Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.

Iodoacetic acid (IAA) is an emerging unregulated iodinated disinfection byproduct with high toxicity and widespread exposure. IAA has potential reproductive toxicity and could damage male reproduction. However, the underlying mechanisms and toxicological targets of IAA on male reproductive impairment are still unclear, and thus Sprague-Dawley rats and Leydig cells were used in this work to decode these pending concerns. Results showed that after IAA exposure, the histomorphology and ultrastructure of rat testes were abnormally changed, numbers of Leydig cells were reduced, the hypothalamic-pituitary-testis (HPT) axis was disordered, and testosterone biosynthesis was inhibited. Proteomics analyses displayed that oxidative stress, endoplasmic reticulum stress, and steroid hormone biosynthesis were involved in IAA-caused reproductive injury. Antioxidant enzymes were depleted, while levels of ROS, MDA, 8-OHdG, and γ-H2A.X were increased by IAA. IAA triggered oxidative stress and DNA damage, and then activated the GRP78/IRE1/XBP1s and cGAS/STING/NF-κB pathways in Leydig cells. The two signaling pathways constructed an interactive network by synergistically regulating the downstream transcription factor CHOP, which in turn directly bound to and negatively modulated steroidogenic StAR, finally refraining testosterone biosynthesis in Leydig cells. Collectively, IAA as a reproductive toxicant has anti-androgenic effects, and the GRP78/IRE1 and cGAS/STING pathway crosstalk through CHOP facilitates IAA-mediated testosterone decline.

BACKGROUND: Various factors, including blood, inflammatory, infectious, and immune factors, can cause ischemic stroke. However, the primary cause is often the instability of cervical arteriosclerosis plaque. It is estimated that 18-25% of ischemic strokes are caused by the rupture of carotid plaque.1 Plaque stability is crucial in determining patient prognosis. Developing a highly accurate, non-invasive, or minimally invasive technique to assess carotid plaque stability is crucial for diagnosing and treating stroke.Previous research by our group has demonstrated that the expression levels of CHOP (C/EBP homologous protein) and GRP78 (glucose-regulated protein 78) are correlated with the stability of atherosclerotic plaques.2 OBJECT: This research assesses changes in GRP78 and CHOP expressions in human umbilical vein endothelial cells(HUVEC) following experiments within the hemodynamic influencing factors test system. Additionally, it includes conducting an empirical study on the impact of blood flow shear force on the stability of human carotid atherosclerotic plaques. The objective is to explore the implications of blood flow shear force on the stability of carotid atherosclerotic plaques.
METHODS: The hemodynamic influencing factors test bench system was configured with low (Group A, 4 dyns/cm²), medium (Group B, 8 dyns/cm²), and high shear force groups (Group C, 12 dyns/cm²). Relative expression levels of GRP78 and CHOP proteins in human umbilical vein endothelial cells were measured using Western blot analysis, and quantitative analysis of GRP78 and CHOP mRNA was conducted using RT-qPCR. Meanwhile, plaques from 60 carotid artery patients, retrieved via Carotid Endarterectomy (CEA), were classified into stable (S) and unstable (U) groups based on pathological criteria. Shear force at the carotid bifurcation was measured preoperatively using ultrasound. Western blot and RT-qPCR were used to analyze the relative expression levels of GRP78 and CHOP proteins and mRNA, respectively, in the plaque specimens from both groups.
RESULTS: Expression levels of GRP78, CHOP proteins, and their mRNAs were assessed in groups A, B, and C via Western blot and RT-qPCR. Results showed that in the low-shear group, all markers were elevated in group A compared to groups B and C. Statistical analysis revealed significantly lower shear forces at the carotid bifurcation in group U compared to group S. In group U plaques, GRP78 and CHOP expressions were significantly higher in group U than in group S.
CONCLUSIONS: Blood flow shear forces variably affect the expression of GRP78 and CHOP proteins, as well as their mRNA levels, in vascular endothelial cells. The lower the shear force and fluid flow rate, the higher the expression of GRP78 and CHOP, potentially leading to endoplasmic reticulum stress(ERS), which may destabilize the plaque.

BACKGROUND: To date, Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disease associated with clinical complications. Dietary fatty acids have been suggested to be involved in preventing or reversing the accumulation of hepatic fat. However, contradicting roles of monounsaturated fatty acids to the liver have been implicated in various human and murine models, mainly due to the insolubility nature of fatty acids.
METHODS: High pressure homogenization methods were used to fabricate oleic acid embedded lipid nanoparticles (OALNs). The in vitro and in vivo models were used to validate the physiological effect of this OALNs via various cellular and molecular approaches including cell viability essay, fluorescent staining, electron microscope, RNAseq, qPCR, Western blots, and IHC staining.
RESULTS: We successfully fabricated OALNs with enhanced stability and solubility. More importantly, lipid accumulation was successfully induced in hepatocytes via the application of OALNs in a dose-dependent manner. Overload of OALNs resulted in ROS accumulation and apoptosis of hepatocytes dose-dependently. With the help of transcriptome sequencing and traditional experimental approaches, we demonstrated that the lipotoxic effect induced by OALNs was exerted via the DDIT3/BCL2/BAX/Caspases signaling. Moreover, we also verified that OALNs induced steatosis and subsequent apoptosis in the liver of mice via the activation of DDIT3 in vivo.
CONCLUSIONS: In all, our results established a potential pathogenic model of NAFLD for further studies and indicated the possible involvement of DDIT3 signaling in abnormal steatosis process of the liver.

C/EBP homologous protein (CHOP) triggers the death of multiple cancers via endoplasmic reticulum (ER) stress. However, the function and regulatory mechanism of CHOP in liver cancer remain elusive. We have reported that late endosomal/lysosomal adapter, mitogen-activated protein kinase and mTOR activator 5 (LAMTOR5) suppresses apoptosis in various cancers. Here, we show that the transcriptional and posttranscriptional inactivation of CHOP mediated by LAMTOR5 accelerates liver cancer growth. Clinical bioinformatic analysis revealed that the expression of CHOP was low in liver cancer tissues and that its increased expression predicted a good prognosis. Elevated CHOP contributed to destruction of LAMTOR5-induced apoptotic suppression and proliferation. Mechanistically, LAMTOR5-recruited DNA methyltransferase 1 (DNMT1) to the CpG3 region (-559/-429) of the CHOP promoter and potentiated its hypermethylation to block its interaction with general transcription factor IIi (TFII-I), resulting in its inactivation. Moreover, LAMTOR5-enhanced miR-182/miR-769 reduced CHOP expression by targeting its 3\'UTR. Notably, lenvatinib, a first-line targeted therapy for liver cancer, could target the LAMTOR5/CHOP axis to prevent liver cancer progression. Accordingly, LAMTOR5-mediated silencing of CHOP via the regulation of ER stress-related apoptosis promotes liver cancer growth, providing a theoretical basis for the use of lenvatinib for the treatment of liver cancer.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple‑negative breast cancer MDA‑MB‑231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription‑quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki‑67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti‑apoptotic protein Bcl‑2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3‑II and DNA damage markers, including cleaved PARP‑1, phosphorylated (p)‑ATM and p‑histone H2AX. The number of caspase 3/7‑positive cells was greater in montelukast‑treated cells compared with zafirlukast‑treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol‑requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast‑induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

Autophagy and Unfolded Protein Response (UPR) can be regarded as the safe keepers of cells exposed to intense stress. Autophagy maintains cellular homeostasis, ensuring the removal of foreign particles and misfolded macromolecules from the cytoplasm and facilitating the return of the building blocks into the system. On the other hand, UPR serves as a shock response to prolonged stress, especially Endoplasmic Reticulum Stress (ERS), which also includes the accumulation of misfolded proteins in the ER. Since one of the many effects of viral infection on the host cell machinery is the hijacking of the host translational system, which leaves in its wake a plethora of misfolded proteins in the ER, it is perhaps not surprising that UPR and autophagy are common occurrences in infected cells, tissues, and patient samples. In this book chapter, we try to emphasize how UPR, and autophagy are significant in infections caused by six major oncolytic viruses-Epstein-Barr (EBV), Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), Human Herpesvirus-8 (HHV-8), Human T-cell Lymphotropic Virus (HTLV-1), and Hepatitis B Virus (HBV). Here, we document how whole-virus infection or overexpression of individual viral proteins in vitro and in vivo models can regulate the different branches of UPR and the various stages of macro autophagy. As is true with other viral infections, the relationship is complicated because the same virus (or the viral protein) exerts different effects on UPR and Autophagy. The nature of this response is determined by the cell types, or in some cases, the presence of diverse extracellular stimuli. The vice versa is equally valid, i.e., UPR and autophagy exhibit both anti-tumor and pro-tumor properties based on the cell type and other factors like concentrations of different metabolites. Thus, we have tried to coherently summarize the existing knowledge, the crux of which can hopefully be harnessed to design vaccines and therapies targeted at viral carcinogenesis.

BACKGROUND: Regorafenib, a multi-targeted kinase inhibitor, has been used in the treatment of Hepatocellular carcinoma (HCC). The purpose of this study is to investigate the mechanism of Regorafenib in HCC.
METHODS: Regorafenib\'s impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments.
RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib.
CONCLUSIONS: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.

The study aims to explore the fluctuating expression of C/EBP Homologous Protein (CHOP) following rat carotid artery injury and its central role in vascular stenosis. Using in vivo rat carotid artery injury models and in vitro ischemia and hypoxia cell models employing human aortic endothelial cells (HAECs) and vascular smooth muscle cells (T/G HA-VSMCs), a comprehensive investigative framework was established. Histological analysis confirmed intimal hyperplasia in rat models. CHOP expression in vascular tissues was assessed using Western blot and immunohistochemical staining, and its presence in HAECs and T/G HA-VSMCs was determined through RT-PCR and Western blot. The study evaluated HAEC apoptosis, inflammatory cytokine secretion, cell proliferation, and T/G HA-VSMCs migration through Western blot, ELISA, CCK8, and Transwell migration assays. The rat carotid artery injury model revealed substantial fibrous plaque formation and vascular stenosis, resulting in an increased intimal area and plaque-to-lumen area ratio. Notably, CHOP is markedly elevated in vessels of the carotid artery injury model compared to normal vessels. Atorvastatin effectively mitigated vascular stenosis and suppresses CHOP protein expression. In HAECs, ischemia and hypoxia-induced CHOP upregulation, along with heightened TNFα, IL-6, caspase3, and caspase8 levels, while reducing cell proliferation. Atorvastatin demonstrated a dose-dependent suppression of CHOP expression in HAECs. Downregulation of CHOP or atorvastatin treatment led to reduced IL-6 and TNFα secretion, coupled with augmented cell proliferation. Similarly, ischemia and hypoxia conditions increased CHOP expression in T/G HA-VSMCs, which was concentration-dependently inhibited by atorvastatin. Furthermore, significantly increased MMP-9 and MMP-2 concentrations in the cell culture supernatant correlated with enhanced T/G HA-VSMCs migration. However, interventions targeting CHOP downregulation and atorvastatin usage curtailed MMP-9 and MMP-2 secretion and suppressed cell migration. In conclusion, CHOP plays a crucial role in endothelial injury, proliferation, and VSMCs migration during carotid artery injury, serving as a pivotal regulator in post-injury fibrous plaque formation and vascular remodeling. Statins emerge as protectors of endothelial cells, restraining VSMCs migration by modulating CHOP expression.