Inhibition of SIRT7 overcomes sorafenib acquired resistance by suppressing ERK1/2 phosphorylation via the DDX3X-mediated NLRP3 inflammasome in hepatocellular carcinoma.

AIMS: Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance. METHODS: Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib. RESULTS: SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1ß inhibition. CONCLUSIONS: SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.

SIRT7 promotes the proliferation and migration of anaplastic thyroid cancer cells by regulating the desuccinylation of KIF23.

OBJECTIVE: This study was designed to investigate the regulatory effects of kinesin family member (KIF) 23 on anaplastic thyroid cancer (ATC) cell viability and migration and the underlying mechanism. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the levels of KIF23 in ATC cells. Besides, the effects of KIF23 and sirtuin (SIRT) 7 on the viability and migration of ATC cells were detected using cell counting kit-8, transwell and wound healing assays. The interaction between SIRT7 and KIF23 was evaluated by co-immunoprecipitation (Co-IP) assay. The succinylation (succ) of KIF23 was analyzed by western blot. RESULTS: The KIF23 expression was upregulated in ATC cells. Silencing of KIF23 suppressed the viability and migration of 8505C and BCPAP cells. The KIF23-succ level was decreased in ATC cells. SIRT7 interacted with KIF23 to inhibit the succinylation of KIF23 at K537 site in human embryonic kidney (HEK)-293T cells. Overexpression of SIRT7 enhanced the protein stability of KIF23 in HEK-293T cells. Besides, overexpression of KIF23 promoted the viability and migration of 8505C and BCPAP cells, which was partly blocked by silenced SIRT7. CONCLUSIONS: SIRT7 promoted the proliferation and migration of ATC cells by regulating the desuccinylation of KIF23.

Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis.

SGLT2 (sodium-glucose cotransporter 2) inhibitors produce a distinctive pattern of benefits on the evolution and progression of cardiomyopathy and nephropathy, which is characterized by a reduction in oxidative and endoplasmic reticulum stress, restoration of mitochondrial health and enhanced mitochondrial biogenesis, a decrease in proinflammatory and profibrotic pathways, and preservation of cellular and organ integrity and viability. A substantial body of evidence indicates that this characteristic pattern of responses can be explained by the action of SGLT2 inhibitors to promote cellular housekeeping by enhancing autophagic flux, an effect that may be related to the action of these drugs to produce simultaneous upregulation of nutrient deprivation signaling and downregulation of nutrient surplus signaling, as manifested by an increase in the expression and activity of AMPK (adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), SIRT3 (sirtuin 3), SIRT6 (sirtuin 6), and PGC1-α (peroxisome proliferator-activated receptor γ coactivator 1-α) and decreased activation of mTOR (mammalian target of rapamycin). The distinctive pattern of cardioprotective and renoprotective effects of SGLT2 inhibitors is abolished by specific inhibition or knockdown of autophagy, AMPK, and sirtuins. In the clinical setting, the pattern of differentially increased proteins identified in proteomics analyses of blood collected in randomized trials is consistent with these findings. Clinical studies have also shown that SGLT2 inhibitors promote gluconeogenesis, ketogenesis, and erythrocytosis and reduce uricemia, the hallmarks of nutrient deprivation signaling and the principal statistical mediators of the ability of SGLT2 inhibitors to reduce the risk of heart failure and serious renal events. The action of SGLT2 inhibitors to augment autophagic flux is seen in isolated cells and tissues that do not express SGLT2 and are not exposed to changes in environmental glucose or ketones and may be related to an ability of these drugs to bind directly to sirtuins or mTOR. Changes in renal or cardiovascular physiology or metabolism cannot explain the benefits of SGLT2 inhibitors either experimentally or clinically. The direct molecular effects of SGLT2 inhibitors in isolated cells are consistent with the concept that SGLT2 acts as a nutrient surplus sensor, and thus, its inhibition causes enhanced nutrient deprivation signaling and its attendant cytoprotective effects, which can be abolished by specific inhibition or knockdown of AMPK, sirtuins, and autophagic flux.

Metabolic Syndrome and Cardiac Remodeling Due to Mitochondrial Oxidative Stress Involving Gliflozins and Sirtuins.

PURPOSE OF REVIEW: To address the mechanistic pathways focusing on mitochondria dysfunction, oxidative stress, sirtuins imbalance, and other contributors in patient with metabolic syndrome and cardiovascular disease. Sodium glucose co-transporter type 2 (SGLT-2) inhibitors deeply influence these mechanisms. Recent randomized clinical trials have shown impressive results in improving cardiac function and reducing cardiovascular and renal events. These unexpected results generate the need to deepen our understanding of the molecular mechanisms able to generate these effects to help explain such significant clinical outcomes. RECENT FINDINGS: Cardiovascular disease is highly prevalent among individuals with metabolic syndrome and diabetes. Furthermore, mitochondrial dysfunction is a principal player in its development and persistence, including the consequent cardiac remodeling and events. Another central protagonist is the renin-angiotensin system; the high angiotensin II (Ang II) activity fuel oxidative stress and local inflammatory responses. Additionally, sirtuins decline plays a pivotal role in the process; they enhance oxidative stress by regulating adaptive responses to the cellular environment and interacting with Ang II in many circumstances, including cardiac and vascular remodeling, inflammation, and fibrosis. Fasting and lower mitochondrial energy generation are conditions that substantially reduce most of the mentioned cardiometabolic syndrome disarrangements. In addition, it increases sirtuins levels, and adenosine monophosphate-activated protein kinase (AMPK) signaling stimulates hypoxia-inducible factor-1ß (HIF-1 beta) and favors ketosis. All these effects favor autophagy and mitophagy, clean the cardiac cells with damaged organelles, and reduce oxidative stress and inflammatory response, giving cardiac tissue protection. In this sense, SGLT-2 inhibitors enhance the level of at least four sirtuins, some located in the mitochondria. Moreover, late evidence shows that SLGT-2 inhibitors mimic this protective process, improving mitochondria function, oxidative stress, and inflammation. Considering the previously described protection at the cardiovascular level is necessary to go deeper in the knowledge of the effects of SGLT-2 inhibitors on the mitochondria function. Various of the protective effects these drugs clearly had shown in the trials, and we briefly describe it could depend on sirtuins enhance activity, oxidative stress reduction, inflammatory process attenuation, less interstitial fibrosis, and a consequent better cardiac function. This information could encourage investigating new therapeutic strategies for metabolic syndrome, diabetes, heart and renal failure, and other diseases.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

SIRT6 ameliorates LPS-induced apoptosis and tight junction injury in ARDS through the ERK1/2 pathway and autophagy.

Sirtuin6 (SIRT6) has been demonstrated to be involved in a range of physiological processes and diseases, while its role in acute respiratory distress syndrome (ARDS) remains unclear. Therefore, this study focused on the role and underlying mechanism of SIRT6 in ARDS with the aim of identifying potential therapeutic targets. In this study, we found that SIRT6 was significantly decreased in lipopolysaccharide (LPS)-induced A549 cells and a murine model. In vitro overexpression of SIRT6 restored the expression of tight junction proteins (ZO-1 and occludin) and alleviated cell apoptosis and inflammation, while knockdown of SIRT6 aggravated the loss of tight junction proteins (ZO-1 and occludin) and promoted cell apoptosis and inflammation in LPS-induced A549 cells. Furthermore, the overexpression of SIRT6 enhanced autophagy and inhibited the ERK1/2 pathway, while the knockdown of SIRT6 inhibited autophagy and activated the ERK1/2 pathway. The autophagy activator rapamycin and the ERK1/2 inhibitor PD98059 rescued the effects of SIRT6 knockdown on tight junction proteins, apoptosis, and inflammation. Mechanistically, SIRT6 deacetylated histone 3 at Lys9 to negatively regulate the ERK1/2 pathway. In vivo, the SIRT6-specific inhibitor OSS_128167 also significantly accelerated LPS-induced loss of tight junction proteins, lung inflammation, and apoptosis. Meanwhile, the SIRT6-specific inhibitor OSS_128167 also activated the ERK1/2 pathway and inhibited lung autophagy. These results suggested that SIRT6 could ameliorate the loss of tight junction proteins, inflammation, and apoptosis in LPS-induced ARDS by inhibiting the ERK1/ 2 pathway and enhancing autophagy, indicating that SIRT6 plays a beneficial role in ARDS and might be a potential therapeutic target for ARDS.

Knockdown of circLRWD1 weakens DDP resistance via reduction of SIRT5 expression through releasing miR-507 in non-small cell lung cancer.

Cisplatin (DDP) is an antineoplastic agent for non-small cell lung cancer (NSCLC). Hsa_circ_0081664 (circLRWD1) is overexpressed in DDP-resistant NSCLC cells, but its function is unclear. Thus, this study is to investigate whether circLRWD1 participates in DDP resistance in NSCLC. Changes in circLRWD1 expression were determined by real-time quantitative PCR. Effects of circLRWD1 inhibition on DDP-resistant NSCLC cell viability, proliferation, migration, invasion, and apoptosis were analyzed. The sponge function of circLRWD1 was predicted by bioinformatics analysis and verified by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. The function of circLRWD1 in DDP resistance was verified by xenograft models. CircLRWD1 was unconventionally overexpressed in DDP-resistant NSCLC samples and cells. Moreover, circLRWD1 silencing decreased IC 50 value, restrained cell proliferation, reduced cell migration and invasion, and facilitated cell apoptosis in DDP-resistant NSCLC cells. Also, circLRWD1 knockdown elevated DDP-resistant NSCLC cell sensitivity to DDP in xenograft models. Furthermore, circLRWD1 regulated SIRT5 expression via adsorbing miR-507. SIRT5 overexpression weakened circLRWD1 silencing-mediated suppression of cell resistance to DDP in DDP-resistant NSCLC cells. In conclusion, circLRWD1 elevated SIRT5 expression via adsorbing miR-507, resulting in promoting NSCLC cell resistance to DDP, providing evidence to explain the significant role of circLRWD1 in DDP resistance in NSCLC.

In vivo exposure to a new 2-cyano-2-p-nitrophenyl-N-benzylthioamide decreases doxorubicin-triggered structural damages in the mature testis.

The use of doxorubicin (DOX) in clinical practice continues to be challenged by its severe toxicity. DOX cytotoxic activity is not only directed against malignant tumours, given that the treatment will damage healthy tissues as well leading to irreversible injuries. This study aimed to address the in vivo effects of DOX and its co-administration with a new analog of thioamide; thiocyanoacetamide (TA) on the germinal epithelium. Thus, male rats received either intravenous injection (iv) of 0.03 mg/kg of body weight/week, 0.9% NaCl and were regarded as the control group (CTR), treated with DOX (3.7 mg/kg/week iv), TA [10 mg/kg/day intragastrically (ig)] or a co-supplementation of DOX and TA. After 50 days, the left testes were dissected and used for toluidine blue, periodic acid-Schiff (PAS) staining (to evaluate the change in polysaccharides/glycoproteins content), and transmission electron microscopy (TEM) (to assess the morphological damages). To estimate the impact of the test compounds on mitochondrial biogenesis, the expression of NAD-dependent deacetylase sirtuin-3 (SIRT-3) and proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were evaluated by immunofluorescence. Apoptotic cells were observed using Hoechst 33324 fluorescent staining. Data showed testicular injuries in the DOX-treated group, manifested by a significant decrease in total germ cell (GC) number, alteration of Sertoli cell (SC) nucleolus, anchoring junction, along with modifications of the basement membrane (BM) regularity and increase in apoptotic cell count. Mitochondrial aspect and SIRT-3 and PGC-1α expression in the testis were unaffected by the DOX. Co-therapy increased GC number, decreased apoptotic cell count, and restored the BM and anchoring junction regular aspects. This study provides novel insights into understanding DOX-mediated impairment in rats' testis and might offer some basis for the emerging new alternative therapeutic schemes in male patients undergoing chemotherapy.

Involvement of sirtuins (Sirt1 and Sirt3) and aryl hydrocarbon receptor (AhR) in the effects of triclosan (TCS) on production of neurosteroids in primary mouse cortical neurons cultures.

Epidemiological studies have shown the presence of triclosan (TCS) in the brain due to its widespread use as an antibacterial ingredient. One of the confirmed mechanisms of its action is the interaction with the aryl hydrocarbon receptor (AhR). In nerve cells, sirtuins (Sirt1 and Sirt3) act as cellular sensors detecting energy availability and modulate metabolic processes. Moreover, it has been found that Sirt1 inhibits the activation of estrogen receptors, regulates the androgen receptor, and may interact with the AhR receptor. It is also known that Sirt3 stimulates the production of estradiol (E2) via the estradiol receptor ß (Erß). Therefore, the aim of the present study was to evaluate the effect of TCS alone or in combination with synthetic flavonoids on the production of neurosteroids such as progesterone (P4), testosterone (T), and E2 in primary neural cortical neurons in vitro. The contribution of Sirt1 and Sirt3 as well as AhR to these TCS-induced effects was investigated as well. The results of the experiments showed that both short and long exposure of neurons to TCS increased the expression of the Sirt1 and Sirt3 proteins in response to AhR stimulation. After an initial increase in the production of all tested neurosteroids, TCS acting for a longer time lowered their levels in the cells. This suggests that TCS activating AhR as well as Sirt1 and Sirt3 in short time intervals stimulates the levels of P4, T, and E2 in neurons, and then the amount of neurosteroids decreases despite the activation of AhR and the increase in the expression of the Sirt1 and Sirt3 proteins. The use of both the AhR agonist and antagonist prevented changes in the expression of Sirt1, Sirt3, and AhR and the production of P4, T, and E2, which confirmed that this receptor is a key in the mechanism of the TCS action.

A study on Wistar Albino rats: investigating protective role of ramelteon on liver damage caused by methotrexate.

Methotrexate is an important immunosuppressive and antineoplastic drug and is widely used for treatment. However, hepatotoxicity is one of the major adverse effects of methotrexate. In this study, it was aimed to investigate whether ramelteon has a possible protective effect on hepatotoxicity induced by methotrexate. Thirty-two Wistar albino rats were equally divided into four groups: control, methotrexate, methotrexate + ramelteon, and ramelteon. Following a single dose of 20 mg/kg, methotrexate (i.p.), either saline or ramelteon 10 mg/kg (orally) was administered for 7 days. After treatment, animals were sacrificed, and histopathological analyses were evaluated with Hematoxylin-eosin (H-E), immunohistological analyses were evaluated with Interleukin-1 Beta (IL-1ß) and Caspase 3 (CAS-3), biochemical analyzes were evaluated with Total Oxidant Status (TOS), Total antioxidants status (TAS), Oxidative Stress Index (OSI), aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, at last genetical analyses were evaluated with Sirtuin-1 (SIRT-1) - P53 gene expressions. In the control and ramelteon groups, normal histological structures were observed, while histopathological findings were observed in the methotrexate group. Increasing levels of IL-1ß staining, CAS-3 staining, p53 gene expression, TOS, OSI, AST and ALT were observed in methotrexate group while were observed decreasing levels of TAS and SIRT-1 gene expression (p < 0.05). However, ramelteon reduced the increased findings in methotrexate-induced hepatotoxicity (p < 0.05). The results of the present study showed that ramelteon protects against methotrexate induced hepatotoxicity in rats via SIRT-1 signaling by histological, immunohistological, biochemical and genetical analyses.

Sirtuin 6 ameliorates alcohol-induced liver injury by reducing endoplasmic reticulum stress in mice.

Alcoholic liver disease (ALD) occurs as a result of chronic and excessive alcohol consumption. It encompasses a wide spectrum of chronic liver abnormalities that range from steatosis to alcoholic hepatitis, progressive fibrosis and cirrhosis. Endoplasmic reticulum (ER) stress induced by ethanol metabolism in hepatocytes has been established as an important contributor to the pathogenesis of ALD. However, whether SIRT6 exerts regulatory effects on ethanol-induced ER stress and contributes to the pathogenesis of ALD is unclear. In this study, we developed and characterized Sirt6 hepatocyte-specific knockout and transgenic mouse models that were treated with chronic-plus-binge ethanol feeding. We observed that hepatic Sirt6 deficiency led to exacerbated ethanol-induced liver injury and aggravated hepatic ER stress. Tauroursodeoxycholic acid (TUDCA) treatment remarkably attenuated ethanol-induced ER stress and ameliorated ALD pathologies caused by Sirt6 ablation. Reciprocally, SIRT6 hepatocyte-specific transgenic mice exhibited reduced ER stress and ameliorated liver injury caused by ethanol exposure. Consistently, knockdown of Sirt6 elevated the expression of ER stress related genes in primary hepatocytes treated with ethanol, whereas overexpression of SIRT6 reduced their expression, indicating SIRT6 regulates ethanol-induced hepatic ER stress in a cell autonomous manner. Collectively, our results suggest that SIRT6 is a positive regulator of ethanol-induced ER stress in the liver and protects against ALD by relieving ER stress.

Melatonin ameliorates Bisphenol S induced testicular damages by modulating Nrf-2/HO-1 and SIRT-1/FOXO-1 expressions.

BPS has detrimental effects on human reproductive health and emerged as an environmental contaminant for global health concern. This study deals with the adverse impact of BPS exposure on testicular oxidative stress, inflammation and apoptosis in adult male golden hamster, Mesocricetus auratus and its amelioration by melatonin. BPS (75 mg/kg BW/day) exposure caused testicular impairment as evident by histological degenerative changes, declined sperm quality (viability and motility), serum levels of testosterone and melatonin with a concomitant decrease in testicular androgen receptor (AR) and melatonin receptor (MT1) expression. The BPS exposure caused marked increase in testicular oxidative load, inflammation (NF-kB/COX-2) and apoptosis (caspase-3). Melatonin (10 mg/kg BW/alternate day) pretreatment to BPS exposed hamsters resumed normal testicular histoarchitecture, sperm quality and decreased testicular oxidative load as evident by enhanced antioxidant enzymes (SOD and catalase) activities and decreased lipid peroxidation (LPO) level. Further, melatonin also stimulated the testicular antioxidant proteins Nrf-2/HO-1, SIRT-1/FOXO-1 and reduced inflammatory proteins NF-kB/COX-2 expression to counteract BPS induced testicular damages. Melatonin administration to the BPS treated hamsters resulted in increased testicular cell proliferation (PCNA), survival (Bcl-2), gap junction (connexin-43) and decreased apoptosis (caspase-3). In conclusion, our study documented the detrimental effects of BPS on testes that compromises male fertility. Further, melatonin was found as a potent molecule that rescued the BPS induced testicular damages in male golden hamster Mesocricetus auratus.

Screening Analysis of Sirtuins Family Expression on Anti-Inflammation of Resveratrol in Endothelial Cells.

BACKGROUND Resveratrol has been shown to possess beneficial activities including antioxidant, anti-inflammatory, and cardioprotective effects through activating a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. The current study was undertaken to investigate the role of sirtuin family members (SIRT1-SIRT7) on the anti-inflammation activities of resveratrol in endothelial cells. MATERIAL AND METHODS Primary human umbilical vein endothelial cells (HUVECs) were pretreated with resveratrol before tumor necrosis factor (TNF)-alpha (10-20 µg/L) stimulation. Cell viability was measured using the Cell Counting Kit-8 method. Total RNA was extracted after different treatments and the NimbleGen Human 12×135K Gene Expression Array was applied to screen and analyze SIRTs expression. Quantitative real-time polymerase chain reaction and western blot were applied to verify the results of the gene expression microarrays. Reactive oxygen species (ROS) production was examined using flow cytometry analysis. RESULTS Microarray analysis showed that the expressions of SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 showed the tendency to increase while SIRT4 showed the tendency to decrease. SIRT1, SIRT2, SIRT5, and SIRT7 gene expression could be upregulated by pretreatment with resveratrol compared with TNF-alpha alone while there were no obvious differences of SIRT3, SIRT4, and SIRT6 expressions observed in TNF-alpha alone treated cells and resveratrol-TNF-alpha co-treated cells. Interestingly, SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 siRNA could reverse the effect of resveratrol on ROS production; SIRT1 and SIRT5 siRNA could significantly increase CD40 expression inhibited by resveratrol in TNF-a treated cells. CONCLUSIONS Our results suggest that resveratrol inhibiting oxidative stress production is associated with SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 pathways; attenuating CD40 expression was only associated with SIRT1 and SIRT5 pathways in TNF-alpha-induced endothelial cells injury.

Inhibitory Effect of Sirtuin6 (SIRT6) on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND The imbalance between bone resorption and formation is the basic mechanism underlying osteoporosis in the elderly. Osteogenesis is the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts. Sirtuin6 (SIRT6) regulates various biological functions, including differentiation. Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a non-selective cation channel that can be activated by physical and chemical stimulation. However, experimental data supporting the role of SIRT6 in osteogenic differentiation (OD) of hMSCs are lacking. MATERIAL AND METHODS Differentiation of hMSCs was induced. The expressions of SIRT6, TRPV1, and CGRP were detected by Q-PCR, Western blot, and ELISA, respectively. SIRT6 was overexpressed in hMSCs by transfection. ALP activity and Alizarin Red staining were utilized to detect the effect of SIRT6 on hMSC OD. Then, capsaicin and capsazepine, the TRPV1 agonist and antagonist, respectively, were administrated to assess the role of TRPV1. RESULTS SIRT6 expression was downregulated during hMSC differentiation. SIRT6 overexpression was accompanied by reduced expression of specific genes and alkaline phosphatase (ALP) activity in osteoblasts. Furthermore, TRPV1 channel was also reduced by SIRT6 overexpression via ubiquitinating TRPV1. Capsaicin was utilized in SIRT6-overexpressed cells. Capsaicin therapy counteracted the effect of SIRT6 overexpression on OD, and markedly decreased OD. CONCLUSIONS The SIRT6-TRPV1-CGRP signal axis is the key to regulating OD in hMSCs, which could be a potential therapeutic target for osteoporosis and bone loss-related diseases.

Depletion of SIRT7 sensitizes human non-small cell lung cancer cells to gemcitabine therapy by inhibiting autophagy.

Anti-metabolic therapy, as a major chemotherapy, is an important option in the treatment of lung cancer. However, tumor resistance to cytotoxic chemotherapy has become more common. It has been reported that autophagy is one of the processes contributing to such resistance. In our study, we find that SIRT7 protein level elevated dramatically in response to an anti-metabolic drug-gemcitabine treatment. Moreover, autophagy induced by gemcitabine in non-small cell lung cancer cells is SIRT7-dependent. Furthermore, depletion of SIRT7 promoted Gemcitabine-induced cell death. Our report also shows that SIRT7 knockdown markedly improves the anti-tumor activity of gemcitabine treatment in mice. These results suggest that SIRT7-elicits an autophagic response that plays a protective role against cell death and the SIRT7-inhibition has a potential to improve the efficacy of anti-metabolic therapy in non-small cell lung cancer cells.

Antioxidant supplementation during in vitro culture improves mitochondrial function and development of embryos from aged female mice.

Maternal aging results in reduced oocyte and blastocyst quality, thought to be due, in part, to mitochondrial dysfunction and accumulation of reactive oxygen species. To reduce oxidative stress, the antioxidants α-lipoic acid (ALA; 10µM), α-tocopherol (250µM), hypotaurine (1mM) and N-acetylcysteine (NAC; 1mM), and sirtuin (100ngmL(-1)) were added to embryo culture medium (AntiOX) and compared with a control (CON) without antioxidants to assess blastocyst development after in vitro maturation and fertilisation of oocytes from aged B6D2F1 female mice (13.5 months). Development to the blastocyst stage increased in the AntiOX compared with CON group (87.6% vs 72.7%, respectively; P<0.01), in addition to higher mitochondrial membrane potential and ATP levels in the AntiOX group. Expression of genes associated with oxidative stress (PI3K, FOXO3A and GLRX2) was upregulated in the CON compared with AntiOX group. In addition to AntiOX, a medium containing only NAC and ALA (rAntiOX) was used to culture embryos from young CF1 females (6-8 weeks). More blastocysts developed in the rAntiOX compared with CON group (64.1% vs 43.3%, respectively; P<0.01), although AntiOX (48.0% blastocysts) did not result in improved development in young mice. Antioxidants improved mitochondrial activity, gene expression and development in embryos of older female mice, whereas a reduced level of antioxidants during culture was beneficial to embryos from young mice.

SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress.

SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.

SIRT3 and SIRT4 double-genes remodeled the mitochondrial network to induce hepatocellular carcinoma cell line differentiation and suppress malignant phenotypes.

Tumor cells with damaged mitochondria undergo metabolic reprogramming, but gene therapy targeting mitochondria has not been comprehensively reported. In this study, plasmids targeting the normal hepatocyte cell line (L-O2) and hepatocellular carcinoma cell line were generated using three genes SIRT3, SIRT4, and SIRT5. These deacetylases play a variety of regulatory roles in cancer and are related to mitochondrial function. Compared with L-O2, SIRT3 and SIRT4 significantly ameliorated mitochondrial damage in HCCLM3, Hep3B and HepG2 cell lines and regulated mitochondrial biogenesis and mitophagy, respectively. We constructed double-gene plasmid for co-express SIRT3 and SIRT4 using the internal ribosome entry site (IRES). The results indicated that the double-gene plasmid effectively expressed SIRT3 and SIRT4, significantly improved mitochondrial quality and function, and reduced mtDNA level and oxidative stress in HCC cells. MitoTracker analysis revealed that the mitochondrial network was restored. The proliferation, migration capabilities of HCC cells were reduced, whereas their differentiation abilities were enhanced. This study demonstrated that the use of IRES-linked SIRT3 and SIRT4 double-gene vectors induced the differentiation of HCC cells and inhibited their development by ameliorating mitochondrial dysfunction. This intervention helped reverse metabolic reprogramming, and may provide a groundbreaking new framework for HCC treatment.

Rosmarinic Acid Activates the Nrf2/ARE Signaling Pathway via the miR-25-3p/SIRT6 Axis to Inhibit Vascular Remodeling.

The vital pathological processes in intimal hyperplasia include aberrant vascular smooth muscle cells (VSMCs) proliferation, migration, and phenotypic switching. Rosmarinic acid (RA) is a natural phenolic acid compound. Nevertheless, the underlying mechanism of RA in neointimal hyperplasia is still unclear. Our analysis illustrated that miR-25-3p mimics significantly enhanced PDGF-BB-mediated VSMCs proliferation, migration, and phenotypic switching while RA partially weakened the effect of miR-25-3p. Mechanistically, we found that miR-25-3p directly targets sirtuin (SIRT6). The suppressive effect of the miR-25-3p inhibitor on PDGF-BB-induced VSMCs proliferation, migration, and phenotypic switch was partially eliminated by SIRT6 knockdown. The suppression of the PDGF-BB-stimulated Nrf2/ARE signaling pathway that was activated by the miR-25-3p inhibitor was exacerbated by the SIRT6 knockdown. In in vivo experiments, RA reduced the degree of intimal hyperplasia while miR-25-3p agomir partially reversed the suppressive effect of RA in vascular remodeling. Our results indicate that RA activates the Nrf2/ARE signaling pathway via the miR-25-3p/SIRT6 axis to inhibit vascular remodeling.

Cisplatin-induced PANDAR-Chemo-EVs contribute to a more aggressive and chemoresistant ovarian cancer phenotype through the SRSF9-SIRT4/SIRT6 axis.

OBJECTIVE: We previously elucidated that long non-coding RNA Promoter of CDKN1A Antisense DNA damage Activated RNA (PANDAR) as a p53-dependent oncogene to promote cisplatin resistance in ovarian cancer (OC). Intriguingly, high level of p53-independent PANDAR was found in cisplatin-resistant patients with p53 mutation. Here, our study probed the new roles and the underlying mechanisms of PANDAR in p53-mutant OC cisplatin-resistance. METHODS: A2780 and A2780-DDP cells were served as OC cisplatin-sensitive and cisplatin-resistant cells. HO-8910PM cells were subjected to construct chemotherapy-induced extracellular vesicles (Chemo-EVs). Transmission electron microscopy (TEM) and nanoparticle tracking analysis were employed to evaluate Chemo-EVs. Cell viability was assessed using cell counting kit-8 and colony formation assays. Cell apoptosis was assessed using Annexin V and propidium iodide staining. The relationships between PANDAR, serine and arginine-rich pre-mRNA splicing factor 9 (SRSF9) were verified by RNA immunoprecipitation and fluorescence in situ hybridization. Tumor xenograft experiment was employed to evaluate the effects of PANDAR-Chemo-EVs on OC cisplatin-resistance in vivo. Immunofluorescent staining and immunohistochemistry were performed in tumor tissue. RESULTS: PANDAR level increased in OC patients with p53-mutation. PANDAR efflux enacted via exosomes under cisplatin conditions. Additionally, exosomes from OC cell lines carried PANDAR, which significantly increased cell survival and chemoresistance in vitro and tumor progression and metastasis in vivo. During cisplatin-induced stress, SRSF9 was recruited to nuclear bodies by increased PANDAR and muted apoptosis in response to cisplatin. Besides, SRSF9 significantly increased the ratio of SIRT4/SIRT6 mRNA in OC. CONCLUSION: Cisplatin-induced exosomes transfer PANDAR and lead to a rapid adaptation of OC cell survival through accumulating SRSF9 following cisplatin stress exposure.