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Aim The present work investigated serum levels of miR-29a, miR-122 and sestrin2 in obese children with/without type-2-diabetes mellitus (T2DM), and their correlations with inflammatory, metabolic and anthropometric parameters. Methods The study included 298 children, divided into: G1 (control, n = 136), G2 (obese without diabetes, n = 90) and G3 (obese with T2DM, n = 72). Metabolic and anthropometric parameters, miR-29a, miR-122 relative expressions, and sestrin2, high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were measured by their specific methods. The data was processed and analyzed by SPSS V.26 using the corresponding tests. After testing the variables’ normality, Kruskal–Wallis one-way-ANOVA, Spearman correlations coefficient were used. Results Significant higher serum miR-29a, miR-122, IL-6, hsCRP and TNF-α and lower sestrin2 levels were found in G2 and G3 than G1 and in G3 than G2 (p= > 0.001 for all). Especially in G3, miR-29a and miR-122 levels correlated positively while sestrin2 levels correlated negatively with waist circumference and BMI percentiles, serum levels of LDL-cholesterol, triacylglycerol, total cholesterol, HbA1c%, glucose, insulin, c-peptide, homeostatic model assessment-insulin resistance (HOMA-IR), IL-6, hsCRP and TNF-α. Conclusion The change in the serum miR-29a, miR-122 and sestrin2 levels in obese children with/without T2DM may suggest a possible role of these biomarkers in the pathogenesis of childhood obesity and their accompanied complications e.g. inflammations and T2DM. Also, further studies are required to test drugs that antagonize the action miR-29a and miR-122 or upregulate sestrin2 in the management of these cases.

Background/Aims: Sestrin 2 is associated with the pathophysiology of several diseases. The aim of this study was to investigate the effects and potential mechanisms of Sestrin 2 in rat hepatic stellate cells (HSCs) during liver fibrogenesis. Methods: In this study, Sestrin 2 protein expression was detected in rat HSC-T6 cells challenged with transforming growth factor-β (TGF-β) and in mice treated with carbon tetrachloride (CCl4), a well-known model of hepatic fibrosis. Next, HSC-T6 cells and fibrotic mice were transfected with lentivirus. The mRNA expression levels of markers of liver fibrosis [alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1)] were analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Cell death and proliferation were evaluated by the MTT assay, and biochemical markers of liver damage in serum [alanine transaminase (ALT) and aspartate transaminase (AST)] were also measured using a biochemical analyzer. Histopathological examination was used to evaluate the degree of liver fibrosis, and protein expression [phospho-adenosine monophosphate-activated protein kinase (p-AMPK), AMPK, phospho-mammalian target of rapamycin (p-mTOR), and mTOR] was determined by western blotting. Results: We found that Sestrin 2 was elevated in both the HSC-T6 cell and hepatic fibrosis models. In vitro, overexpression of Sestrin 2 attenuated the mRNA levels of α-SMA and Col1A1, suppressed α-SMA protein expression, and modulated HSC-T6 cell proliferation. In vivo, overexpression of Sestrin 2 reduced the ALT and AST levels as well as the α-SMA and Col1A1 protein expression in the CCl4 model of liver fibrosis. Moreover, the degree of liver fibrosis was ameliorated. Interestingly, overexpression of Sestrin 2 increased p-AMPK but decreased p-mTOR protein expression. Conclusion: Our findings indicate that Sestrin 2 may attenuate the activation of HSCs and ameliorate liver fibrosis, most likely via upregulation of AMPK phosphorylation and suppression of the mTOR signaling pathway.

Fisetin was reported to have an anti-proliferative and apoptotic activity as a novel anti-cancer agent in various cancer cell lines. However, the possible molecular targets for the anti-cancer effect of fisetin in human head and neck cancer (HNCC) have not yet been clarified. In this study, the influence of fisetin on the growth and apoptosis of HNCCs were examined. In HSC3 cells, fisetin treatment reduced the viability and induced apoptosis. Through the results from the screening of the expression profile of apoptosis-related genes, sestrin 2 (SESN2) was functionally involved in fisetin-mediated apoptosis showing the knockdown of SESN2 by siRNA clearly restored fisetin-induced apoptosis. In addition, fisetin reduced the protein expression levels of phospho-mTOR (p-mTOR) and Mcl-1, which are the downstream molecules of SESN2. It also induced PARP cleavage by inducing an increase in the expression levels of SESN2 together with reducing mTOR and Mcl-1 proteins in other three HNCCs (MC3, Ca9.22, and HN22). Taken together, our findings suggest that the anti-cancer effect of fisetin on HNCCs is associated with SESN2/mTOR/Mcl-1 signaling axis.

Summary Proper regulation of mTORC1 and mTORC2 upon nutrient starvation is critical for cancer cell survival. Upregulation of Sestrin2 in response to glutamine deprivation rescues cell death by suppressing mTORC1. However, the contribution of mTORC2 to Sestrin2-mediated mTORC1 suppression remains unclear. Here, we report that both Sestrin2 and mTORC2 are upregulated in glutamine-depleted lung cancer cells. Moreover, glutamine depletion caused Sestrin2 to associate with mTORC2, which was required for the increase in Sestrin2 protein stability and the reduction in mTORC1 activity. Ultimately, differential regulation of mTORC1 and 2 by Sestrin2 reprogramed lipid metabolism and enabled glutamine-depleted lung cancer cells to survive by maintaining energy and redox balance. Importantly, combined inhibition of glutamine utilization and Sestrin2 induced lung cancer cell death both in vitro and in vivo. This study shows that differential Sestrin2-mediated regulation of mTORC1 and mTORC2 is necessary for the survival of glutamine-depleted lung cancer cells.

// Jin-Lai Wei 1 , Min Fang 2 , Zhong-Xue Fu 1 , Shou–Ru Zhang 1 , Jin-Bao Guo 3 , Rong Wang 1 , Zhen-Bing Lv 1 and Yong-Fu Xiong 1 1 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China 2 Department of Emergency and Intensive Care Unit, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China 3 Department of Thoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Correspondence to: Zhong-Xue Fu, email: fzx033@163.com Keywords: colorectal cancer, sestrin 2, AMPK, mTORC1, proliferation Received: February 09, 2017 Accepted: April 17, 2017 Published: May 03, 2017 ABSTRACT Sestrin 2 is a conserved antioxidant protein that reduces reactive oxygen species (ROS) and inhibits mammalian target of rapamycin complex 1 (mTORC1). We previously showed that sestrin 2 is abnormally decreased in colorectal cancer (CRC). To elucidate the molecular mechanism behind the potential contribution of sestrin 2 to CRC, we used a lentiviral expression vector system to determine the effects of sestrin 2 overexpression on human CRC cells. We found that sestrin 2 overexpression decreased ROS production, inhibited cell growth, and stimulated apoptosis in two CRC cell lines. In parallel, expression of the proliferation marker PCNA was decreased, proapoptotic caspase 3, 7, and 9 levels were increased, and expression of the anti-apoptotic protein survivin was reduced. Sestrin 2 overexpression also activated the adenosine monophosphate-activated protein kinase (AMPK) pathway, and suppressed mTORC1 signaling. Treating CRC cells with compound C, an AMPK inhibitor, reversed or attenuated changes in proliferation, apoptosis, and signaling proteins of the AMPK/mTORC1 axis. In a xenograft mouse model, CRC growth was attenuated by sestrin 2 overexpression. These results suggest that sestrin 2 suppresses CRC cell growth through activation of the AMPK/mTORC1 pathway and induction of apoptosis, and could be a novel pharmacological target for the treatment of CRC.

Oxaliplatin is an anticancer drug administered to colorectal cancer (CRC) patients in combination with 5-fluorouracil and antibodies (bevacizumab and cetuximab), thereby significantly improving the survival rate of CRC. However, due to various side effects associated with the above treatment strategy, the need for combinatorial therapeutic strategies has emerged. Based on the demand for new combinatorial therapies and the known antitumor effects of the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), we investigated the Oxaliplatin and DHA combination for its effect. Our results indicated that DHA further enhanced Oxaliplatin-induced cell viability and autophagic cell death, in vitro and in vivo. Oxaliplatin and DHA also increased the expression of Sestrin 2 (SESN2) and endoplasmic reticulum (ER) stress related C/EBP homologous protein (CHOP). Additionally, treatment with Oxaliplatin and DHA enhanced the binding of CHOP to the promotor region of SESN2, increasing SESN2 expression. These results suggested that DHA enhanced Oxaliplatin-induced reduction in cell viability and increase in autophagy via activating SESN2 and increasing ER stress. Thus, SESN2 may be an effective preclinical target for CRC treatment.

Rosemary extract (RE) is an approved food preservative in the European Union and contains dietary phytochemicals that are beneficial for gastrointestinal health. This study investigated the effects of RE on dextran sodium sulfate (DSS)-induced colitis and also determined the pharmacokinetics of dietary phytochemicals administered to mice via oral gavage. Individual components of rosemary extract were separated and identified by LC–MS/MS. The pharmacokinetics of two major diterpenes from RE, carnosic acid (CA) and carnosol (CL), administered to mice via oral gavage were determined. Then, the effect of RE pre-treatment on the disease activity index (DAI) of DSS-induced colitis in mice was investigated. The study determined that 100 mg/kg RE significantly improved DAI in DSS-induced colitis compared to negative control. Sestrin 2 protein expression, which increased with DSS exposure, was reduced with RE treatment. Intestinal barrier integrity was also shown to improve via fluorescein isothiocyanate (FITC)–dextran administration and Western blot of zonula occludens-1 (ZO-1), a tight junction protein. Rosemary extract was able to improve the DAI of DSS-induced colitis in mice at a daily dose of 100 mg/kg and showed improvement in the intestinal barrier integrity. This study suggests that RE can be an effective preventative agent against IBD.

Upregulation of SESTRIN 2 (SESN2) has been reported in response to diverse cellular stresses. In this study we demonstrate SESTRIN 2 induction following endoplasmic reticulum (ER) stress. ER stress-induced increases in SESTRIN 2 expression were dependent on both PERK and IRE1/XBP1 arms of the unfolded protein response (UPR). SESTRIN 2 induction, post ER stress, was responsible for mTORC1 inactivation and contributed to autophagy induction. Conversely, knockdown of SESTRIN 2 prolonged mTORC1 signaling, repressed autophagy and increased ER stress-induced cell death. Unexpectedly, the increase in ER stress-induced cell death was not linked to autophagy inhibition. Analysis of UPR pathways identified prolonged eIF2 alpha, ATF4 and CHOP signaling in SESTRIN 2 knockdown cells following ER stress. SESTRIN 2 regulation enables UPR derived signals to indirectly control mTORC1 activity shutting down protein translation thus preventing further exacerbation of ER stress.

Made available in DSpace on 2018-11-26T15:45:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Sestrins and autophagy deficiencies are associated with several aging-related organic dysfunctions and metabolic disorders. Here we evaluate the effects of acute exercise on Sestrin 2 (Sesn2) protein content and autophagy markers in the skeletal muscle of experimental models of aging. Twenty-four months-old C57BL/6 J male mice were submitted to a single bout of swimming exercise and the gastrocnemius muscle was evaluated by Western blot. Transcriptomic and phenotypic analysis were also performed by using strains of genetically-diverse BXD mice. The bioinformatics analysis showed a negative correlation between Sesn2 mRNA levels in the skeletal muscle and body weight gain, plasma triglycerides and fasting glucose and positive correlation with several autophagic markers in the muscle of BXDmice. Consistent with these findings, low levels of Sesn2 protein content were observed in the gastrocnemius muscle of C57BL/6 J old mice when compared to young group. Interestingly, the acute aerobic exercise induced Sesn2 accumulation and modulated several markers of autophagy in the gastrocnemius muscle old mice, including unc-51-like kinase-1 (Ulk1) phosphorylation and the protein levels of Atg5, Atg7, p62 and LC3-II. Finally, exercise increased insulin sensitivity in old animals, as demonstrated by kITT. Taken together, these findings demonstrated the acutely, aerobic physical exercise recovers Sestrin 2 protein content and induces autophagy in the skeletal muscle of old mice, contributing with the improvement of insulin sensitivity an aging animal model. (C) 2017 Elsevier Inc. All rights reserved. Univ Campinas UNICAMP, Sch Appl Sci, Lab Mol Biol Exercise LaBMEx, Limeira, SP, Brazil Univ Campinas UNICAMP, Fac Med Sci, Dept Internal Med, Campinas, SP, Brazil Univ Estadual Campinas, Sch Appl Sci, Biotechnol Lab, BR-13484350 Limeira, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rio Claro, SP, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Postgrad Program Rehabil & Funct Performance, Ribeirao Preto, SP, Brazil Univ Sao Paulo, Sch Phys Educ & Sport Ribeirao Preto, Ribeirao Preto, SP, Brazil Univ Estadual Campinas, Sch Appl Sci, Lab Nutr Genom LabGeN, Limeira, SP, Brazil Univ Campinas UNICAMP, Sch Appl Sci, CEPECE Ctr Res Sport Sci, Limeira, SP, Brazil Sao Paulo State Univ, Inst Biosci, Rio Claro, SP, Brazil FAPESP: FAPESP- 2016/01089-3