RESUMO
CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteína 9 Associada à CRISPR/genética , Sinais de Localização Nuclear/genéticaRESUMO
BACKGROUND: Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. METHODS: A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. RESULTS: After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. CONCLUSIONS: Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.
RESUMO
Vascular endothelial growth factor A (VEGFA) plays a critical role as a potent angiogenesis factor and is highly expressed in hepatocellular carcinoma (HCC). Although the expression of VEGFA has been strongly linked to the aggressive nature of HCC, the specific posttranscriptional modifications that might contribute to VEGFA expression and HCC angiogenesis are not yet well understood. In this study, we aimed to investigate the epitranscriptome regulation of VEGFA in HCC. A comprehensive analysis integrating MeRIP-seq, RNA-seq, and crosslinking-immunprecipitation-seq data revealed that VEGFA was hypermethylated in HCC and identified the potential m6A regulators of VEGFA including a m6A methyltransferase complex component RBM15 and the two readers, YTHDF2 and IGF2BP3. Through rigorous cell and molecular biology experiments, RBM15 was validated as a key component of methyltransferase complex responsible for m6A methylation of VEGFA, which was subsequently recognized and stabilized by IGF2BP3 and YTHDF2, leading to enhanced VEGFA expression and VEGFA-related functions such as human umbilical vascular endothelial cells (HUVEC) migration and tube formation. In the HCC xenograft model, knockdown of RBM15, IGF2BP3, or YTHDF2 resulted in reduced expression of VEGFA, accompanied by significant inhibition of tumor growth closely associated with VEGFA expression and angiogenesis. Furthermore, our analysis of HCC clinical samples identified positive correlations between the expression levels of VEGFA and the regulators RBM15, IGF2BP3, and YTHDF2. Collectively, these findings offer novel insights into the posttranscriptional modulation of VEGFA and provide potential avenues for alternative approaches to antiangiogenesis therapy targeting VEGFA.
RESUMO
Hepatocellular carcinoma (HCC) is a hypervascular tumor, and accumulating evidence has indicated that stimulation of angiogenesis by hepatitis B virus (HBV) may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV protein and has a close clinical association with HCC; however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that the forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD counts in HCC patients' specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, the overexpression of SHBs increased vascular endothelial growth factor A (VEGFA) expression at both the mRNA and protein levels. Higher VEGFA expression levels were also observed in xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues than in their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVEC migration and vessel formation. Furthermore, all three unfolded protein response (UPR) sensors, inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), and activating transcription factor 6 (ATF6), associated with ER stress were found to be activated in SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention for HBV-related HCC and its malignant progression. IMPORTANCE Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to the overexpression of angiogenic factors like vascular endothelial growth factor A (VEGFA). However, a detailed mechanism of HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV protein, i.e., small surface antigen (SHBs), can enhance the angiogenic capacity of HCC cells by the upregulation of VEGFA expression both in vitro and in vivo. Mechanistically, SHBs induced endoplasmic reticulum (ER) stress, which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important proangiogenic role in HBV-associated HCC and may represent a potential target for antiangiogenic therapy in HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Estresse do Retículo Endoplasmático , Antígenos de Superfície da Hepatite B/metabolismo , Neoplasias Hepáticas/patologia , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/virologia , Transdução de Sinais , Resposta a Proteínas não Dobradas , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.
Assuntos
Gluconeogênese , Antígenos de Superfície da Hepatite B , Hepatite B Crônica , Animais , Camundongos , Antígenos de Superfície/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucagon/metabolismo , Glucagon/farmacologia , Gluconeogênese/genética , Glucose/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) has been identified as a crucial immune suppressor in human cancers, comparable to programmed cell death 1 ligand (PD-L1). However, the regulatory mechanisms underlying its transcriptional upregulation in human cancers remain largely unknown. Here, we show that the transcription factors ETS-1 and ETS-2 bound to the Siglec-15 promoter to enhance transcription and expression of Siglec-15 in hepatocellular carcinoma (HCC) cells and that transforming growth factor ß-1 (TGF-ß1) upregulated the expression of ETS-1 and ETS-2 and facilitated the binding of ETS-1 and ETS-2 to the Siglec-15 promoter. We further demonstrate that TGF-ß1 activated the Ras/C-Raf/MEK/ERK1/2 signaling pathway, leading to phosphorylation of ETS-1 and ETS-2, which consequently upregulates the transcription and expression of Siglec-15. Our study defines a detailed molecular profile of how Siglec-15 is transcriptionally regulated which may offer significant opportunity for therapeutic intervention on HCC immunotherapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Linhagem Celular , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
BACKGROUND: COVID-19 is an extremely severe infectious disease. However, few studies have focused on the epidemiological and clinical characteristics of pediatric COVID-19. This study conducted a retrospective review of the epidemiological and clinical features of COVID-19 in children. METHODS: A retrospective study was conducted on children with a definite diagnosis of COVID-19 in mainland China using the web crawler technique to collect anonymous COVID-19 updates published by local health authorities. RESULTS: Three hundred forty-one children aged 4 days to 14 years with a median age of 7 years were included. Sixty-six percent of pediatric patients were infected via family members with COVID-19. The median incubation period was 9 days (interquartile range, 6 to 13). Asymptomatic cases accounted for 5.9%, of which 30% had abnormal chest radiologic findings. A majority of pediatric COVID-19 cases showed mild to moderate clinical features, and only a few developed severe or critical diseases (0.6% and 0.3%, respectively). Fever (77.9%) and cough (32.4%) were the predominant presenting symptoms of pediatric COVID-19. The pediatric patients had fewer underlying diseases and complications than adults. The treatment modalities for pediatric COVID-19 patients were not as complex as those of adult COVID-19 patients. The overall prognosis of pediatric COVID-19 was benign with a decent recovery. The median time from onset to cure was 16 days (interquartile range, 13 to 21). CONCLUSIONS: Compared to adults, COVID-19 in children has distinct features of epidemiology and clinical manifestations. The findings from this study might help to guide the development of measures to prevent and treat this ongoing global pandemic. TRIAL REGISTRATION: Chinese Clinical Trial Registry ( chictr.org.cn ) identifier: ChiCTR2000030464.
Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Adolescente , COVID-19 , Criança , Pré-Escolar , China/epidemiologia , Tosse/etiologia , Feminino , Febre/etiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pandemias , Estudos Retrospectivos , SARS-CoV-2RESUMO
The Fas receptor/ligand system plays a prominent role in hepatic apoptosis and hepatocyte death. Although hepatitis B virus (HBV) surface Ag (HBsAg) is the most abundant HBV protein in the liver and peripheral blood of patients with chronic HBV infection, its role in Fas-mediated hepatocyte apoptosis has not been disclosed. In this study, we report that HBsAg sensitizes HepG2 cells to agonistic anti-Fas Ab CH11-induced apoptosis through increasing the formation of SDS-stable Fas aggregation and procaspase-8 cleavage but decreasing both the expression of cellular FLIPL/S and the recruitment of FLIPL/S at the death-inducing signaling complex (DISC). Notably, HBsAg increased endoplasmic reticulum stress and consequently reduced AKT phosphorylation by deactivation of phosphoinositide-dependent kinase-1 (PDPK1) and mechanistic target of rapamycin complex 2 (mTORC2), leading to enhancement of Fas-mediated apoptosis. In a mouse model, expression of HBsAg in mice injected with recombinant adenovirus-associated virus 8 aggravated Jo2-induced acute liver failure, which could be effectively attenuated by the AKT activator SC79. Based on these results, it is concluded that HBsAg predisposes hepatocytes to Fas-mediated apoptosis and mice to acute liver failure via suppression of AKT prosurviving activity, suggesting that interventions directed at enhancing the activation or functional activity of AKT may be of therapeutic value in Fas-mediated progressive liver cell injury and liver diseases.
Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Hepatócitos/fisiologia , Falência Hepática Aguda/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor fas/metabolismo , Acetatos/administração & dosagem , Acetatos/farmacologia , Adenoviridae/genética , Animais , Anticorpos Monoclonais/metabolismo , Apoptose , Benzopiranos/administração & dosagem , Benzopiranos/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Hep G2 , Hepatite B/patologia , Hepatócitos/virologia , Humanos , Falência Hepática Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/agonistas , Receptor fas/imunologiaRESUMO
Tumor necrosis factor-α (TNF-α) is a highly pleiotropic cytokine executing biological functions as diverse as cell proliferation, metabolic activation, inflammatory responses, and cell death. TNF-α can induce multiple mechanisms to initiate apoptosis in hepatocytes leading to the subsequent liver injury. Since the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway is known to have a protective role in death factor-mediated apoptosis, it is our hypothesis that activation of Akt may represent a therapeutic strategy to alleviate TNF-α-induced hepatocyte apoptosis and liver injury. We report here that the Akt activator SC79 protects hepatocytes from TNF-α-induced apoptosis and protects mice from d-galactosamine (d-Gal)/lipopolysaccharide (LPS)-induced TNF-α-mediated liver injury and damage. SC79 not only enhances the nuclear factor-κB (NF-κB) prosurvival signaling in response to TNF-α stimulation, but also increases the expression of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein L and S (FLIPL/S), which consequently inhibits the activation of procaspase-8. Furthermore, pretreatment of the PI3K/Akt inhibitor LY294002 reverses all the SC79-induced hepatoprotective effects. These results strongly indicate that SC79 protects against TNF-α-induced hepatocyte apoptosis and suggests that SC79 is likely a promising therapeutic agent for ameliorating the development of liver injury. NEW & NOTEWORTHY SC79 protects hepatocytes from TNF-α-mediated apoptosis and mice from Gal/LPS-induced liver injury and damage. Cytoprotective effects of SC79 against TNF-α act through both AKT-mediated activation of NF-κB and upregulation of FLIPL/S.
Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Crônica Induzida por Substâncias e Drogas/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Hepatócitos/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis; however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-mediated apoptosis is thought to be one of the major promotors for hepatitis B virus (HBV) chronicity and malignancy. The purpose of this study was to investigate whether HBSP could protect hepatocytes against Fas-initiated apoptosis. We showed here that HBSP mediated resistance of hepatoma cells or primary human hepatocytes (PHH) to agonistic anti-Fas antibody (CH11)- or FasL-induced apoptosis. Under Fas signaling stimulation, expression of HBSP inhibited Fas aggregation and prevented recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 (or FADD-like interleukin-1ß-converting enzyme [FLICE]) into the death-inducing signaling complex (DISC) while increasing recruitment of cellular FLICE-inhibitory protein L (FLIPL) into the DISC. Those effects may be mediated through activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway as evidenced by increased cellular phosphatidylinositol (3,4,5)-trisphosphate (PIP3) content and PI3K activity and enhanced phosphorylation of mTORC2 and PDPK1 as well as Akt itself. Confirmedly, inhibition of PI3K by LY294002 reversed the effect of HBSP on Fas aggregation, FLIPL expression, and cellular apoptosis. These results indicate that HBSP functions to prevent hepatocytes from Fas-induced apoptosis by enhancing PI3K/Akt activity, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.IMPORTANCE Our study revealed a previously unappreciated role of HBSP in Fas-mediated apoptosis. The antiapoptotic activity of HBSP is important for understanding hepatitis B virus pathogenesis. In particular, HBV variants associated with hepatoma carcinoma may downregulate apoptosis of hepatocytes through enhanced HBSP expression. Our study also found that Akt is centrally involved in Fas-induced hepatocyte apoptosis and revealed that interventions directed at inhibiting the activation or functional activity of Akt may be of therapeutic value in this process.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Hepatócitos/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Virais/metabolismo , Receptor fas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Células Tumorais Cultivadas , Proteínas Virais/genética , Receptor fas/genéticaRESUMO
Acute liver failure is a serious clinical problem of which the underlying pathogenesis remains unclear and for which effective therapies are lacking. The Fas receptor/ligand system, which is negatively regulated by AKT, is known to play a prominent role in hepatocytic cell death. We hypothesized that AKT activation may represent a strategy to alleviate Fas-induced fulminant liver failure. We report here that a novel AKT activator, SC79, protects hepatocytes from apoptosis induced by agonistic anti-Fas antibody CH11 (for humans) or Jo2 (for mice) and significantly prolongs the survival of mice given a lethal dose of Jo2. Under Fas-signaling stimulation, SC79 inhibited Fas aggregation, prevented the recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 [or FADD-like IL-1ß-converting enzyme (FLICE)] into the death-inducing signaling complex (DISC), but SC79 enhanced the recruitment of the long and short isoforms of cellular FLICE-inhibitory protein at the DISC. All of the SC79-induced hepatoprotective and DISC-interruptive effects were confirmed to have been reversed by the Akt inhibitor LY294002. These results strongly indicate that SC79 protects hepatocytes from Fas-induced fatal hepatic apoptosis. The potent alleviation of Fas-mediated hepatotoxicity by the relatively safe drug SC79 highlights the potential of our findings for immediate hepatoprotective translation.
Assuntos
Acetatos/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Hepatócitos/efeitos dos fármacos , Falência Hepática Aguda/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor fas/metabolismo , Animais , Caspases/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Falência Hepática Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The PI3K/AKT signaling pathway is one of the most frequently activated signaling networks in human cancers and has become a valuable target in anticancer therapy. However, accumulating reports suggest that adverse effects such as severe liver injury and inflammation may accompany treatment with pan-PI3K and pan-AKT inhibitors. Our prior work has demonstrated that activation of the PI3K/AKT pathway has a protective role in Fas- or TNFα-induced hepatocytic cell death and liver injury. We postulated that PI3K or AKT inhibitors may exacerbate liver damage via the death factor-mediated hepatocyte apoptosis. In this study we found that several drugs targeting PI3K/AKT either clinically used or in clinical trials sensitized hepatocytes to agonistic anti-Fas antibody- or TNFα-induced apoptosis and significantly shortened the survival of mice in in vivo liver damage models. The PI3K or AKT inhibitors promoted Fas aggregation, inhibited the expression of cellular FLICE-inhibitory protein S and L (FLIPL/S), and enhanced procaspase-8 activation. Conversely, cotreatment with the AKT specific activator SC79 reversed these effects. Taken together, these findings suggest that PI3K or AKT inhibitors may render hepatocytes hypersensitive to Fas- or TNFα-induced apoptosis and liver injury.
Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Aminopiridinas/toxicidade , Animais , Anticorpos/toxicidade , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Imidazóis/toxicidade , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Purinas/toxicidade , Quinazolinonas/toxicidade , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Microcystin-LR (MC-LR) is a type of cyclic heptapeptide toxin produced by cyanobacteria during bloom events. MC-LR-induced cell death is critically involved in its potent specific hepatotoxicity. Many studies have demonstrated that prototypical apoptosis as a form of programmed cell death after MC-LR is associated with liver injury. However, whether another form of programmed cell death exists and the underlying mechanism have not been reported. Here, we demonstrate that MC-LR can induce necroptosis via ROS overactivation in primary mouse hepatocytes. Various potential pathways of programmed cell death induced by MC-LR were evaluated by annexin V/PI dual staining for flow cytometric analysis, image-based PI staining analysis and western blot analysis. Cell viability was determined by the CCK8 assay. Rupture of the plasma membrane was indicated by lactate dehydrogenase release. ROS was evaluated with the carboxy-H2DCFDA fluorescent probe. It was found that in MC-LR-treated cells, as the plasma membrane was damaged, annexin V/PI-stained double-positive cells were significantly induced and PI-stained nuclei were more diffuse. Western blot analysis showed that MC-LR treatment significantly upregulated the expression of necroptotic and apoptotic proteins. Mechanistically, MC-LR induced ROS overproduction by dysregulating the expression and activity of the pro-oxidants SOD1, MAOA, and NOX4 and the antioxidant GPX1. These results indicate the presence of a novel mechanism for MC-LR-mediated liver injury and present a novel target in the treatment of MC-LR-exposed patients.
Assuntos
Hepatócitos/efeitos dos fármacos , Microcistinas/toxicidade , Necroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Proteínas Reguladoras de Apoptose/biossíntese , Membrana Celular/efeitos dos fármacos , Eutrofização , Hepatócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/metabolismo , Cultura Primária de Células , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Gastric cancer (GC) is one of the most common human cancers with the high rate of recurrence, metastasis and mortality. Aberrantly expressed microRNAs (miRNAs) are associated with invasion and metastasis in various human cancers. Recently, miR-188-5p has been indicated as an oncogene in GC since it promotes GC cell growth and metastasis. However, the underlying molecular mechanism remains to be fully defined. METHODS: Using Significance Analysis of Microarrays (SAM) screening, we identified that miR-188-5p is associated with overall survival and lymph node metastasis in patients with GC. The functional impact of miR-188-5p on GC metastasis was validated using in vitro and in vivo assays. The regulatory function of miR-188-5p on Wnt/ß-catenin signaling activation through directly targeting PTEN was proven using quantitative real-time PCR, western blot analysis, a dual-luciferase assay, a Transwell assay, and immunofluorescence. Immunohistochemical analyses further confirmed the clinical significance of miR-188-5p in GC. RESULTS: MiR-188-5p diminishes tumor suppressor PTEN expression, and further increases phospho-Ser9 of GSK3ß to activate Wnt/ß-catenin signaling in GC. Consequently, miR-188-5p enhanced the migration and invasion of GC cells in vitro and tumor metastasis in vivo, whereas inhibition of miR-188-5p had the opposite effects. Moreover, miR-188-5p was negatively correlated with PTEN expression but positively correlated with nuclear ß-catenin staining in GC samples. CONCLUSIONS: Our findings revealed a model of the miR-188-5p-PTEN-ß-catenin axis in GC, which mediates the constitutive activation of Wnt/ß-catenin signaling and promotes tumor metastasis, inferring that miR-188-5p is a potential therapeutic target to treat GC.
Assuntos
Metástase Linfática/genética , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Gástricas/patologia , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , PTEN Fosfo-Hidrolase/metabolismo , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Análise de Sobrevida , Regulação para CimaRESUMO
BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. METHODS: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. RESULTS: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. CONCLUSION: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Desoxiguanosina/análogos & derivados , Monoéster Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Transativadores/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Metilação de DNA , Enzimas Reparadoras do DNA/genética , Desoxiguanosina/metabolismo , Hepatite B/metabolismo , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monoéster Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Pirofosfatases/genética , Espécies Reativas de Oxigênio/metabolismo , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND: Gastric cancer (GC) accounts for the fourth most occurring malignancy and the third major cause of cancer death. Identifying novel molecular signaling pathways participating in gastric tumorigenesis and progression is pivotal for rational design of targeted therapies to improve advanced GC outcome. Recently, the endoplasmic reticulum (ER) protein 29 (ERp29) has been shown to inversely associate with primary tumor development and function as a tumor suppressor in breast cancer. However, the role of ERp29 in GC patients' prognosis and its function in GC progression is unknown. METHODS: Clinical importance of ERp29 in the prognosis of GC patients was assessed by examining its expression in 148 GC tumor samples and correlation with clinicopathological characteristics and survival of the patients. The function and underlying mechanisms of ERp29 in GC growth, invasion and metastasis were explored both in vitro and in vivo. RESULTS: Downregulation of ERp29 was commonly found in GC tissues and highly correlated with more aggressive phenotypes and poorer prognosis. Functional assays demonstrated that knockdown of ERp29 increased GC cell migration and invasion and promoted metastasis. Conversely, ectopic overexpression of ERp29 produced opposite effects. Mechanistic studies revealed that loss of ERp29 induced an epithelial-to-mesenchymal transition (EMT) in the GC cells through activation of PI3K/Akt pathway signaling. CONCLUSION: These findings suggest that downregulation of ERp29 is probably one of the key molecular mechanisms responsible for the development and progression of GC.
Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas de Choque Térmico/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Expressão Ectópica do Gene , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Neoplasias Gástricas/patologiaRESUMO
Cyclodextrin glycosyltransferase (CGTase) is an important industrial enzyme for production of cyclodextrins (CDs) from starch by intramolecular transglycosylation. CGTase consists of five domains labeled A to E. For optimizing catalytic activity of CGTase, CGTase of Geobacillus sp. was fused with the family 20 carbohydrate-binding module (CBM) of the Bacillus circulans strain 251 CGTase. The CBMbc251 that has a low binding free energy with maltohexaose, was selected by in silico design. Then the fusion enzyme, CGTΔE-CBMbc251, was constructed by fusing the CBMbc251 to the C-terminal region of CGTΔE. The fusion enzyme displayed an even greater enhancement of total α-cyclization activity (40.2%) and γ-cyclization activity (181.58%). Optimal reaction pH range was wilder and the thermal stability was better under 50 and 60 °C. Compared to the wild-type CGTase, the fusion enzyme showed a remarkable decrease in Km and a slight alteration in Vmax. The enhancement of soluble starch catalytic efficiency might be due to the changes of substrate binding ability in the critical substrate binding sites between the CBM and starch granule.
Assuntos
Geobacillus/enzimologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Amido/metabolismo , Bacillus/enzimologia , Bacillus/genética , Sítios de Ligação , Biocatálise , Carboidratos , Simulação por Computador , Ciclodextrinas/metabolismo , Geobacillus/genética , Glucosiltransferases/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oligossacarídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Amido/químicaRESUMO
Objective: This study was aimed to enhance the extracellular enzymes activities and soluble expression of CGTase from Geobacillus sp. B1 by directed evolution. Methods: A library of CGTase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved extracellular enzyme activities and soluble expression. After induction, expression and purification, the mutant enzyme was characterized. Results: After screening, two optimum mutants ds-6 and ep-9 with extracellular alpha-cyclization activity are respectively 1.72 times and 2.18 times of the original enzyme. The sequence of ep-9 cgt gene showed that three nucleotides substitution, G2005A, A2037G and T2081G were observed, and two of them caused amino acid changes. According to the 3D structure of Geobacillus sp. B1 cyclodextrin glucosetransferase mimicked by SWISS-MODEL Repository, two amino acid mutations were in rotating angle between beta angle and the random coil. The wild-type CGTase or ep-9 genes was ligated with pET-28 (a)-OmpA vector, and expressed in E. coli BL21 (DE3). After induced by lactose, CGTases were purified and characterized. The results showed that the specific ß-cyclization activity of the evolved CGTase was 1.31-fold than that of the wild-type CGTase, and the Km decreased from 4.3 to 3.74 g/L. The pH stability of the evolved CGTase was better than wild-type CGTase. Site-directed mutagenesis demonstrated the key to improve the soluble expression level and extracellular enzyme activity was G2005A. Conclusion: Directed evolution by error-prone PCR of Geobacillus sp. B1 CGTase gene is effective to improve extracellular enzyme activities and soluble expression, in particular mutation occurred in the G2005A.
Assuntos
Proteínas de Bactérias/genética , Geobacillus/enzimologia , Glucosiltransferases/genética , Reação em Cadeia da Polimerase/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada , Estabilidade Enzimática , Geobacillus/química , Geobacillus/genética , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , TemperaturaRESUMO
BACKGROUND: The EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased ß-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2. RESULTS: We found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and ß-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-ß-catenin upstream of PI3K and AKT. CONCLUSIONS: We conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of ß-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.