Metformin Regulates SIRT1 To Cause Cancer Cell Pyroptosis And Apoptosis And Inhibit Cell Migration

Metformin is a biguanide compound widely prescribed for the treatment of type 2 diabetes owing to its high efficacy and minimal side effects.In recent years,there has been significant interest in metformin as a potential cancer chemo-preventive or therapeutic agent.As a complex I inhibitor,metformin directly induces mitochondrial dysfunction in epithelial cancer cell compartment.It also activates AMP-activated protein kinase(AMPK),a major sensor of cellular energy status in multiple types of cancer and human fibroblasts.AMPK activation raises the intracellular NAD~+concentrations and activates SIRT1 to cause modulations of its downstream targets,such as NF-κB and Fox Os,regulating a variety of physiological processes,including cell proliferation,cell death(apoptosis and autophagy-dependent necrosis),migration and aging.The absence of apoptosis and autophagy-dependent necrosis pathways may activate a new programmed cell death,pyroptosis.Pyroptosis is triggered primarily by activation of the inflammatory caspases(caspase1/4/5/11),which cleaves a cellular substrate called gasdermin D(GSDMD),generating a necrotic N-terminal fragment capable of forming pore.The pore-forming activity disturbs osmotic potential,resulting in cell swelling with large bubbles blowing from the plasma membrane,finally induces pyroptosis.NF-κB activates GSDMD to induce cell pyroptosis in mouse small intestinal epithelial and adipose tissue cells,causing systemic inflammation.In 2017,Shao et al report that gasdermin E(GSDME)is specifically cleaved by caspase3 and releases its N-terminal pore-forming domain for pore formation when treating with certain apoptotic stimuli(such as chemotherapy drugs),eventually causing pyroptotic death in cancer cells.In 2018,activation of the mitochondrial apoptotic pathway(including Bax accumulation,cytochrome c release and activation of caspase3)in the GSDME-expressing cells leads to induction of pyroptosis.So,we propose that metformin activates AMPK/SIRT1/NF-κB pathway and induces mitochondrial dysfunction to drive caspase3/GSDME-mediated cancer cell pyroptosis.In this study,Hep G2,MCF-7,HCT-15 and HT-29 cancer cells were used to explore whether metformin induces cancer cell pyroptosis.MTT assay was performed to examine that metformin inhibited cell proliferation in a time-dependent manner(2,4,8,12,16 and 24 h).The phase-contrast images displayed that the dying cells blew characteristic large bubbles from the plasma membrane,causing typical swelling when 20 m M metformin treatment for 4,8 and 12 h in Hep G2 cells.As an indicator of pyroptotic cell cytotoxicity,the release of lactate dehydrogenase(LDH)was detected when Hep G2,HCT-15 and HT-29 cells were exposed to metformin for 4,8 and 12h,and the results showed that metformin enhanced LDH release.We then detected the expression of pyroptosis-associated proteins by western blot,and results showed that metformin increased cleavage of caspase3 and GSDME-N protein levels,suggesting metformin induced cancer cell pyroptotic death.Next,we explore the molecular mechanisms for metformin to induce pyroptosis.When Hep G2 and MCF-7 cells were exposed to metformin for 2,4,8 and 12 h,and result demonstrated that metformin led to a significant increase in p-AMPK and SIRT1 protein levels,it also enhanced expression of NF-κB p65,Bax and cytochrome c,finally activating cleavage of pyroptosis-associated proteins caspase3 and GSDME.To investigate the role of SIRT1 and NF-κB in metformin-induced pyroptosis of cancer cells,EX527 treatment and knocking down SIRT1 by si RNA reversed the increase of NF-κB p65,Bax,cytochrome c and pyroptosis-associated protein expression induced by metformin,and these proteins were downstreams of SIRT1.Next,when NF-κB inhibitor BAY 11-7082 was added to MCF-7 and Hep G2 cells,NF-κB p65 was inhibited and expression of Bax,caspase3 and GSDME metformin-increased were suppressed.Thus,metformin activated AMPK/SIRT1/NF-κB pathway to induce cancer cell pyroptosis.Metformin induces mitochondrial dysfunction,we thus explore whether metformin induces mitochondrial dysfunction to drive cancer cell pyroptosis.Metformin decreased mitochondrial membrane potential,elevated ROS level and inhibited expression of PGC-1α to induce mitochondrial dysfunction.While CCCP was added to Hep G2 and MCF-7 cells,PGC-1α protein level was further repressed efficiently,and cleaved-caspase3 and GSDME-N levels were enhanced markedly,suggesting metformin promoted cancer cell pyroptosis by inducing mitochondrial dysfunction.When CCCP was added to Hep G2 and MCF-7 cells,the expression of SIRT1,Bax,cytochrome c and pyroptosis-associated proteins metformin-increased were enhanced dramatically,and AMPK inhibitor Compound C(CC)has no effect on mitochondrial membrane potential.These results show that metformin-induced mitochondrial dysfunction activated AMPK/SIRT1 to drive cancer cell pyroptosis.Therefore,metformin activated AMPK/SIRT1 to increase NF-κB p65 expression,facilitating Bax accumulation and cytochrome c release to trigger activation of caspase3 and cleavage of GSDME in cancer cells.Meanwhile,metformin induced mitochondrial dysfunction to stimulate AMPK/SIRT1 pathway-mediated pyroptosis.Previous studies have implicated that SIRT1 locates in cytoplasm as well as nucleus.The subcellular localization of SIRT1 may regulate its anticancer effects.However,few reports that metformin regulates SIRT1 localization to exert anticancer effects have been identified to date.First,breast and lung cancer cells were treated with different concentrations of metformin(5,10,15,20 and 25 m M)for 24 h.MTT assay was performed to demonstrate that metformin inhibited cell proliferation,and cell viability decreased in a concentration-dependent manner.Hoechst and propidium iodide nuclear staining assay were further carried out,and results showed that metformin dramatically increased cancer cell apoptotic rates;metformin also increased cleaved-caspase3 expression to induce cancer cell apoptosis.Meanwhile,we explored the effects of metformin on regulating EMT marker expression and cell migration.These data support that metformin increased the expression of epithelial marker E-cadherin significantly and decreased mesenchymal markers(N-cadherin,β-catenin and Vimentin)level.Wound healing assay demonstrated that metformin inhibited cancer cell migration.Next,we detect the molecular mechanisms that metformin induced cell apoptosis and inhibited cell migration.The result showed metformin increased AMPK protein expression,decreased NADH level and enhanced SIRT1 activity totally.NADH level and SIRT1 activity was enhanced when AMPK inhibitor CC was added to the cancer cells.These results indicate that metformin enhanced SIRT1 activity in an AMPK-dependent manner.To detect the effects of metformin regulates nuclear/cytoplasmic SIRT1 activity,the nuclear and cytoplasmic fractions of MCF-7 and MDA-MB-231 cancer cells were isolated and fluorometric SIRT1 assay was performed.Results showed that metformin decreased nuclear SIRT1 activity and increased cytoplasmic SIRT1 activity,and nuclear SIRT1activity was enhanced while cytoplasmic SIRT1 activity was suppressed when CC was added to cancer cells.SIRT1 was silenced in cancer cells to explore the role of SIRT1 in regulating cancer cell apoptosis and migration.Results showed that there were an enhanced induction of cell apoptosis,and an increased closure of the wound area compared with control cells.These results indicate that metformin decreased nuclear SIRT1 activity and increased cytoplasmic SIRT1activity to induce apoptosis and inhibit cell migration by activating AMPK in cancer cells.It is reported that tumor suppressor gene DLC1 is pivotal for inhibition of cell migration in the cytoplasm and for induction of cell apoptosis in the nucleus of gastric cancer cells,the underlying mechanisms are unclear.Our group has shown that resveratrol increases SIRT1 and DLC1 expression in Nothobranchius guentheri to promote tumor cell apoptosis.In this study,our results showed that metformin increased DLC1 protein level in MCF-7 and H1299 cells totally.DLC1 localized in the cytoplasm as well as nucleus,and both of them were up-regulated treatment with metformin.DLC1 nuclear/cytoplasmic expression ratio was elevated upon metformin treatment,indicating that metformin induced DLC1 nucleus import.DLC1 was overexpressed in MCF-7 cells and the data presented demonstrate that it markedly increased cleaved-caspase3/9 expression,and elevated expression of an epithelial marker(claudin-1)and decreased level of mesenchymal markers(N-cadherin,β-catenin,Vimentin and Snail)to suppress cell migration.To detect the effects of SIRT1 in DLC1 regulation,SIRT1 was knocked down in MCF-7 cells and results reveal that SIRT1 silencing increased DLC1 expression.SIRT1inhibitor EX527 were exposed to cancer cells,nuclear DLC1 level was increased significantly,indicating that DLC1 is a downstream target of SIRT1,and SIRT1 negatively regulates protein level of nuclear DLC1.In gastric and prostate cancer cells,histone deacetylase inhibitor TSA activates DLC1 promoter by regulating histone acetylation level.We suppose that SIRT1 as a histone deacetylase,it is possible for SIRT1 to regulate DLC1 promoter.In addition,DLC1 could be phosphorylated by PKC and PKD,which in turn represses the interaction with 14-3-3 and fails to stimulate DLC1 nuclear import.To further explore How SIRT1 regulates expression of DLC1 induced by metformin.We performed semi-PCR assay,and found that metformin and SIRT1 inhibitor EX527 increased DLC1 m RNA level.Ch IP assay identified the presence of the interacting DLC1 promoter fragments with SIRT1,and metformin reduced this interaction to elevate nuclear DLC1 expression.Our results also showed that metformin significantly decreased PKC and 14-3-3 expression,inhibited interaction of DLC1 with 14-3-3 protein to promote DLC1 nuclear import.In addition to PKC,DLC1 was phosphorylated by AKT(PKB)to reduce its activity,finally inhibits cell growth and migration without nuclear import in hepatocellular carcinoma cells.Our previous study show that SIRT1 activator,resveratrol,regulates AKT phosphorylation of DLC1 to inhibit hepatocellular carcinoma cell growth.We next explore whether metformin regulates posttranslational modulations of DLC1 via SIRT1 to inhibit cell migration.Co-IP assay showed that metformin enhanced the interaction of SIRT1 and DLC1,and decreased DLC1 acetylation levels;it also reduced the presence of active AKT(p-AKT)and inhibited DLC1 phosphorylation levels(PAS).Depletion of SIRT1 by si RNA or EX527 treatment in MCF-7 cells to detect the effects of SIRT1 in regulating phosphorylation of DLC1 induced by metformin.Results showed that phosphorylation and acetylation levels of DLC1 increased by inhibiting SIRT1,which indicate that metformin increased SIRT1deacetylation of DLC1 to disturb AKT-mediated DLC1 phosphorylation,promoting DLC1activity.These results display that metformin regulated SIRT1 activity in nucleus and cytoplasm to promote DLC1-mediated cell apoptosis induction and migration inhibition in cancer cells.FoxO3a is a downstream target of SIRT1,and FoxO3a deacetylation would enhance its transcriptional activity.AKT phosphorylation of FoxO3a promotes its interaction with 14-3-3protein,then results in cytoplasmic retention of FoxO3a,negatively regulating FoxO3a activity.Next,we detect how metformin regulates SIRT1-mediated posttranslational modulations of FoxO3a to inhibit cancer cell proliferation.Western blot analysis showed that metformin elevated total FoxO3a levels.The nuclear and cytoplasmic fractions of MCF-7 and MDA-MB-231 cancer cells were isolated and Western blot assay displayed that metformin up-regulated nuclear FoxO3a expression;the nucleus/cytoplasm expression ratio of FoxO3a increased,indicating that metformin induced FoxO3a nuclear import.Western blot and Co-IP assay also revealed that metformin decreased expression of p-AKT,14-3-3 and p-FoxO3a,inhibited the interaction between FoxO3a and 14-3-3.To detect the effects of SIRT1 in metformin-regulated phosphorylation of FoxO3a,we employ Co-IP assay,results showed that metformin elevated the interaction of SIRT1 and FoxO3a to reduce FoxO3a acetylation level.Silencing SIRT1 or EX527 treatment up-regulated the acetylation of FoxO3a,enhanced p-FoxO3a expression and its association with 14-3-3.These results indicate that metformin promoted SIRT1 deacetylation of FoxO3a to suppress its phosphorylation AKT-mediated,resulting in FoxO3a nucleus import.FoxO3a activation involves in cancer cell proliferation,we performed Western blot to detect expression of PCNA,and result exhibited that metformin decreased PCNA expression.These findings support that metformin stimulated FoxO3a nuclear localization through SIRT1-mediated posttranslational modulations to inhibit cancer cell proliferation.Finally,we used the chicken chorioallantoic membrane(CAM)model to detect the effects of metformin on tumor growth in vivo,results showed that metformin reduced MCF-7 and A549tumor volume.To further investigate the mechanisms that metformin suppressed tumor growth in vivo,immunohistochemical experiment was performed on frozen sections of tumor,and results showed that metformin promoted protein levels of DLC1 and apoptotic marker cleaved-caspase3,and reduced EMT markerβ-catenin expression in tumor tissues;it also increased protein level of FoxO3a and declined PCNA expression in tumor tissues.Above results demonstrated that metformin promoted expression of DLC1 and FoxO3a to inhibit tumor growth in vivo.Thus,metformin decreased nuclear SIRT1 activity and increased cytoplasmic SIRT1activity in an AMPK-dependent manner to induce cancer cell apoptosis and inhibit cell migration.Specifically,in nucleus,metformin decreased SIRT1 binding to DLC1 promoter to elevate nuclear DLC1 m RNA and protein levels,and metformin inhibited PKC pathway to trigger DLC1nuclear import,which promoted caspase-induced cell apoptosis.In cytoplasm,metformin increased SIRT1 activity,the higher activity of SIRT1 decreased DLC1 and FoxO3a acetylation to disturb AKT-mediated their phosphorylation,therefore promoted DLC1 suppression of EMT-associated cancer cell migration as well as FoxO3a inhibition of cancer cell proliferation.This study implicates a novel anticancer mechanism for metformin to activate DLC1 and FoxO3a by regulating cytoplasmic/nuclear SIRT1 activity.