Resistance exercise preconditioning prevents disuse muscle atrophy by inhibiting apoptosis and protein degradation via SESN2 in C57BL/6J mice.

AIM: To compare the effects of different exercise preconditioning in the context of skeletal muscle atrophy and to investigate the potential involvement of Sestrin2 (SESN2), a stress-inducible protein that can be regulated by exercise, in exercise preconditioning on preventing disuse muscle atrophy. METHODS: Eight-week-old male C57BL/6J mice were randomly assigned to sedentary groups (SD), aerobic exercise groups (AE), resistance exercise groups (RE), and combined exercise groups (CE) with or without 7 days of immobilization. The duration of the exercise intervention was 10 weeks. The effects of different exercise preconditioning to prevent muscle atrophy were analyzed by evaluating skeletal muscle function and mass. Additionally, to investigate the potential underlying mechanism of exercise-induced protection of skeletal muscle, wild-type and SESN2--/-- mice were randomly divided into sedentary group and resistance exercise preconditioning group. C2C12 cells were treated with SESN2 adenoviruses and MK2206 (an AKT inhibitor) for 48 h to elucidate the underlined mechanism. RESULTS: RE was more effective in preserving skeletal muscle function, muscle mass and maintaining skeletal muscle protein homeostasis than AE and CE under immobilized condition. Importantly, exercise performance, muscle mass to body weight ratio, and the cross-sectional area of muscle fibers were significantly lower in SESN2-/- mice than wild-type mice after resistance exercise preconditioning. Mechanistically, the absence of SESN2 led to activation of the ubiquitin-proteasome system and induction of apoptosis. In vitro experiments showed that MK2206 treatment mitigated the regulatory effects of overexpression-SESN2 on protein hydrolysis and apoptosis. CONCLUSION: RE was more effective than AE or CE in preventing disuse muscle atrophy. SESN2 mediated the protective effects of resistance exercise preconditioning on skeletal muscle atrophy.

SESN1 functions as a new tumor suppressor gene via Toll-like receptor signaling pathway in neuroblastoma.

AIMS: Neuroblastoma (NB) is the most common extracranial solid tumor in children, with a 5-year survival rate of <50% in high-risk patients. MYCN amplification is an important factor that influences the survival rate of high-risk patients. Our results indicated MYCN regulates the expression of SESN1. Therefore, this study aimed to investigate the role and mechanisms of SESN1 in NB. METHODS: siRNAs or overexpression plasmids were used to change MYCN, SESN1, or MyD88's expression. The role of SESN1 in NB cell proliferation, migration, and invasion was elucidated. Xenograft mice models were built to evaluate SESN1's effect in vivo. The correlation between SESN1 expression and clinicopathological data of patients with NB was analyzed. RNA-Seq was done to explore SESN1's downstream targets. RESULTS: SESN1 was regulated by MYCN in NB cells. Knockdown SESN1 promoted NB cell proliferation, cell migration, and cell invasion, and overexpressing SESN1 had opposite functions. Knockdown SESN1 promoted tumor growth and shortened tumor-bearing mice survival time. Low expression of SESN1 had a positive correlation with poor prognosis in patients with NB. RNA-Seq showed that Toll-like receptor (TLR) signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway in cancer were potential downstream targets of SESN1. Knockdown MyD88 or TLRs inhibitor HCQ reversed the effect of knockdown SESN1 in NB cells. High expression of SESN1 was significantly associated with a higher immune score and indicated an active immune microenvironment for patients with NB. CONCLUSIONS: SESN1 functions as a new tumor suppressor gene via TLR signaling pathway in NB.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Sestrin 2 protects human lens epithelial cells from oxidative stress and apoptosis induced by hydrogen peroxide by regulating the mTOR/Nrf2 pathway.

OBJECTIVE: We aimed to explore the effect and potential mechanism of Sestrin 2 (SESN2) in human lens epithelial cells (HLECs). METHODS: To mimic the oxidative stress environment, SAR01/04 cells were treated with 200 µM hydrogen peroxide (H2O2) for 24 h. Cell viability and apoptosis were checked by cell counting kit-8 and flow cytometry. Western blot was taken to check the protein changes of SESN2, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, ribosomal protein S6 kinase B1 (p70S6K), p-p70S6K, and nuclear factor erythroid 2-related factor 2 (Nrf2). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected via the corresponding reagent kit. The levels of interleukin (IL)-1ß, IL-18, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. RESULTS: SESN2 was down-regulated in cataract lens tissue and up-regulated in SAR01/04 cells treated with H2O2. Under treatment of H2O2, up-regulation of SESN2 improved cell viability, enhanced the activity of SOD and CAT, inhibited cell apoptosis, and reduced the levels of MDA, ROS, IL-1ß, IL-18, and TNF-α, while down-regulation of SESN2 caused the contrary effects. Further bioinformatics analysis suggested that SESN2 regulated the mTOR signaling pathway. Treatment of H2O2 inhibited p-mTOR and p-p70S6K protein expression, while overexpression of SESN2 increased p-mTOR and p-p70S6K protein expression in the H2O2 group and down-regulation of SESN2 further decreased p-mTOR and p-p70S6K protein expression in the H2O2 group. Additionally, H2O2 increased Nrf2 protein expression, and overexpression of SESN2 further increased Nrf2 protein expression in the H2O2 group. Importantly, rapamycin (an inhibitor of mTOR signaling pathway) and knockdown of Nrf2 reversed the promotive effects of SESN2 on cell viability and the inhibitive effects of SESN2 on cell apoptosis, oxidative stress, and inflammatory reaction. CONCLUSION: SESN2 protected HLECs damage induced by H2O2, which was related to the activation of mTOR/Nrf2 pathway.

Investigating the Potential Mechanisms of Ferroptosis and Autophagy in the Pathogenesis of Gestational Diabetes.

Ferroptosis and autophagy are two different cellular processes that have recently been highlighted for their potential roles in the pathogenesis and progression of gestational diabetes (GD). This research sought to uncover the crucial genes tied to ferroptosis and autophagy in GD, further investigating their mechanisms. Differentially expressed genes (DEGs) linked to ferroptosis and autophagy in GD were identified using publicly available data. Pathway enrichment, protein interactions, correlation with immune cell infiltration, and diagnostic value of DEGs were analyzed. HTR-8/SVneo cells were subjected to varying glucose levels to evaluate cell viability and the expression of markers related to ferroptosis and proteins associated with autophagy. Crucial DEGs were validated in vitro. A total of 12 DEGs associated with ferroptosis and autophagy in GD were identified, enriched in the PI3K-AKT signaling pathway. These genes exhibited significant correlations with monocyte infiltration, resting CD4 memory T cells, and follicular helper T cells. They exhibited high diagnostic value for GD (AUC: 0.77-0.97). High glucose treatment inhibited cell viability, induced ferroptosis, and activated autophagy in HTR-8/SVneo cells. Validation confirmed altered expression of SNCA, MTDH, HMGB1, TLR4, SOX2, SESN2, and HMOX1 after glucose treatments. In conclusion, ferroptosis and autophagy may play a role in GD development through key genes (e.g., TLR4, SOX2, SNCA, HMOX1, HMGB1). These genes could serve as promising biomarkers for GD diagnosis.

Orientin alleviates ox-LDL-induced oxidative stress, inflammation and apoptosis in human vascular endothelial cells by regulating Sestrin 1 (SESN1)-mediated autophagy.

Endothelial cells are a crucial component of the vessel-tissue wall and exert an important role in atherosclerosis (AS). To explore the role of Orientin in AS, human vascular endothelial cells (HUVECs) were induced by oxidized low-density lipoprotein (ox-LDL) to simulate the vascular endothelial injury during AS. Cell viability was detected by CCK-8 assay. Oxidative stress and inflammation related markers were measured using kits, RT-qPCR or western blot. Besides, cell apoptosis was assessed with TUNEL staining and cell autophagy was evaluated by LC3 immunofluorescent staining. Additionally, western blot was utilized to evaluate the expression of Sestrin 1 (SESN1) and proteins in AMPK/mTOR signaling. Afterwards, SESN1 was silenced to determine the expression of autophagy-related proteins. The further application of autophagy inhibitor 3-methyladenine (3-MA) was used to clarify the regulatory mechanism of Orientin on autophagy. Results showed that the decreased viability of HUVECs caused by ox-LDL induction was elevated by Orientin. Oxidative stress and inflammation were also attenuated after Orientin addition in HUVECs under ox-LDL condition. Moreover, Orientin suppressed apoptosis and induced autophagy of HUVECs stimulated by ox-LDL, accompanied by enhanced level of phospho (p)-AMPK and declined level of p-mTOR. Interestingly, SESN1 level was elevated by Orientin, and SESN1 depletion alleviated autophagy and reduced p-AMPK expression but enhanced p-mTOR expression. The further experiments indicated that SESN1 silencing or 3-MA addition reversed the inhibitory effects of Orientin on the oxidative stress, inflammation and apoptosis of HUVECs. Collectively, Orientin could induce autophagy by activating SESN1 expression, thereby regulating AMPK/mTOR signaling in ox-LDL-induced HUVECs.

DNMT3a-mediated upregulation of the stress inducible protein sestrin-2 contributes to malignant transformation of human bronchial epithelial cells following nickel exposure.

BACKGROUND: Nickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. METHODS: We employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. RESULT: Exposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, ß-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3ß/ß-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3ß/ß-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. CONCLUSION: Our results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/ß-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.

MicroRNA-29a and microRNA-122 expressions and other inflammatory markers among obese children with diabetes.

OBJECTIVES: This study was conducted to study the expression of both microRNA-29a and microRNA-122, and serum levels of sestrin-2, interleukin-6 (IL-6), and other inflammatory markers among obese children with/and without diabetes mellitus. METHODS: One hundred obese children with diabetes in addition to 100 age- and sex-matched obese children without diabetes, and 100 age- and sex-matched apparently healthy children were included in the study. Expressions of both microRNA-29a and microRNA-122, and serum levels of sestrin-2, IL-6, tumor necrosis factor-α (TNF-α), and high sensitive-CRP (hsCRP) were measured for all included study populations. RESULTS: Study results showed that the expressions of both microRNA-29a and microRNA-122, serum levels of IL-6, TNF-α, and hsCRP were significantly higher among obese children with diabetes in comparison to both obese children without diabetes and healthy children. In contrast, serum sestrin level was significantly low among obese children with diabetes in comparison to the other study populations. Expressions of both microRNA-29a and microRNA-122 were correlated with waist circumference, BMI, total cholesterol, triglycerides, LDL-cholesterol, HbA1c, c-peptide, glucose, insulin, homeostatic model assessment-insulin resistance (HOMA-IR), IL-6, hsCRP, and TNF-α among obese children with diabetes. However, serum sestrin-2 level was correlated inversely with these parameters. Higher expressions of both microRNA-29a and microRNA-122 among obese children either with or without diabetes mellitus (DM) can suggest their roles in the development of obesity among children. CONCLUSIONS: The study results can hypothesize that down-regulation of these micro-RNAs may solve this health problem with its sequelae, a hypothesis that needs more studies.

Epigenetic regulation of autophagy-related genes: Implications for neurodevelopmental disorders.

Macroautophagy/autophagy is an evolutionarily highly conserved catabolic process that is important for the clearance of cytosolic contents to maintain cellular homeostasis and survival. Recent findings point toward a critical role for autophagy in brain function, not only by preserving neuronal health, but especially by controlling different aspects of neuronal development and functioning. In line with this, mutations in autophagy-related genes are linked to various key characteristics and symptoms of neurodevelopmental disorders (NDDs), including autism, micro-/macrocephaly, and epilepsy. However, the group of NDDs caused by mutations in autophagy-related genes is relatively small. A significant proportion of NDDs are associated with mutations in genes encoding epigenetic regulatory proteins that modulate gene expression, so-called chromatinopathies. Intriguingly, several of the NDD-linked chromatinopathy genes have been shown to regulate autophagy-related genes, albeit in non-neuronal contexts. From these studies it becomes evident that tight transcriptional regulation of autophagy-related genes is crucial to control autophagic activity. This opens the exciting possibility that aberrant autophagic regulation might underly nervous system impairments in NDDs with disturbed epigenetic regulation. We here summarize NDD-related chromatinopathy genes that are known to regulate transcriptional regulation of autophagy-related genes. Thereby, we want to highlight autophagy as a candidate key hub mechanism in NDD-related chromatinopathies.Abbreviations: ADNP: activity dependent neuroprotector homeobox; ASD: autism spectrum disorder; ATG: AutTophaGy related; CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; EHMT: euchromatic histone lysine methyltransferase; EP300: E1A binding protein p300; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; H3K4me3: histone 3 lysine 4 trimethylation; H3K9me1/2/3: histone 3 lysine 9 mono-, di-, or trimethylation; H3K27me2/3: histone 3 lysine 27 di-, or trimethylation; hiPSCs: human induced pluripotent stem cells; HSP: hereditary spastic paraplegia; ID: intellectual disability; KANSL1: KAT8 regulatory NSL complex subunit 1; KAT8: lysine acetyltransferase 8; KDM1A/LSD1: lysine demethylase 1A; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NDD: neurodevelopmental disorder; PHF8: PHD finger protein 8; PHF8-XLID: PHF8-X linked intellectual disability syndrome; PTM: post-translational modification; SESN2: sestrin 2; YY1: YY1 transcription factor; YY1AP1: YY1 associated protein 1.

Role of sestrins in metabolic and aging-related diseases.

Sestrins are a type of highly conserved stress-inducing protein that has antioxidant and mTORC1 inhibitory functions. Metabolic dysfunction and aging are the main risk factors for development of human diseases, such as diabetes, neurodegenerative diseases, and cancer. Sestrins have important roles in regulating glucose and lipid metabolism, anti-tumor functions, and aging by inhibiting the reactive oxygen species and mechanistic target of rapamycin complex 1 pathways. In this review, the structure and biological functions of sestrins are summarized, and how sestrins are activated and contribute to regulation of the downstream signal pathways of metabolic and aging-related diseases are discussed in detail with the goal of providing new ideas and therapeutic targets for the treatment of related diseases.

MicroRNA-34a-5p promotes the progression of osteoarthritis secondary to developmental dysplasia of the hip by restraining SESN2-induced autophagy.

Osteoarthritis (OA), a late-stage complication of developmental dysplasia of the hip (DDH), is a key factor leading to further degeneration of joint function. Studies have shown that Sestrin2 (SESN2) is a positive regulator in protecting articular cartilage from degradation. However, the regulatory effects of SESN2 on DDH-OA and its upstream regulators remain obscure. Here, we first identified that the expression of SESN2 significantly decreased in the cartilage of DDH-OA samples, with an expression trend negatively correlated with OA severity. Using RNA sequencing, we identified that the upregulation of miR-34a-5p may be an important factor for the decrease in SESN2 expression. Further exploring the regulation mechanism of miR-34a-5p/SESN2 is of great significance for understanding the mechanism of DDH occurrence and development. Mechanistically, we showed that miR-34a-5p could significantly inhibit the expression of SESN2, thereby promoting the activity of the mTOR signaling pathway. We also found that miR-34a-5p significantly inhibited SESN2-induced autophagy, thereby suppressing the proliferation and migration of chondrocytes. We further validated that knocking down miR-34a-5p in vivo resulted in a significant increase in SESN2 expression and autophagy activity in DDH-OA cartilage. Our study suggests that miR-34a-5p is a negative regulator of DDH-OA, and may provide a new target for the prevention of DDH-OA.

Overexpression of SESN1 improves mitochondrial damage and mitophagy, a potential therapeutic strategy for cognitive dysfunction after anaesthesia.

Postoperative cognitive dysfunction (POCD) is a prevalent central nervous system complication predominantly observed in elderly patients. Sevoflurane, a general anaesthetic agent, has been implicated in the development of POCD, yet the underlying regulatory mechanisms potentially involving Sestrin1 (SESN1), a stress-responsive protein that plays a critical role in cellular homeostasis and protection against stress-induced damage, including oxidative stress and DNA damage, remain elusive. This study endeavoured to elucidate the impact of SESN1 on sevoflurane-induced cognitive impairment in rats. Employing a model in which SESN1 was transfected into SD male rats and cognitive dysfunction was induced by sevoflurane. The Morris Water Maze test was used for behavioural evaluation, Enzyme-Linked Immunosorbent Assay, Western blotting and immunofluorescence were applied to assess the influence of SESN1 on the inflammatory response and mitophagy in the rat hippocampus. The study further aimed to uncover the putative mechanism by which SESN1, through SIRT1, might modulate cognitive function. Concurrently, levels of malondialdehyde, superoxide dismutase and mitochondrially produced ATP within the rat hippocampus were quantified. Experimental outcomes suggested that SESN1 overexpression significantly mitigated the deleterious effects of sevoflurane anaesthesia, ameliorated neuroinflammation and inflammasome activation, modified mitochondrial function and facilitated mitophagy. Additionally, SESN1, via the activation of SIRT1, may suppress inflammasome activation and mitochondrial dysfunction. Collectively, these findings underscore SESN1's integral role in counteracting sevoflurane-induced cognitive impairment, impeding inflammasome activation, enhancing mitochondrial function and fostering mitophagy, which appear to be intricately linked to SESN1-mediated SIRT1 activation. SESN1 is a novel therapeutic target for POCD, potentially advancing neuroprotective strategies in clinical settings.

Maternal serum interleukin-1ß, FoxO1 and Sestrin2 levels in predicting preterm delivery.

The study aimed to investigate whether serum IL-1ß, FoxO1and Sesn2 concentrations differed between threatened preterm labor (TPL) and uncomplicated pregnancies. This study was conducted on 54 women with TPL pregnancies and 26 healthy pregnant women. The TPL group was further divided into two subgroups according to the gestational age at delivery. Patients who gave birth within 48-72 hours after the hospitalization were referred to as preterm delivery (PD) and those who gave birth at ≥37 weeks were referred to as term delivery (TD). Maternal levels of serum IL-1ß, FoxO1 and Sesn2 were measured with the use of enzyme-linked immunosorbent assay kits. The mean maternal serum IL-1ß and FoxO1 of PD were significantly higher than TD (p<.000*) and the control group (p < .000*). The mean maternal serum IL-1ß, FoxO1 level of TD was significantly higher than the control group (p<.000*). The mean maternal serum Sesn2 levels of TD and the control group were significantly higher than the preterm group (p<.000*). The mean maternal serum Sesn2 level of the control group was significantly higher than the TD group (p <.000*). A negative correlation was found between serum concentration of serum IL-1ß, and FoxO1 with the gestational week of delivery (r= -0.722, p< .000*for, IL-1ß; r = -0.625, p < .000* for FoxO1). A positive correlation was found between the serum concentration of serum Sesn2 with the gestational week of delivery (r = 0.507, p<.000* for sesn2). High serum IL-1ß, FoxO1 levels, and low Sesn2 levels may have the potential to be used as biomarkers for the differentiation of PD and TD.

Metformin Caused Radiosensitivity of Breast Cancer Cells through the Expression Modulation of miR-21-5p/SESN1axis.

OBJECTIVE: In this research we evaluated molecular mechanism of effect of metformin in radio sensitivity of breast cancer cells. METHODS: This research was done in cellular and molecular research center of Qazvin university of Medical science in 1399 to 1401. Studied samples were two breast cancer cell lines (MCF-7 and MDA-MB-231) they are derived from primary and secondary tumors resected from a single patient. We exposed them to cumulative 50 Gy radiation and constructed radio resistant cell lines. Then resistant cell lines were treated with 50µm of metformin. Our studied groups were resistant cells treated and un treated with metformin. Then, the expression rate of miR-21-5p and SESN1 gene in resistant and control cells was checked by Quantitative Real-time PCR(qRTPCR). After the cell lines were treated with different concentrations of metformin at different intervals, the rate of cell proliferation and cell death was checked by CCK-8 assay and flow cytometry. The Western blot method was also used to confirm the expression of some genes. RESULTS: Our results showed that the expression of miR-21-5p was upregulated in radiation-resistant cancer cells (1.8±0.65) (P<0.0001) MCF-7 cell line and (1.6±0.42)(P<0.001) MBA-MD-231 cell line, while the expression of SESN1 was down regulated (0.46±0.12) (P<0.0001) MCF-7 cell line and (0.42±0.22) (P<0.001) MBA-MD-231 cell line. Metformin enhanced the radio sensitivity of breast cancer cells in a dose and time-dependent manner. Also, metformin treatment decreased the expression of miR-21-5p (0.47±0.32) (P<0.0001) MCF-7 Cell line and (0.45±0.21)(P<0.001) MBA-MD-231 cell line and increased the expression of SESN1 (1.65±0.72)(P<0.0001)MCF-7 cell line and (1.73±0.52)(P<0.0001) MBA-MD-231 cell line. The function of metformin was reversed by miR-21-5p inhibitors or the transfection of SESN1 overexpressing plasmids. CONCLUSION: In conclusion, based on this research results, metformin enhanced the radio sensitivity of breast cancer cells via modulating  the expression of miR-21-5p and SESN1.

Resistance exercise alleviates the prefrontal lobe injury and dysfunction by activating SESN2/AMPK/PGC-1α signaling pathway and inhibiting oxidative stress and inflammation in mice with myocardial infarction.

OBJECTIVES: Myocardial infarction (MI) induces inflammatory response and oxidative stress in the brain, which would be one of the causes of cardiac dysfunction. Exercise training is viewed as a feasible strategy to improve cardiac function of the infarcted heart. The aim of this study was to investigate whether exercise training could alleviate MI-induced prefrontal lobe injury via activating Sestrin2 (SESN2) signaling and inhibiting oxidative stress and inflammation. METHODS: Male C57BL/6 mice were divided into five groups: control group (CON), aerobic exercise group (AE), resistance exercise group (RE), whole-body vibration group (WBV) and electrical stimulation group (ES); and three groups: sham-operated group (S), sedentary MI group (MI) and MI with resistance exercise group (MRE). After four weeks of training, sensorimotor function, spatial learning, long-term and spatial memory, and cardiac function were detected. Then, mice were euthanized, and the prefrontal areas were separated for HE, Nissl, SESN2, microtubule-associated protein 2 (MAP2), neuron-specific nucleoprotein (NeuN), and TUNEL staining. Activation of SESN2/adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) signaling pathway and expression of proteins related to oxidative stress, inflammation and apoptosis in the prefrontal lobe were detected by western blotting. RESULTS: Different types of exercise training all activated the SESN2/AMPK/PGC-1α signaling pathway, and the effect of RE is the best. RE improved sensorimotor, learning, and memory impairments, increased the expressions of antioxidant, anti-inflammatory and anti-apoptotic proteins, reduced oxidative stress, inflammation and apoptosis, ultimately alleviated the prefrontal lobe injury and dysfunction in mice with MI. CONCLUSION: RE alleviates MI-indued prefrontal lobe injury and dysfunction by inhibiting the levels of oxidative stress, inflammation and apoptosis, partially via activating SESN2/AMPK/PGC-1α signaling pathway.

Inhibition of the NOTCH and mTOR pathways by nelfinavir as a novel treatment for T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T­ALL), a neoplasm derived from T cell lineage­committed lymphoblasts, is characterized by genetic alterations that result in activation of oncogenic transcription factors and the NOTCH1 pathway activation. The NOTCH is a transmembrane receptor protein activated by γ­secretase. γ­secretase inhibitors (GSIs) are a NOTCH­targeted therapy for T­ALL. However, their clinical application has not been successful due to adverse events (primarily gastrointestinal toxicity), limited efficacy, and drug resistance caused by several mechanisms, including activation of the AKT/mTOR pathway. Nelfinavir is an human immunodeficiency virus 1 aspartic protease inhibitor and has been repurposed as an anticancer drug. It acts by inducing endoplasmic reticulum (ER) stress and inhibiting the AKT/mTOR pathway. Thus, it was hypothesized that nelfinavir might inhibit the NOTCH pathway via γ­secretase inhibition and blockade of aspartic protease presenilin, which would make nelfinavir effective against NOTCH­associated T­ALL. The present study assessed the efficacy of nelfinavir against T­ALL cells and investigated mechanisms of action in vitro and in preclinical treatment studies using a SCL­LMO1 transgenic mouse model. Nelfinavir blocks presenilin 1 processing and inhibits γ­secretase activity as well as the NOTCH1 pathway, thus suppressing T­ALL cell viability. Additionally, microarray analysis of nelfinavir­treated T­ALL cells showed that nelfinavir upregulated mRNA levels of CHAC1 (glutathione­specific γ­glutamylcyclotransferase 1, a negative regulator of NOTCH) and sestrin 2 (SESN2; a negative regulator of mTOR). As both factors are upregulated by ER stress, this confirmed that nelfinavir induced ER stress in T­ALL cells. Moreover, nelfinavir suppressed NOTCH1 mRNA expression in microarray analyses. These findings suggest that nelfinavir inhibited the NOTCH1 pathway by downregulating NOTCH1 mRNA expression, upregulating CHAC1 and suppressing γ­secretase via presenilin 1 inhibition and the mTOR pathway by upregulating SESN2 via ER stress induction. Further, nelfinavir exhibited therapeutic efficacy against T­ALL in an SCL­LMO1 transgenic mouse model. Collectively, these findings highlight the potential of nelfinavir as a novel therapeutic candidate for treatment of patients with T­ALL.

Resistance exercise alleviates dexamethasone-induced muscle atrophy via Sestrin2/MSTN pathway in C57BL/6J mice.

AIM: It has long been recognized that resistance exercise can substantially increase skeletal muscle mass and strength, but whether it can protect against glucocorticoid-induced muscle atrophy and its potential mechanism is yet to be determined. This study aimed to investigate the protective effects of resistance exercise in dexamethasone-induced muscle atrophy and elucidate the possible function of exercise-induced protein Sestrin2 in this process. METHODS: Eight-week-old male C57BL/6J mice carried out the incremental mouse ladder exercise for 11 weeks. Two weeks before the end of the intervention, mice were daily intraperitoneally injected with dexamethasone. Body composition, muscle mass, and exercise performance were examined to evaluate muscle atrophy. In vitro, C2C12 cells were used for RT-qPCR, Western Blot, and immunofluorescence experiments to elucidate the potential mechanism. RESULTS: Our results showed that long-term resistance exercise is an effective intervention for dexamethasone-induced muscle atrophy. We also found that Sestrin2 plays a vital role in dexamethasone-induced muscle atrophy. In both animal (P = .0006) and cell models (P = .0266), dexamethasone intervention significantly reduced the protein expression of Sestrin2, which was increased (P = .0112) by resistance exercise. Inversely, overexpression of Sestrin2 improved (P < .0001) dexamethasone-induced myotube cell atrophy by reducing the activation of the ubiquitin-proteasome pathway via inhibiting Forkhead box O3 (FoxO3a) and myostatin (MSTN)/small mother against decapentaplegic (Smad) signaling pathways. CONCLUSION: Taken together, our results indicated that Sestrin2 may serve as an effective molecule that mimics the protective effect of resistance exercise on dexamethasone-induced muscle atrophy.

Flavonoids Quercetin and Kaempferol Are NR4A1 Antagonists and Suppress Endometriosis in Female Mice.

Nuclear receptor 4A1 (NR4A1) plays an important role in endometriosis progression; levels of NR4A1 in endometriotic lesions are higher than in normal endometrium, and substituted bis-indole analogs (NR4A1) antagonists suppress endometriosis progression in mice with endometriosis. In addition, the flavonoids kaempferol and quercetin are natural products that directly bind NR4A1 and significantly repress the intrinsic NR4A1-dependent transcriptional activity in human endometriotic epithelial and stromal cells and Ishikawa endometrial cancer cells. NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin suppressed proliferation of human endometriotic epithelial cells and Ishikawa cells by inhibiting epidermal growth factor receptor/c-Myc/survivin-mediated growth-promoting and survival pathways, The mammalian target of rapamycin (mTOR) signaling and αSMA/CTGF/COL1A1/FN-mediated fibrosis signaling but increasing Thioredoxin domain Containing 5/SESN2-mediated oxidative/estrogen receptors stress signaling. In human endometriotic stromal cells, NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin primarily inhibited mTOR signaling by suppressing proliferation of human endometrial stromal cells. In addition, kaempferol and quercetin treatment also effectively suppressed the growth of endometriotic lesions in mice with endometriosis compared with the vehicle without any body weight changes. Therefore, kaempferol and quercetin are NR4A1 antagonists with potential as nutritional therapy for endometriosis.

Gallic acid as a Sestrin (SESN2) activator and potential obesity therapeutic agent: A molecular docking study.

Sestrins (SESNs) are a family of evolutionarily conserved proteins among mammals. They have several body homeostatic functions such as antioxidant, metabolic, and anti-aging, and are required to regenerate hyperoxidized forms of peroxiredoxins and reactive oxygen species. Sestrin 2 has been studied as a therapeutic agent in obesity treatment. Gallic acid (GA) is a triphenolic compound with beneficial biological activities including anti-inflammatory, antidiabetic, antihypertensive, and antioxidant effects. Recent studies demonstrated the GA's ability to reduce body weight gain and improve glycemic parameters. In this sense, the present study aims to investigate the GA activating potential of Sestrin using the molecular docking method. The 3D structure of gallic acid was retrieved from the NCBI PubChem database and the chemical structure of the Sestrin2 protein from the RCSB Protein Data Bank (5DJ4). The docking calculus was performed via UCSF Chimera and AutoDock Vinaprograms. The results showed that amino acids Arg390, Glu451, Trp444, Thr386, Arg448, Thr374, Tyr375, Asn376, Thr377, Leu389, His454, Ser450, His86, and Val455 are very important for GA stabilization, resembling the interactions that permit Leucine to activate SESN2. In this context, the obesity therapeutic property of GA can be understood from a Sestrin activating process through amino acid metabolism.