RESUMEN
B-cell lymphoma 2 (Bcl-2)-associated transcription factor 1 (Bclaf1) is known to be involved in diverse biological processes, but, to date, there has been no evidence for any functional role of Bclaf1 in hepatocellular carcinoma (HCC) progression. Here, we demonstrate that Bclaf1 is frequently up-regulated in HCC and that Bclaf1 up-regulation is associated with Edmondson grade, lower overall survival rates, and poor prognosis. Overexpression of Bclaf1 in HCC cell lines HepG2 and Huh7 promoted proliferation considerably, whereas Bclaf1 knockdown had the opposite effect. Xenograft tumors grown from Bclaf1 knockdown Huh7 cells had smaller tumor volumes than tumors grown from control cells. Furthermore, our study describes MYC proto-oncogene (c-Myc) as a downstream target of Bclaf1, given that Bclaf1 regulates c-MYC expression posttranscriptionally by its RS domain. To exert this function, Bclaf1 must interact with the molecular chaperone, heat shock protein 90 alpha (Hsp90α). In HCC tissue samples, Hsp90α levels were also increased significantly and Hsp90α-Bclaf1 interaction was enhanced. Bclaf1 interacts with the C-terminal domain of Hsp90α, and this interaction is disrupted by the C-terminal domain inhibitor, novobiocin (NB), resulting in proteasome-dependent degradation of Bclaf1. Moreover, NB-induced disruption of Hsp90α-Bclaf1 interaction dampened the production of mature c-MYC mRNA and attenuated tumor cell growth in vitro and in vivo. Conclusion: Our findings suggest that Bclaf1 affects HCC progression by manipulating c-MYC mRNA stability and that the Hsp90α/Bclaf1/c-Myc axis might be a potential target for therapeutic intervention in HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , China/epidemiología , Femenino , Genes myc , Proteínas HSP90 de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Masculino , Ratones Desnudos , Persona de Mediana Edad , Estabilidad Proteica , Proto-Oncogenes MasRESUMEN
Cadmium is a toxic heavy metal distributed broadly in the environment and manufactory industry. Long-term exposure to cadmium, considered as a risk for kidney injury, leads to chronic kidney disease eventually. Phospholipase D1 (PLD1) promotes cell proliferation and inhibits apoptosis, and might be involved in cadmium-induced kidney injury. In this study, we used miRNA microarray assays and bioinformatics analysis to identify miRNAs, which may regulate PLD1 expression and exert an impact on cadmium-induced kidney injury. MiR-122-5p and miR-326-3pï¼selected as candidates, were explored for their regulatory functions in kidney injury, using NRK-52E cells. Both of these two miRNAs exhibited higher expression in kidneys of SD rats after exposure to cadmium for 6 weeks. Cadmium treatment also increased miR-122-5p and miR-326-3p and decreased PLD1 in NRK-52E cells. Both of miR-122-5p and miR-326-3p could downregulate PLD1 expression through targeting its 3'UTR and enhance cadmium-induced apoptosis, while inhibiting either of these two miRNAs could reverse such effects. In conclusion, our results suggest that miR-122-5p and miR-326-3p might enhance cadmium-induced NRK-52E cell apoptosis through downregulating PLD1 expression.
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Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Células Epiteliales/efectos de los fármacos , MicroARNs/genética , Fosfolipasa D/genética , Regiones no Traducidas 3' , Animales , Apoptosis/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
This study was aimed to investigate the regulatory mechanism of heat shock protein 90 (Hsp90) on transcription factor EB (TFEB) during autophagy in liver cancer cells. Human hepatocellular carcinoma cell line HepG2 was treated with Hsp90 N- and C-terminal inhibitors (STA9090 and Novobiocin), respectively. Western blot and RT-PCR were used to detect the expression levels of TFEB and autophagy-related proteins. Chromatin immunoprecipitation (ChIP) assay was used to observe the ability of Hsp90α binding to the TFEB proximal promoter region. The double-luciferase gene reporter experiment was used to determine the activity of TFEB promoter. The results showed that hypoxia induced up-regulation of TFEB protein and mRNA expression levels in the HepG2 cells. The protein expression levels of TFEB, LC3 and P62 were down-regulated significantly by either STA9090 or Novobiocin, under both normoxic and hypoxic conditions. Transfection of Hsp90α-overexpressing plasmids up-regulated TFEB protein levels in either wild-type or Hsp90α knockout HepG2 cells. Hsp90 bound to the TFEB proximal promoter region and was involved in regulating TFEB transcriptional process. Whereas both STA9090 and Novobiocin inhibited Hsp90 to bind to the TFEB proximal promoter region, and decreased the activity of TFEB promoter. These results suggest that Hsp90 promotes TFEB transcription in human hepatocellular carcinoma cells by binding to the proximal promoter region, thereby up-regulating the expression levels of autophagy-related proteins.
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Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma Hepatocelular/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias Hepáticas/patología , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Regiones Promotoras GenéticasRESUMEN
Background: Heavy metal exposure is an important cause of reduced bone mineral density (BMD). Epidemiological studies focusing on the effects of mixed heavy metal exposure on BMD in middle-aged and older people are scarce. In single-metal studies, men and women have shown distinct responses of BMD to environmental metal exposure. This study therefore aimed to elucidate the association between mixed heavy metal exposure and BMD and to investigate whether it is sex-specific. Methods: Data from the 2017-2020 National Health and Nutrition Examination Survey were selected for this cross-sectional study. The study used three statistical methods, i.e., linear regression, Bayesian kernel machine regression (BKMR) modeling, and weighted quartiles (WQS) regression, to explore the association between the urinary concentrations of 11 metals (barium, cadmium, cobalt, cesium, manganese, molybdenum, lead, antimony, tin, thallium, and Tungsten), either individually or as a mixture, and total femoral BMD. Results: A total of 1,031 participants were included in this study. Femoral BMD was found to be higher in men than women. A significant negative correlation between the urinary concentrations of the 10 metals and femoral BMD was found in the overall cohort. Further gender sub-stratified analyses showed that in men, urinary metal concentrations were negatively correlated with femoral BMD, with cobalt and barium playing a significant and non-linear role in this effect. In women, although urinary metal concentrations negatively modulated femoral BMD, none of the correlations was statistically significant. Antimony showed sex-specific differences in its effect. Conclusion: The urinary concentrations of 10 mixed heavy metals were negatively correlated with femoral BMD in middle-aged and older participants, and this effect showed gender differences. These findings emphasize the differing role of mixed metal exposure in the process of BMD reduction between the sexes but require further validation by prospective studies.
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Densidad Ósea , Fémur , Metales Pesados , Encuestas Nutricionales , Humanos , Femenino , Masculino , Estudios Transversales , Anciano , Metales Pesados/orina , Persona de Mediana Edad , Factores Sexuales , Exposición a Riesgos Ambientales , Teorema de Bayes , Anciano de 80 o más AñosRESUMEN
Micro-LiNiCoMnO2 (MNCM), a cathode material with highest market share, has increasing demand with the growth of lithium battery industry. However, whether MNCM exposure brings adverse effects to workers remains unclear. This study aimed to explore the association between MNCM exposure with systemic inflammation and cardiac function. A cross-sectional study of 347 workers was undertaken from the MNCM production industry in Guangdong province, China in 2020. Metals in urine were measured using ICP-MS. The associations between metals, systemic inflammation, and cardiac function were appraised using a linear or logistic regression model. Bayesian kernel machine regression (BKMR) and generalized weighted quantile sum (gWQS) models were used to explore mixed metal exposures. The analysis of interaction and mediation was adopted to assess the role of inflammation in the relation between urinary metals and cardiac function. We observed that the levels of lithium (Li) and cobalt (Co) were positively associated with systemic inflammation and heart rate. The amount of Co contributed the highest weight on the increased systemic immune-inflammation index (SII) (59.8%), the system inflammation response index (SIRI) (44.3%), and heart rate (65.0%). Based on the mediation analysis, we estimated that SII mediated 32.3% and 20.9% of the associations between Li and Co with heart rate, and SIRI mediated 44.6% and 22.2% of the associations between Li and Co with heart rate, respectively. This study demonstrated for the first time that MNCM exposure increased the risk of workers' systemic inflammation and elevated heart rate, which were contributed by the excessive Li and Co exposure. Additionally, it indicates that systemic inflammation was a major mediator of the associations of Li and Co with cardiac function in MNCM production workers.
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Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Apoptosis , Novobiocina/farmacología , Neoplasias Hepáticas/genética , Ubiquitinación , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Heat shock protein 90 (Hsp90) has been an important therapeutic target for cancer therapy for decades. Unexpectedly, the monotherapy of N-terminal Hsp90 inhibitor STA9090 related clinical trials halted in phase III, and metastases were reported in animal models with the treatment of N-terminal Hsp90 inhibitors. Vacuolar protein sorting-associated protein 35 (VPS35) plays a vital role in endosome-derived EV (extracellular vesicle) traffic in neurodegeneration diseases, but no vps35 related EV were reported in tumors till now. Since tumor derived EVs contributes to metastasis and VPS35 is recently found to be involved in the invasion and metastasis of hepatocellular carcinoma (HCC), whether N-terminal Hsp90 inhibitor STA9090 induced EVs generation and the role of VPS35 in it were explored in this study. We found that N-terminal Hsp90 inhibitor STA9090 upregulated Bclaf1 and VPS35 levels, increased the secretion of EVs, and STA9090-induced-EVs promoted the invasion of HepG2 cells. As the clinical data suggested that the increased Bclaf1 and VPS35 levels correlated with increased metastasis and poorer prognosis in HCC, we focused on the Bclaf1-VPS35-EVs axis to further explore the mechanism of VPS35-related metastasis. The results demonstrated that Bclaf1 facilitated the transcription of VPS35 via bZIP domain, and knockdown of Bclaf1 or VPS35 alleviated pro-metastatic capability of STA9090-induced-EVs. All the results revealed the role of Bclaf1-VPS35-EVs axis on metastasis of HCC, and VPS35 knockdown decreased Hsp90 Inhibitor STA9090 induced extracellular vesicle release and metastasis, which provided a new combination therapeutic strategy to inhibit the metastasis of HCC caused by N-terminal Hsp90 inhibitor induced extracellular vesicles.
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We previously determined that specific microRNAs (miRNAs) are involved in renal pathophysiological occurrences induced by cadmium (Cd) in rats. This study expands our studies on miRNAs, determining their role in Cd-induced nephrotoxicity in occupational workers. We performed miRNA microarray analyses of blood and urine samples from patients diagnosed as occupational chronic Cd poisoning (OCCP) with abnormally elevated concentrations of urinary beta-2-microglobulin (U-ß2-MG), an indicator of tubular proteinuria. We also performed in vitro bioinformatics-based investigations of apoptosis-related genes targeted by miRNAs involved in the biological response to Cd exposure. We tested five differentially expressed miRNAs and determined a significant increase of sera miR-363-3p. Also, we determined that miR-363-3p increase is associated with phosphoinositide 3-kinase (PI3K) down-regulation and the suppressed proliferation and enhanced apoptosis of renal tubule epithelial cells. The obtained results suggest miR-363-3p involvement in the pathophysiology of Cd-induced renal injury in humans and maybe considered for possible interventional therapeutic strategies for Cd-associated kidney damage.
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Apoptosis/efectos de los fármacos , Cadmio/efectos adversos , Enfermedades Renales/inducido químicamente , Túbulos Renales Proximales/efectos de los fármacos , MicroARNs/metabolismo , Exposición Profesional/efectos adversos , Fosfatidilinositol 3-Quinasa/metabolismo , Adulto , Animales , Estudios de Casos y Controles , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Enfermedades Renales/enzimología , Enfermedades Renales/genética , Enfermedades Renales/patología , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/patología , Masculino , MicroARNs/genética , Persona de Mediana Edad , Salud Laboral , Fosfatidilinositol 3-Quinasa/genética , Ratas , Transducción de SeñalRESUMEN
As a conserved molecular chaperone, heat shock protein 90 (Hsp90) maintains the stability and homeostasis of oncoproteins and helps cancer cells survive. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a pivotal role in the non-homologous end joining pathway for DNA double-strand breaks (DSB) repair. Tumor cells contain higher levels of DNA-PKcs to survive by the hostile tumor microenvironment and various antitumor therapies. Here, we showed that increased levels of Hsp90α, Hsp90ß, and DNA-PKcs correlated with a poor overall survival in hepatocellular carcinoma (HCC). We revealed that Hsp90 N-terminal domain and C-terminal domain have different effects on DNA-PKcs protein and mRNA levels. The stability of DNA-PKcs depended on Hsp90α N-terminal nucleotide binding domain. Transcription factor SP1 regulates the transcription of PRKDC (gene name of DNA-PKcs) and is a client protein of Hsp90. Inhibition of Hsp90 N-terminal by STA9090 decreased the location of Hsp90α in nucleus, Hsp90α-SP1 interaction, SP1 level, and the binding of Hsp90α/SP1 at the proximal promoter region of PRKDC Because hyperthermia induces DSBs with increases level of DNA-PKcs, combined STA9090 treatment with hyperthermia effectively delayed the tumor growth and significantly decreased DNA-PKcs levels in xenografts model. Consistently, inhibition of Hsp90 increased the number of heat shock-induced γ-H2AX foci and delayed the repair of DSBs. Altogether, our results suggest that Hsp90 inhibitor STA9090 decreases DNA-PKcs protein stability and PRKDC mRNA level, which provide a theoretical basis for the promising combination therapy of hyperthermia and Hsp90 inhibitor in HCC.
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Carcinoma Hepatocelular/tratamiento farmacológico , Daño del ADN , Proteína Quinasa Activada por ADN/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Hipertermia Inducida/efectos adversos , ARN Mensajero/genética , Animales , Apoptosis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Reparación del ADN , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Pronóstico , Estabilidad Proteica , Tasa de Supervivencia , Triazoles , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Exposure to cadmium (Cd), a common heavy metal used in industry, can result in long-term chronic toxicity. It has been well characterized that kidneys are the main organs that are targeted by toxicity, which can cause apoptosis, necrosis, and atrophy of renal tubular epithelial cells. However, the molecular mechanisms associated with Cd toxicity remain unclear. In this study, the expression of renal proteins in Sprague-Dawley rats exposed to chronic Cd was analyzed with iTRAQ proteomics. Bioinformatics analysis indicated that phospholipase D1 (PLD1) was significantly underexpressed and may correlate strongly with Cd-induced chronic kidney impairment. Previous studies have shown that PLD1 promotes cell proliferation and inhibits apoptosis, indicating that PLD1 may be implicated in the pathogenesis of kidney injury induced by Cd. Studies in vivo and in vitro all demonstrate that the mRNA and protein levels of PLD1 decrease significantly both in kidney tissue and in proximal tubular cell lines exposed to Cd. Overexpression of PLD1 and its downstream product PA could ameliorate Cd-induced apoptosis. Moreover, we identified that miR-122-5p was a regulatory miRNA of PLD1. miR-122-5p was overexpressed after Cd exposure and promoted cell apoptosis by downregulating PLD1 through binding the 3'UTR of the locus at 1761-1784 nt. In conclusion, our results indicated that PLD1 and its downstream PA were strongly implicated in Cd-induced chronic kidney impairment and could be a novel player in the defense against Cd-induced nephrotoxicity.
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Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Riñón/efectos de los fármacos , Fosfolipasa D/genética , Insuficiencia Renal/tratamiento farmacológico , Regiones no Traducidas 3' , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica , Riñón/citología , Riñón/metabolismo , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Fosfolipasa D/metabolismo , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/etiologíaRESUMEN
Cadmium is a common environmental and occupational pollutant and can produce toxic effects in a range of organs, especially in kidneys, after long-term exposure. MicroRNAs are ideal candidate biomarkers for various types of disorders, including renal diseases. In this study, we profiled the global miRNA expressions in rat kidneys using miRNA microarrays and found a collection of differentially expressed miRNAs induced by cadmium exposure. Among all of the candidate miRNAs, we identified miR-122-5p and miR-326-3p as early biomarkers for cadmium-induced nephrotoxicity. The two-miRNA signature was validated by quantitative real-time PCR in HK-2 and NRK-52E cells, rat kidney, serum and urine samples, and serum of an occupational population. Our results indicate that miR-122-5p and miR-326-3p may be potential biomarkers for cadmium exposure.
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Cadmio/toxicidad , Biomarcadores Ambientales/genética , Exposición a Riesgos Ambientales/análisis , Riñón/efectos de los fármacos , MicroARNs/análisis , Adulto , Animales , Estudios de Casos y Controles , Contaminantes Ambientales/toxicidad , Femenino , Humanos , Riñón/metabolismo , Masculino , MicroARNs/sangre , Exposición Profesional/análisis , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , TranscriptomaRESUMEN
The development of hepatocellular carcinomas (HCC) depends on their local microenvironment and the induction of neovascularization is a decisive step in tumor progression, since the growth of solid tumors is limited by nutrient and oxygen supply. Hypoxia is the critical factor that induces transcription of the hypoxia inducible factor-1α (HIF-1α) encoding gene HIF1A and HIF-1α protein accumulation to promote angiogenesis. However, the basis for the transcriptional regulation of HIF1A expression in HCC is still unclear. Here, we show that Bclaf1 levels are highly correlated with HIF-1α levels in HCC tissues, and that knockdown of Bclaf1 in HCC cell lines significantly reduces hypoxia-induced HIF1A expression. Furthermore, we found that Bclaf1 promotes HIF1A transcription via its bZIP domain, leading subsequently to increased transcription of the HIF-1α downstream targets VEGFA, TGFB, and EPO that in turn promote HCC-associated angiogenesis and thus survival and thriving of HCC cells. Moreover, we demonstrate that HIF-1α levels and microvessel density decrease after the shRNA-mediated Bclaf1 knockdown in xenograft tumors. Finally, we found that Bclaf1 levels increase in hypoxia in a HIF-1α dependent manner. Therefore, our study identifies Bclaf1 as a novel positive regulator of HIF-1α in the hypoxic microenvironment, providing new incentives for promoting Bcalf1 as a potential therapeutic target for an anti-HCC strategy.