Unravelling the role of NFE2L1 in stress responses and related diseases.

The nuclear factor erythroid 2 (NF-E2)-related factor 1 (NFE2L1, also known as Nrf1) is a highly conserved transcription factor that belongs to the CNC-bZIP subfamily. Its significance lies in its control over redox balance, proteasome activity, and organ integrity. Stress responses encompass a series of compensatory adaptations utilized by cells and organisms to cope with extracellular or intracellular stress initiated by stressful stimuli. Recently, extensive evidence has demonstrated that NFE2L1 plays a crucial role in cellular stress adaptation by 1) responding to oxidative stress through the induction of antioxidative responses, and 2) addressing proteotoxic stress or endoplasmic reticulum (ER) stress by regulating the ubiquitin-proteasome system (UPS), unfolded protein response (UPR), and ER-associated degradation (ERAD). It is worth noting that NFE2L1 serves as a core factor in proteotoxic stress adaptation, which has been extensively studied in cancer and neurodegeneration associated with enhanced proteasomal stress. In these contexts, utilization of NFE2L1 inhibitors to attenuate proteasome "bounce-back" response holds tremendous potential for enhancing the efficacy of proteasome inhibitors. Additionally, abnormal stress adaptations of NFE2L1 and disturbances in redox and protein homeostasis contribute to the pathophysiological complications of cardiovascular diseases, inflammatory diseases, and autoimmune diseases. Therefore, a comprehensive exploration of the molecular basis of NFE2L1 and NFE2L1-mediated diseases related to stress responses would not only facilitate the identification of novel diagnostic and prognostic indicators but also enable the identification of specific therapeutic targets for NFE2L1-related diseases.

2-Hydroxypropyl-ß-cyclodextrin Regulates the Epithelial to Mesenchymal Transition in Breast Cancer Cells by Modulating Cholesterol Homeostasis and Endoplasmic Reticulum Stress.

Cholesterol metabolism affects endoplasmic reticulum (ER) stress and modulates epithelial-mesenchymal transition (EMT). Our previous study demonstrated that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) attenuated EMT by blocking the transforming growth factor (TGF)-ß/Smad signaling pathway and activating ER stress in MDA-MB-231 cells. To further assess the detailed mechanisms between cholesterol metabolism, ER stress, and EMT, LXR-623 (an agonist of LXRα) and simvastatin were used to increase and decrease cholesterol efflux and synthesis, respectively. Here, we found that high HP-ß-CD concentrations could locally increase cholesterol levels in the ER by decreasing LXRα expression and increasing Hydroxymethylglutaryl-Coenzyme A reductase (HMGCR) expression in MDA-MB-231 and BT-549 cells, which triggered ER stress and inhibited EMT. Meanwhile, tunicamycin-induced ER stress blocked the TGF-ß/Smad signaling pathway. However, low HP-ß-CD concentrations can decrease the level of membrane cholesterol, enhance the TGF-ß receptor I levels in lipid rafts, which helped to activate TGF-ß/Smad signaling pathway, inhibit ER stress and elevate EMT. Based on our findings, the use of high HP-ß-CD concentration can lead to cholesterol accumulation in the ER, thereby inducing ER stress, which directly suppresses TGF-ß pathway-induced EMT. However, HP-ß-CD is proposed to deplete membrane cholesterol at low concentrations and concurrently inhibit ER stress and induce EMT by promoting the TGF-ß signaling pathways.

Tunicamycin-Induced Endoplasmic Reticulum Stress Promotes Breast Cancer Cell MDA-MB-231 Apoptosis through Inhibiting Wnt/ß-Catenin Signaling Pathway.

Triple negative breast cancer (TNBC) has significantly threatened human health. Many aspects of TNBC are closely related to Wnt/ß-catenin pathway, and cell apoptosis induced by endoplasmic reticulum stress (ER stress) in TNBC may act as a potential target of non-chemotherapy treatment. However, how ER stress interacts with this pathway in TNBC has not yet been understood. Here, the tunicamycin and LiCl have been applied to MDA-MB-231. The related proteins' expression was measured by western blotting. Moreover, acridine orange/ethidium bromide (AO/EB) staining was applied to test the apoptosis degree of the cells, and cell viability was tested by MTT experiment. Then, we found the ER stress and apoptosis degree of MDA-MB-231 were induced after treatment with tunicamycin. Besides, tunicamycin dose dependently inhibited both Wnt/ß-catenin pathway and cells viability. Licl, an activator of Wnt/ß-catenin signaling pathway, could significantly inhibit cell apoptosis. In conclusion, our study found that the activation of ER stress could promote the MDA-MB-231 apoptosis by repressing Wnt/ß-catenin pathway, which provides some promising prospects and basic mechanism to the further research.

Endoplasmic Reticulum Stress Affects Cholesterol Homeostasis by Inhibiting LXRα Expression in Hepatocytes and Macrophages.

Atherosclerosis (AS) is the most common cardiovascular disease, and reverse cholesterol transport (RCT) plays an important role in maintaining cholesterol homeostasis. Both endoplasmic reticulum (ER) stress and LXRα can affect the metabolism of cholesterol. However, whether ER stress can modulate cholesterol metabolism by LXRα in hepatocytes and macrophages remains unclear. Therefore, in this study, we aimed to explore the relationship between ER stress induced by tunicamycin and LXRα in hepatocytes and macrophages and clarify their possible mechanisms and roles in AS. C57BL/6 mice and Huh-7 and THP-1 cells were treated with tunicamycin and LXR-623 (an agonist of LXRα) alone or in combination. Tunicamycin-induced ER stress caused liver injury; promoted the accumulation of cholesterol and triglycerides; inhibited the expression of LXRα, ABCA1 and ABCG1 in the livers of mice, thus reducing serum high-density lipoprotein (HDL)-C, low-density lipoprotein (LDL)-C, total cholesterol and triglyceride levels; however, LXR-623 could attenuate ER stress and reverse these changes. We also obtained the same results in Huh-7 and THP-1 cells. ER stress induced by tunicamycin could clearly be reversed by activating LXRα because it promoted cholesterol efflux by enhancing the expression of ABCA1 and ABCG1 in hepatocytes and macrophages, contributing to attenuation of the development of AS.

Sphingomyelin Synthase 2 Participate in the Regulation of Sperm Motility and Apoptosis.

Sphingomylin participates in sperm function in animals, and also regulates the Akt and ERK signaling pathways, both of which are associated with the asthenospermia. Sphingomyelin synthase 2 (SMS2) is involved in the biosynthesis of sphingomylin. To determine the relationship between SMS2 and human sperm function, we analyzed the distribution of SMS2 in human sperm and testes, and SMS2 expression in patients with asthenospermia and normozoospermia; human sperm were treated with anti-SMS2, and the sperm motility, penetration ability into methylcellulose, capacitation and acrosome reaction, and sperm [Ca2+]i imaging were evaluated, while the Akt and ERK pathway and cleaved caspase 3 were also analyzed. Results showed that SMS2 was localized in the testis and human sperm, and the protein levels of normozoospermia were higher than asthenospermia. Inhibition of SMS2 activity significantly decreased sperm motility and penetration ability into methylcellulose, but had no influence on capacitation and acrosome reaction, or on intracellular [Ca2+]i compared to IgG-treated control groups. Moreover, the phosphorylation level of Akt was decreased, whereas the phosphorylation of ERK and cleaved-caspase 3 levels were significantly increased. Taken together, SMS2 can affect sperm motility and penetration ability into methylcellulose, and participate in apoptosis associated with the Akt and ERK signaling pathways.

Hydroxypropyl­ß­cyclodextrin attenuates the epithelial­to­mesenchymal transition via endoplasmic reticulum stress in MDA­MB­231 breast cancer cells.

The epithelial­to­mesenchymal transition (EMT) has been reported to serve vital roles in regulating the progress of cancer metastasis. In addition, lipid rafts enriched in sphingolipids and cholesterol serve important roles in physiological and biochemical processes as a signaling platform. The present study explored the effects of hydroxypropyl­ß­cyclodextrin (HP­ß­CD), a cholesterol­depleting agent of lipid rafts, on the transforming growth factor (TGF)­ß/Smad signaling pathway and endoplasmic reticulum (ER) stress in mediating EMT in MDA­MB­231 breast cancer cells. HP­ß­CD treatment inhibited TGF­ß1­induced EMT, based on increased expression of E­cadherin and decreased expression of vimentin. HP­ß­CD reduced the expression of the TGF receptor TßRI and blocked the phosphorylation of Smad2. In addition, HP­ß­CD increased the expression of ER stress­related proteins (binding immunoglobulin protein and activating transcription factor 6), but TGF­ß1 could reverse these changes. Sodium 4­phenylbutyrate, an inhibitor of ER stress, suppressed these effects of HP­ß­CD on EMT and TGF­ß/Smad signaling pathway inhibition in breast cancer cells. Thus, HP­ß­CD can block the TGF­ß/Smad signaling pathway via diminishing the expression of TßRI which helps to activate ER stress and attenuate EMT in MDA­MB­231 cells, highlighting a potential target of lipid rafts for breast cancer treatment.

Individual factors define the overall effects of dietary genistein exposure on breast cancer patients.

As an endocrine disruptor, tyrosine kinase inhibitor, and DNA methyltransferase inhibitor, genistein can interfere with breast cancer development. However, as the results of numerous studies are contradictory, it is unclear whether genistein plays a positive or negative role. Retrospective epidemiological studies have indicated that high genistein intake is related to reduced breast cancer risk, but this protective effect has not been reported in clinical trials. Additionally, rodent and cellular studies show that genistein promoted breast cancer progression. Obviously, genistein's bioactivities do not solely depend upon the dose, and simply discussing the overall effects of genistein without considering individual factors is unrealistic. The purpose of this review was to collect relevant studies (over 164) on genistein and breast cancer that were published on PubMed from 1984 to 2019 and to summarize the impact of key individual factors on the bioactivities of genistein in breast cancer prevention and treatment. Furthermore, the related potential molecular mechanisms were explored to explain the contradictions in genistein-breast cancer studies. Our results showed that the intake mode and metabolic characteristics of genistein, as well as the menopausal status, estrogen receptor expression pattern, and gene mutations of the patient, are important factors that should be included when discussing the bioactivities of genistein. A better understanding of the influence of individual factors may enable the precise prediction of personalized responses to dietary genistein exposure. Given that the current information on genistein is mostly restricted to the cellular level, more comprehensive human studies should be performed to clarify the relationship between genistein and breast cancer.

Sphingomyelin synthase 1 regulates the epithelial­to­mesenchymal transition mediated by the TGF­ß/Smad pathway in MDA­MB­231 cells.

Breast cancer is the most common cancer in women and a leading cause of cancer­associated mortalities in the world. Epithelial­to­mesenchymal transition (EMT) serves an important role in the process of metastasis and invasive ability in cancer cells, and transforming growth factor ß1 (TGF­ß1) have been investigated for promoting EMT. However, in the present study, the role of the sphingomyelin synthase 1 (SMS1) in TGF­ß1­induced EMT development was investigated. Firstly, bioinformatics analysis demonstrated that the overexpression of SMS1 negatively regulated the TGFß receptor I (TßRI) level of expression. Subsequently, the expression of SMS1 was decreased, whereas, SMS2 had no significant difference when MDA­MB­231 cells were treated by TGF­ß1 for 72 h. Furthermore, the present study constructed an overexpression cells model of SMS1 and these cells were treated by TGF­ß1. These results demonstrated that overexpression of SMS1 inhibited TGF­ß1­induced EMT and the migration and invasion of MDA­MB­231 cells, increasing the expression of E­cadherin while decreasing the expression of vimentin. Furthermore, the present study further confirmed that SMS1 overexpression could decrease TßRI expression levels and blocked smad family member 2 phosphorylation. Overall, the present results suggested that SMS1 could inhibit EMT and the migration and invasion of MDA­MB­231 cells via TGF­ß/Smad signaling pathway.

Synergistic effect of receptor-interacting protein 140 and simvastatin on the inhibition of proliferation and survival of hepatocellular carcinoma cells.

Hepatocellular carcinoma is the sixth most prevalent malignant tumor and the third most common cause of cancer-associated mortality. Statins have been investigated for carcinoma prevention and treatment. In addition, receptor-interacting protein 140 (RIP140) has been observed to inhibit the Wnt/ß-catenin signaling pathway and cell growth. The present study aimed to investigate whether simvastatin (SV) is able to induce SMCC-7721 cell apoptosis through the Wnt/ß-catenin signaling pathway. Initially, a cell model of RIP140 overexpression was established, and then cells were treated with SV. The cell growth, viability and apoptosis were measured by cell counting kit-8 and flow cytometry. Furthermore, the expression levels of RIP140, ß-catenin, c-myc and cyclin D1 were detected by reverse transcription-quantitative polymerase chain, western blot analysis and immunofluorescence. The results demonstrated that SV significantly increased the expression of RIP140 in SMCC-7721 cells; however, ß-catenin, c-myc and cyclin D1 levels were significantly decreased. Furthermore, the immunofluorescence assay of ß-catenin confirmed that SV decreased the content of this protein in SMCC-7721 cells. Notably, RIP140 exerted a synergistic effect on the apoptosis rate induced by SV (RIP140 + SV group), while the alteration in RIP140, ß-catenin, c-myc and cyclin D1 levels was more evident in the combination group as compared with the RIP140 or SV alone groups. In conclusion, these results suggested that SV is able to induce the apoptosis of SMCC-7721 cells through the Wnt/ß-catenin signaling pathway, as well as that RIP140 and SV exert a synergistic effect on the inhibition of cell proliferation and survival.

Sphingomyelin synthase 2 overexpression promotes cisplatin-induced apoptosis of HepG2 cells.

Hepatoblastoma (HB) is the most type of common pediatric liver cancer. The primary chemotherapy drug for HB is cisplatin (DDP). However, patients readily develop intrinsic and acquired resistance, and severe side effects to treatment. Sphingomyelin synthase 2 (SMS2) is a key enzyme involved in the generation of sphingomyelin (SM), which is able to regulate cell proliferation, apoptosis and differentiation. The death receptors (DRs) have important functions in DDP-induced apoptosis. However, whether SMS2 is able to modulate cell apoptosis through the DR signaling pathway remains unknown. To investigate this question, SMS2 was overexpressed in HepG2 cells and treated with 3.5 mg/l cisplatin in the present study. After 24 h, the expression of SMS2, avian myelocytomatosis viral oncogene homolog (c-Myc), DR4, DR5 and caspase-3 was analyzed. Furthermore, cell viability was quantified, and apoptosis was assessed by western blot and flow cytometry analysis as well as Cell Counting kit-8. The results of the present study revealed that overexpression of SMS2 was able to increase the expression of c-Myc, cleaved caspase-3, DR4 and DR5 compared with the control group (P<0.05, n=3), and increase the levels of apoptosis in the SMS2 + DDP group, compared with the control (P<0.001, n=3). These results indicate that overexpression of SMS2 is able to improve sensitivity of HepG2 cells to DDP by increasing the expression of c-Myc, DR4 and DR5 in HepG2 cells. This increased sensitivity may decrease intrinsic and acquired resistance of chemotherapy in HB, and reduce the associated severe side effects in pediatric patients.

Sphingomyelin synthase overexpression increases cholesterol accumulation and decreases cholesterol secretion in liver cells.

BACKGROUND: Studies have shown that plasma high density lipoprotein cholesterol levels are negatively correlated with the development of atherosclerosis, whereas epidemiological studies have also shown that plasma sphingomyelin level is an independent risk factor for atherosclerosis. METHODS: To evaluate the relationship between cellular sphingomyelin level and cholesterol metabolism, we created two cell lines that overexpressed sphingomyelin synthase 1 or 2 (SMS1 or SMS2), using the Tet-off expression system. RESULTS: We found that SMS1 or SMS2 overexpression in Huh7 cells, a human hepatoma cell line, significantly increased the levels of intracellular sphingomyelin, cholesterol, and apolipoprotein A-I and decreased levels of apolipoprotein A-I and cholesterol in the cell culture medium, implying a defect in both processes. CONCLUSIONS: Our findings indicate that the manipulation of sphingomyelin synthase activity could influence the metabolism of sphingomyelin, cholesterol and apolipoprotein A-I.