBM) is a highly aggressive and therapy-resistant brain tumor. In our earlier study, we demonstrated that the antihyperlipidemic drug Gemfibrozil, when repurposed, exerts strong cytotoxic effects on GBM cells by modulating autophagy. In the present study, we further investigated the complex interplay among key homeostatic pathways, specifically autophagy, endoplasmic reticulum (ER) stress, and calcium signalling, following Gemfibrozil exposure in the context of GBM progression and treatment resistance. Our findings reveal that Gemfibrozil-induced inhibition of autophagy triggers the unfolded protein response (UPR), leading to activation of ER stress pathways, as evidenced by altered expression of canonical markers, including IRE1α, PERK, and CHOP. Notably, CHOP silencing reduced levels of cleaved Caspase-3 and Caspase-9, confirming the involvement of ER stress-mediated apoptosis following disruption of autophagy. Moreover, the induction of ER stress and inhibition of autophagy were accompanied by disturbances in calcium homeostasis, demonstrated by reduced expression of the calcium-binding proteins Calmodulin and Calreticulin. This imbalance resulted in mitochondrial calcium overload, loss of mitochondrial membrane potential, and elevated reactive oxygen species (ROS) production, ultimately culminating in caspase activation and cell death. Mechanistically, PGC-1α emerged as a key regulator of Gemfibrozil-mediated anti-tumor activity. Collectively, our findings uncover a critical molecular cascade involving suppression of autophagy, induction of ER stress, calcium dysregulation, mitochondrial dysfunction, and oxidative stress that drives GBM cell death. These insights not only highlight Gemfibrozil as a potential therapeutic agent but also emphasize the importance of targeting homeostatic vulnerabilities in GBM.
Asprosin is a profibrillin-1-derived protein hormone involved in energy metabolism, glucose homeostasis, and appetite. Although asprosin can cross the blood-brain barrier and act within the central nervous system, its direct role in hippocampal neurons remains unclear. This study examined asprosin system components and the effects of asprosin on metabolic activity, proliferation, reactive oxygen species (ROS) production, cell cycle progression, apoptosis, and intracellular signaling in mouse hippocampal HT-22 cells. Asprosin, Furin, and Olfr734 expression was assessed by Western blot and immunocytochemistry, whereas intracellular and secreted asprosin levels were quantified by ELISA. Cells were treated with asprosin over 0.01-100 nM, and low nanomolar doses were selected for detailed cell cycle, apoptosis, and signaling analyses. Metabolic activity, proliferation, ROS production, cell cycle distribution, and apoptosis were evaluated using alamarBlue, bromodeoxyuridine incorporation, ROS detection, and flow cytometry assays. Selected ROS-, cell cycle-, and apoptosis-related markers were analyzed by reverse transcription quantitative polymerase chain reaction and Western blot. PKA and ERK1/2 involvement was assessed by phosphorylation analysis and pharmacological inhibition. HT-22 cells expressed Asprosin, Furin, and Olfr734. Asprosin regulated cellular responses in a dose- and time-dependent manner: low nanomolar doses generally supported metabolic and proliferative activity, whereas 10 and 100 nM reduced these parameters. Asprosin did not markedly increase total ROS production but modulated Hmox1 and Sod2 expression. It also altered cell cycle distribution, apoptosis-related parameters, PKA and ERK1/2 phosphorylation, and inhibitor-sensitive expression of Ccne1, Bax, and Bcl2. These findings identify, for the first time, a functional asprosin system in mouse HT-22 hippocampal neurons.
Genistein (GEN) exhibits concentration- and cell-type-dependent biological activities; however, the mechanisms underlying its cytotoxicity remain incompletely understood. This study investigated whether oxidative stress-mediated mitochondrial dysfunction contributes to GEN-induced apoptosis in TM4 mouse Sertoli cells and ELT3 rat leiomyoma cells. Cells were exposed to GEN (30 or 100 µM) for 48 h. Cytotoxicity, oxidative stress, mitochondrial function, and apoptosis were assessed by measuring cell viability, lactate dehydrogenase release, reactive oxygen species (ROS), malondialdehyde, glutathione reductase activity, mitochondrial membrane potential (ΔΨm), intracellular ATP, apoptosis-related gene expression, and caspase-3/-9 activities. The involvement of oxidative stress was examined using N-acetyl-L-cysteine (NAC). GEN reduced cell viability, antioxidant capacity, ΔΨm, and ATP content while increasing membrane damage, ROS accumulation, and lipid peroxidation in both cell lines. These changes were accompanied by upregulation of Bax, Tp53, caspase-3, and caspase-9; downregulation of Bcl-2; and increased caspase-3/-9 activities. NAC pretreatment attenuated these alterations, supporting ROS as an upstream mediator. Notably, TM4 cells were approximately 4.4-fold more sensitive to GEN than ELT3 cells at 48 h. Collectively, the findings support a ROS-dependent mitochondrial apoptotic response under elevated in vitro GEN exposure but do not establish selective antitumor cytotoxicity or direct physiological relevance.
Background Progressive loss of pancreatic β-cell function in type 2 diabetes mellitus (T2DM) is linked to endoplasmic reticulum (ER) stress–induced apoptosis. Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) have glucose-lowering and potential cytoprotective effects. We investigated whether empagliflozin protects β-cells from ER stress–mediated apoptosis and the underlying pathway. Methods Mouse Beta-TC-6 (BTC-6) and hamster HIT-T15 pancreatic β-cell lines were exposed to tunicamycin (5 or 10 μg/mL) to induce ER stress, with or without empagliflozin (10−8, 10−9, 10−10 M). SGLT-1 and SGLT-2 mRNA were assessed by RT-qPCR. Expression of ER stress markers (GRP-94, BiP, PERK, eIF-2α, IRE-1, ATF-4, CHOP) was measured by RT-qPCR; eIF-2α, phospho-eIF-2α and CHOP protein levels were analyzed by Western blot. Cell proliferation and apoptosis were quantified by XTT and Annexin V-FITC assays. Results BTC-6 cells expressed SGLT-1 but not SGLT-2. Empagliflozin (10−8 and 10−9 M) increased BTC-6 proliferation (p < 0.01 and p < 0.05 respectively). Tunicamycin caused significant apoptosis after 48h (p < 0.001). Co-treatment with empagliflozin (10−8, 10−9 M) significantly reduced tunicamycin-induced apoptosis, especially at 5 μg/ml tunicamycin (p < 0.01). Empagliflozin co-incubation lowered PERK, eIF-2α, IRE-1α and CHOP mRNA levels versus tunicamycin alone (all p < 0.05), and decreased phospho-eIF-2α (p < 0.01) and CHOP (p < 0.05) protein levels. Similar anti-apoptotic effects were observed in HIT-T15 cells. Conclusions Empagliflozin enhances β-cell proliferation and attenuates ER stress–induced apoptosis in vitro, primarily via downregulation of the PERK–eIF-2α–CHOP pathway. These findings support further investigation of SGLT-2 inhibitors for preservation of β-cell survival and function.
