RESUMEN
Hexavalent chromium [Cr (VI)] is a well-established carcinogen. Cr (VI)-treated cells are phenotypically characterized by aberrant levels of growth and migration. Curcumin, a polyphenolic compound from the plant turmeric, has been found to possess antiproliferation, anti-inflammation, and antioxidant properties. In this study, the effect of curcumin on Cr (VI)-induced cell survival and migration and the underlying mechanism were investigated. Cell viability assay on A549 and human embryonic lung fibroblast cells showed that curcumin at the concentration of 10 µM could significantly attenuate Cr (VI)-induced viability in both cell lines. Following Western blot assay and metabolomics assays, cotreatment with curcumin and Cr (VI) resulted in the suppression of Cr (VI)-induced glycolysis-, autophagy-, and migration-related proteins. Meanwhile, curcumin increased Cr (VI)-reduced oxidative phosphorylation (OXPHOS)-related proteins, COXIV and ND1. Moreover, curcumin suppressed Cr (VI)-induced mitochondrial dysfunction, mitochondrial mass decrease, and mitochondrial membrane potential loss. Treatment with curcumin for 24 h significantly attenuated pcATG4B-induced autophagy and the subsequent expression of glucose transporter 1, hexokinase II, and pyruvate kinase M2. Wound healing and transwell assay demonstrated that curcumin reduced Cr (VI)-induced cell migration. Taken together, these results showed that curcumin was able to attenuate Cr (VI)-induced cell viability and migration by targeting autophagy-dependent reprogrammed metabolism from OXPHOS to glycolysis.
Asunto(s)
Curcumina , Humanos , Curcumina/farmacología , Autofagia , Potencial de la Membrana Mitocondrial , Mitocondrias , Ciclo CelularRESUMEN
Chromium (Cr) compounds are markedly toxic and carcinogenic. Previously, we found that Cr (VI) induced autophagy in A549 cells. Here, the effect of mitochondrial dysfunction and endoplasmic reticulum (ER) stress on inducing mitophagy was investigated in both A549 and H1299 cells. Exposure to Cr (VI) for 6 h significantly enhanced reactive oxygen species (ROS) production and reduced mitochondrial membrane potential (MMP). Transmission electron microscopy showed that Cr (VI) induced mitochondrial morphological changes, such as, mitochondrial swelling and vacuolization. The elevated expression of GRP78 and p-PERK suggested that Cr (VI) resulted in ER stress. Both mitochondrial dysfunction and ER stress played an important role in Cr (VI)-induced mitophagy, as the mitochondrial function inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) induced PINK1 and PARK2 and increased the expression of GRP78 and p-PERK while the levels of Cr (VI)-induced PINK1, PARK2, LC3-II were reduced after ER stress inhibitor, phenylbutyric acid (4PBA) pretreatment. When A549 cells were treated with CCCP and 4-PBA simultaneously, CCCP-induced expressions of PINK1, PARK2 and LC3-II decreased significantly compared with that of only CCCP-treated cells, indicating that there was a crosstalk between mitochondria and ER in inducing mitophagy. Additionally, the crosstalk between mitochondrial dysfunction and ER stress modulated the expression of Cr (VI)-induced ATF4, which resulted in mitophagy. Collectively, our data demonstrated that Cr (VI)-induced mitophagy mediated by ATF4 via the crosstalk between ER stress and mitochondrial dysfunction.
Asunto(s)
Estrés del Retículo Endoplásmico , Mitofagia , Cromo/toxicidad , Mitocondrias , Especies Reactivas de OxígenoRESUMEN
Cr (VI) is mutagenic and carcinogenic, but the mechanism is unclear. In this study, the involvement of high mobility group A2 (HMGA2) in Cr (VI)-induced autophagy was investigated. Cr (VI) treatment induced formation of autophagosomes, increased expression of LC3II, Atg12-Atg5, Atg4, Atg10, HMGA1 and HMGA2 proteins, and decreased the expression of p62 in A549 cells. Silencing of HMGA2 gene by siRNA blocked Cr (VI)-induced formation of autophagosomes, expression of LC3II, Atg12-Atg5, Atg10 and reduction of p62. Overexpression of HMGA2 in HEK 293 and HeLa cells could induce the expression of LC3II, Atg12-Atg5 and Atg10, and decrease the expression of p62. Although the protein level of Atg12-Atg5 conjugation changed after Cr (VI) treatment, silencing of HMGA2 and overexpression of HMGA2, both the proteins and mRNA levels of Atg12 and Atg5 were not changed significantly. ChIP assay demonstrated that HMGA2 protein directly bound to the promoter sequence of Atg10 gene, which modulated the conjugation of Atg12-Atg5. Interestingly, 3-MA markedly prevented Cr (VI)-induced cell growth of A549 cells. Our further in vivo study confirmed that the expression of HMGA1, HMGA2, LC3II, Atg12-Atg5, Atg4, Atg5, Atg7, Atg10, Atg12, Beclin 1 were increased and p62 was reduced in lung tissues of Cr (VI)-treated BALB/c mice. Combining, our data demonstrated that HMGA2 plays an important role in Cr (VI)-induced autophagy and the mechanism underlies Atg12-Atg5 conjugation modulated by HMGA2-dependent transcriptional regulation of Atg10. This suggests that HMGA2 might be an important biomarker in Cr (VI)-induced autophagy, cell-growth or other toxicities.
Asunto(s)
Autofagia/efectos de los fármacos , Autofagia/fisiología , Cromo/toxicidad , Proteína HMGA2/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause-and-effect between apoptosis of pancreatic ß-cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic-induced pancreatic ß-cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 µM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH4 Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V-FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic ß-cell apoptosis through activation of the lysosome-mitochondrial pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsénico/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Lisosomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cloruro de Amonio/farmacología , Animales , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Citocromos c/metabolismo , Citometría de Flujo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We hypothesize that citreoviridin (CIT) induces DNA damage in human liver-derived HepG2 cells through an oxidative stress mechanism and that N-acetyl-l-cysteine (NAC) protects against CIT-induced DNA damage in HepG2 cells. CIT-induced DNA damage in HepG2 cells was evaluated by alkaline single-cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT-induced ROS formation and CIT-induced DNA damage were evaluated in HepG2 cells. A significant dose-dependent increment in DNA migration was observed at tested concentrations (2.50-10.00 µM) of CIT. The levels of ROS, 8-OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT-induced ROS formation and CIT-induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT-induced DNA damage in HepG2 cells.
Asunto(s)
Aurovertinas/toxicidad , Daño del ADN , Micotoxinas/toxicidad , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Acetilcisteína/farmacología , Desoxiguanosina/análogos & derivados , Glutatión/metabolismo , Células Hep G2 , Humanos , Lisosomas/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The objective of the present study was to investigate the effect of the fucoxanthin (FUCO) alone and in combination with glucosamine hydrochloride (GAH) on carrageenan/kaolin-induced inflammatory arthritis model in rats and to explore its underlying mechanisms. Joint swelling, muscle weight ratio (%), histopathological examination and scoring, and proteoglycan degradation were examined. Pro-inflammatory interleukin (IL-1ß) and tumor necrosis (TNF-α) levels, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase(iNOS) protein expression and nitric oxide (NO) level in knee synovial tissue extract were analyzed using enzyme-linked immunosorbent assay, western blotting analysis, and Griess reagent assay, respectively. FUCO and FUCO + GAH not only may significantly reduce degrees of knee joint swelling and prevent against muscle atrophy, but also may significantly attenuate inflammation in synovial tissue, cartilage erosion, and proteoglycan loss. The efficacies of FUCO + GAH were stronger than that of GAH or FUCO. FUCO alone and FUCO + GAH can significantly inhibit upregulation of COX-2 and iNOS protein expressions, decrease of IL-1ß and TNF-α levels, and reduce NO production in knee synovial tissue extract. These results indicated that FUCO is an effective anti-arthritis agent through an antiinflammation mechanism. FUCO may enhance therapeutic effect of GAH on rat arthritis through mechanism of antiinflammation.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Glucosamina/farmacología , Xantófilas/farmacología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/metabolismo , Carragenina , Ciclooxigenasa 2/metabolismo , Quimioterapia Combinada , Ensayo de Inmunoadsorción Enzimática , Glucosamina/análogos & derivados , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Caolín , Articulación de la Rodilla/patología , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEA) is a hepatotoxic mycotoxin with estrogenic and anabolic activity found in cereal grains worldwide. ZEA affects hematological and immunological parameters in humans and rodents. The compound can induce cell death, cause lipid peroxidation, inhibit protein and DNA synthesis, and exert genotoxic effects. ZEA may cause increased phagolysosomal fragility in the kidney. Our research showed that exposure of human embryonic kidney (HEK293) cells to ZEA (10 or 20µM) resulted in a concentration-dependent increase in DNA strand breaks measured with the comet assay. Damage was reduced in cells pretreated with NH4Cl, pepstatin A, or desipramine for 1h. Production of reactive oxygen species (ROS) was increased in cells exposed to ZEA, but DNA strand break induction could not be inhibited by the antioxidant hydroxytyrosol (HT). These results suggest that oxidative stress does not play a key role in DNA strand breaks induced by ZEA, that lysosomal injury precedes DNA strand breaks, and that the lysosome may be a primary target for ZEA in HEK293 cells.
Asunto(s)
Daño del ADN/efectos de los fármacos , Estrógenos no Esteroides/farmacología , Lisosomas/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Zearalenona/farmacología , Catepsina D/farmacología , Ensayo Cometa , Células HEK293 , Humanos , Lisosomas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Olive leaf extract (OLE) has antioxidant and antiinflammatory actions. However, the role of OLE in mechanical inflammatory arthritis (osteoarthritis, OA) is unclear. This study investigated the effect of OLE on the development of kaolin and carrageenan-induced arthritis, a murine model of OA. Administration of OLE significantly ameliorated paw swelling, the paw Evans blue content and the histopathological scores. In the human monocyte cell line, THP-1, the OLE reduced the LPS-induced TNF-α production and was dose dependent. Croton oil-induced ear edema in mice also revealed that treatment with OLE suppressed ear edema, myeloperoxidase (MPO) production and was dose dependent. These results indicated that OLE is an effective antiarthritis agent through an antiinflammation mechanism. Also OLE may be beneficial for the treatment of OA in humans.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Carragenina/efectos adversos , Caolín/efectos adversos , Olea/química , Extractos Vegetales/uso terapéutico , Hojas de la Planta/química , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Artritis Experimental/inducido químicamente , Artritis Experimental/patología , Línea Celular Tumoral , Aceite de Crotón/efectos adversos , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Edema/tratamiento farmacológico , Edema/patología , Azul de Evans/química , Humanos , Inflamación/tratamiento farmacológico , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos BALB C , Peroxidasa/química , Fitoterapia , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
6-Gingerol, a major phenolic compound derived from ginger, has been known to possess anticarcinogenic activities. However, the mechanisms are not well understood. In our previous study, it was demonstrated that lysosome and mitochondria may be the primary targets for 6-gingerol in HepG2 cells. Therefore, the aim was to evaluate lysosome-mitochondria cross-signaling in 6-gingerol-induced apoptosis. Apoptosis was detected by Hoechst 33342 and TUNEL assay after 24 h treatment, and the destabilization of lysosome and mitochondria were early upstream initiating events. This study showed that cathepsin D played a crucial role in the process of apoptosis. The release of cathepsin D to the cytosol appeared to be an early event that preceded the release of cytochrome c from mitochondria. Moreover, inhibition of cathepsin D activity resulted in suppressed release of cytochrome c. To further determine the involvement of oxidative stress in 6-gingerol-induced apoptosis, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined. Taken together, these results suggest that cathepsin D may be a positive mediator of 6-gingerol induced apoptosis in HepG2 cells, acting upstream of cytochrome c release, and the apoptosis may be associated with oxidative stress.
Asunto(s)
Apoptosis , Catecoles/farmacología , Catepsina D/metabolismo , Alcoholes Grasos/farmacología , Neoplasias Hepáticas/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Citocromos c/metabolismo , Glutatión/metabolismo , Células Hep G2 , Humanos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Ortho-phenylphenol (OPP) has been found to cause carcinomas in the urinary tract of rats. Since OPP is a potent genotoxic compound, and used as fungicides and antibacterial agents in fruits and fruit products, search for newer, better agents for protection against toxicity of OPP is required. In this study, the chemoprotective effect of hydroxytyrosol (HT) against OPP-induced DNA damage in HepG2 cells was investigated. Comet assay was used to detect the DNA damage induced by OPP. To elucidate the possible mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, intracellular generation of reactive oxygen species (ROS), and reduced glutathione (GSH). Results showed that HT significantly reduced the DNA strand breaks caused by OPP. Moreover, HT effectively suppressed OPP-induced ROS formation, and increased the GSH level. Lysosomal membrane and mitochondrial membrane were also protected when cells were pretreated with HT. These results suggested that the disruption of lysosomal membrane integrity and the oxidative stress, leading to DNA fragmentation, may be the mechanism of DNA damage induced by OPP. The antioxidant activity of HT may play an important part in attenuating the DNA damage of OPP.
Asunto(s)
Compuestos de Bifenilo/toxicidad , Daño del ADN/efectos de los fármacos , Animales , Línea Celular Tumoral , Ensayo Cometa , Glutatión/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: To study mechanism of the apoptosis of rat pancreas islet ß cell strain (INS-1 cells) induced by sodium arsenite. METHODS: INS-1 cells were exposed to sodium arsenite at the different concentrations. MTT assay was used to detect the viability of INS-1 cells. The potentials on mitochondrial membrane and lysosome membrane of INS-1 cells were determined with the fluorescence spectrophotometer. The apoptotic levels of INS-1 cells exposed to sodium arsenite were observed by a fluorescence microscope and flow cytometry. RESULTS: After exposure to sodium arsenite, the viability of INS-1 cells significantly decreased with the doses of sodium arsenite. At 24 h after exposure, the OD values of the mitochondrial membrane potentials declined observably with the doses of sodium arsenite (P < 0.01). At 48 h after exposure, the OD values of the lysosome membrane potentials significantly increased with the doses of sodium arsenite (P < 0.01). At 72 h after exposure, the apoptotic cells were observed under a fluorescence microscope and enhanced with the doses of sodium arsenite. The apoptosis cells with light blue, karyopyknosis, karyorrhexis, apoptotic body and chromatin concentration appeared. The results detected with flow cytometry indicated that after exposure, the apoptotic INS-1E cells significantly increased with the doses of sodium arsenite. CONCLUSIONS: The sodium arsenite can induce the apoptosis of INS-1 cells through the mitochondria-lysosome pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsenitos/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Compuestos de Sodio/toxicidad , Animales , Células Cultivadas , Lisosomas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , RatasRESUMEN
ß-Elemene is a broad-spectrum antitumor agent. In China, several studies have indicated that ß-elemene enhances the cytotoxic effect of radiation in vitro and in vivo. In this study, the alkaline comet assay and neutral comet assay were used to measure both DNA strand breaks and DNA repair activity in A549 cells exposed to ß-elemene, irradiation or combination treatment. The overall object of the study was to test whether ß-elemene radiosensitization is associated with an enhancement in radiation-induced DNA damage or with a decrease in the repair of radiation-induced damage. The results revealed high levels of DNA single strand breaks (SSB) and double strand breaks (DSB) in A549 cells after exposure to the combination of ß-elemene and irradiation. To assess SSB and DSB repair, alkaline comet assay and neutral comet assay were performed at 24 h postirradiation. The damage induced by the combination of ß-elemene and irradiation was repaired at a slower rate. These findings suggest that ß-elemene can enhance A549 cell radiosensitivity through the enhancement of DNA damage and the suppression of DNA repair.
Asunto(s)
Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neoplasias Pulmonares/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Sesquiterpenos/farmacología , Línea Celular Tumoral , Ensayo Cometa , HumanosRESUMEN
Patulin (PAT) is a mycotoxin produced by several Penicillium, Aspergillus and Byssochlamys species. Since PAT is a potent genotoxic compound, and PAT contamination is common in fruits and fruit products, the search for newer, better agents for protection against genotoxicity of PAT is required. In this study, the chemoprotective effect of 6-gingerol against PAT-induced genotoxicity in HepG2 cells was investigated. The comet assay and micronucleus test (MNT) were used to monitor genotoxic effects. To further elucidate the underlying mechanisms, the intracellular generation of reactive oxygen species (ROS) and level of reduced glutathione (GSH) were tested. In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The results showed that 6-gingerol significantly reduced the DNA strand breaks and micronuclei formation caused by PAT. Moreover, 6-gingerol effectively suppressed PAT-induced intracellular ROS formation and 8-OHdG level. The GSH depletion induced by PAT in HepG2 cells was also attenuated by 6-gingerol pretreatment. These findings suggest that 6-gingerol has a strong protective ability against the genotoxicity caused by PAT, and the antioxidant activity of 6-gingerol may play an important part in attenuating the genotoxicity of PAT.
Asunto(s)
Catecoles/farmacología , Roturas del ADN/efectos de los fármacos , Alcoholes Grasos/farmacología , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Mutágenos/toxicidad , Patulina/toxicidad , Extractos Vegetales/farmacología , Zingiber officinale/química , Antioxidantes/farmacología , Microbiología de Alimentos , Glutatión/metabolismo , Células Hep G2 , Humanos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , RizomaRESUMEN
OBJECTIVES: We evaluated the role of oxidative stress in Sudan IV-induced DNA damage, using human liver-derived HepG2 cells. METHODS: The DNA damaging effects of Sudan IV in HepG2 cells were evaluated by alkaline single cell gel electrophoresis assay and micronucleus test (MNT). To clarify the underlying mechanisms, we monitored the intracellular generation of reactive oxygen species (ROS) by 2, 7-dichlorofluorescein diacetate assay and the level of oxidative DNA damage by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG). Furthermore, the intracellular glutathione (GSH) level was moderated by pretreatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor. RESULTS: A significant dose-dependent increment in DNA migration was detected at all tested concentrations (25-100 µM) of Sudan IV. And in the MNT, a significant increase of the frequency of micronuclei was found at higher tested concentrations (50-100 µM). The data suggested that Sudan IV caused DNA strand breaks and chromosome breaks. In addition, significantly increased levels of ROS, 8-OHdG formation were observed in HepG2 cells. It was also found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to Sudan IV-induced DNA damage. CONCLUSIONS: Based on these data we believe that Sudan IV exerts toxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.
Asunto(s)
Compuestos Azo/toxicidad , Colorantes/toxicidad , Citotoxinas/toxicidad , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Células Hep G2 , Humanos , Hígado/metabolismo , Pruebas de Micronúcleos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
High mobility group A2 (HMGA2) is closely related to the occurrence, development and prognosis of tumors. But the mechanism is unclear. Metabolic reprogramming is a dominant way to meet anabolic and energy requirements of tumor cells for their survival, growth and proliferation. Here, we investigated the role of metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis mediated by HMGA2/autophagy axis in cadmium (Cd, CdCl2)-induced migration. First, we found that Cd induced glycolysis and reduced OXPHOS in vivo (0.5 and 1 mg/kg, i.p. or 0.8 and 1.6 µM, i.t.) and in vitro (2 µM in A549 cells and 0.05 µM in HELF cells). Then, genetic knockdown of HMGA2 restored Cd-reduced mitochondrial mass and OXPHOS and inhibited Cd-increased glycolysis, indicating that HMGA2 was involved in Cd-induced metabolic reprogramming. 2-Deoxy-d-glucose (2DG, 5 mM), the inhibitor of glycolysis decreased Cd/HMGA2-induced cell migration and restored Cd/HMGA2-decreased OXPHOS and mitochondrial mass. Inhibition of autophagy by 3-Methyladenine (3MA, 3 mM) elucidated an essential role of autophagy in HMGA2-induced glycolysis, migration, and HMGA2-reduced OXPHOS. Overall, our study demonstrated that autophagy was required for HMGA2-mediated metabolic reprogramming, which was critical for Cd-induced migration. Targeting HMGA2 and autophagy-dependent reprogrammed metabolism may be an effective way to inhibit Cd-induced cell migration.
Asunto(s)
Autofagia/efectos de los fármacos , Cadmio/toxicidad , Movimiento Celular/efectos de los fármacos , Proteína HMGA2/genética , Fosforilación Oxidativa/efectos de los fármacos , Células A549 , Animales , Cadmio/administración & dosificación , Línea Celular , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Glucólisis/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacosRESUMEN
The chemoprotective effect of hydroxytyrosol (HT) against UVB-induced DNA damage was investigated in a human skin keratinocyte cell line, HaCaT. The comet assay was used to monitor DNA strand breaks. Intracellular reactive oxygen species (ROS) formation was measured by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA). The levels of oxidatively generated damage to DNA were estimated by immunocytochemistry analysis of 8-hydroxy-2'-deoxyguanosine (8-OHdG). The protein expression of p53 and NF-kappaB was estimated by western blotting. The results showed that HT significantly reduced the DNA strand breaks caused by UVB. It was also found that HT reduced intracellular ROS formation and 8-OHdG level caused by UVB. Furthermore, HT attenuated the expression of p53 and NF-kappaB in a concentration-dependent manner. These results strongly suggest that HT has a significant protective ability against UVB-induced DNA damage and that oxidative stress plays an important part in it.
Asunto(s)
Daño del ADN , Estrés Oxidativo/efectos de los fármacos , Alcohol Feniletílico/análogos & derivados , Rayos Ultravioleta , 8-Hidroxi-2'-Desoxicoguanosina , Línea Celular , Ensayo Cometa , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Humanos , FN-kappa B/metabolismo , Alcohol Feniletílico/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cadmium (Cd) is a pervasive harmful metal in the environment. It is a well-known inducer of tumorigenesis, but its mechanism is still unclear. We have previously reported that Cd-induced autophagy was apoptosis-dependent and prevents apoptotic cell death to ensure the growth of A549 cells. In this study, the mechanism was further investigated. Cd treatment increased glucose uptake and lactate release significantly. Meanwhile, the protein level of GLUT1ï¼HKIIï¼PKM2 and LDHA increased in a time-dependent manner, indicating that Cd induced aerobic glycolysis in A549 and HELF cells. The inhibitors of autophagy, 3MA, and CQ, repressed Cd-induced glycolysis-related proteins, indicating that autophagy was involved in Cd-induced glycolysis in A549 and HELF cells. Knockdown of ATG4B or ATG5 by siATG4B and siATG5 decreased Cd-induced glycolysis, while overexpression of ATG4B enhanced glycolysis. These results demonstrated that Cd-induced glycolysis was autophagy-dependent. Then, glycolysis inhibitor, 2DG and siPKM2 could inhibit Cd-induced cell viability and cell cycle progression compared to only Cd treatment, indicating that glycolysis played an important role in Cd-induced cell growth. Finally, co-treatment of transfection of ATG4B-DNA plasmids with 2DG or siPKM2 further demonstrated that the autophagy-glycolysis axis played an important role in Cd-induced cell cycle progression. Taken together, our results suggested that Cd-induced glycolysis is autophagy-dependent and the autophagy-glycolysis axis underlies the mechanism of Cd-induced cell growth in A549 and HELF cells.
Asunto(s)
Autofagia/efectos de los fármacos , Cadmio/toxicidad , Proliferación Celular/efectos de los fármacos , Glucólisis/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismoRESUMEN
Chromium (Cr) (VI) is a proven toxin, mutagen and carcinogen. Here, the role of high mobility group A2 (HMGA2) mediating Cr (VI)-induced mitophagy was investigated. Cr (VI)-treatment caused the formation of double membrane autophagic vesicles (AVs) engulfing mitochondria and increased the expression of PINK1, PARK2, LC3 as well as HMGA2 particularly in mitochondria in A549 cells. Silencing of HMGA2 by siRNA decreased expression of PINK1, PARK2 and LC3 II especially in mitochondria, while over-expression of HMGA2 increased the expression of them in A549 cells. It indicated that HMGA2 played a critical role in Cr (VI)-induced mitophagy. Most importantly, the results of co-immunoprecipitation showed for the first time that HMGA2 could bind to PARK2 in mitochondria to activate the mitophagy pathway. In BALB/c mice, Cr (VI) increased the expression of PINK1 and PARK2 in lung tissues. Furthermore, over-expression of HMGA2 in BALB/c mice by transfection of plasmid HMGA2 significantly increased the levels of PINK1, PARK2 and LC3 II in lung tissues. Collectively, our data demonstrated that HMGA2 plays an important role in Cr (VI)-induced mitophagy through direct interaction with PARK2 in A549 cells and lung tissue.
Asunto(s)
Cromo/toxicidad , Contaminantes Ambientales/toxicidad , Proteína HMGA2/metabolismo , Pulmón/efectos de los fármacos , Mitofagia/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Células A549 , Animales , Expresión Génica/efectos de los fármacos , Células HEK293 , Proteína HMGA2/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Transporte de Proteínas , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Chromium (Cr) (VI) compounds are known to be serious toxic and carcinogenic, but the mechanism is not clear. In our previous study, we found that Cr (VI)-induced ER stress plays an important role in the crosstalk between apoptosis and autophagy, while autophagy was apoptosis-dependent and subsequently prevents apoptosis cell death to keep A549 cells resistant to Cr (VI)-induced toxicity. In this study, we found that Cr (VI) could induce aerobic glycolysis in A549 cells. Both ER stress inhibitor, phenylbutyric acid (4-PBA) and the inhibitor of autophagy, 3-MA, repressed Cr (VI)-induced glycolysis, indicating that both ER stress and autophagy were involved in Cr (VI)-induced glycolysis in A549 cells. Co-treatment of the inhibitor of aerobic glycolysis, 2-DG and Cr (VI) for 24 h increased Cr (VI)-induced cleaved caspase-3, caspase-9 and the number of apoptotic cells, demonstrating that aerobic glycolysis played an important role in attenuating Cr (VI)-induced apoptosis. Furthermore, knockdown of ATF4 by siATF4 significantly decreased Cr (VI)-induced aerobic glycolysis and apoptosis, suggesting that ATF4 was involved in Cr (VI)-induced aerobic glycolysis and its effect of attenuating apoptosis in A549 cells. Taken together, our results demonstrated that autophagy-dependent glycolysis played a role in attenuating Cr (VI)-induced apoptosis. ER stress was involved in facilitating glycolysis, whose induction was mediated by ATF4. These findings open a window for the development of therapeutic interventions to prevent Cr (VI)-induced toxicity.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Cromo/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Glucólisis/efectos de los fármacos , Células A549 , Factor de Transcripción Activador 4/genética , Apoptosis/genética , Autofagia/genética , Estrés del Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Glucólisis/genética , HumanosRESUMEN
Patulin (PAT), a mycotoxin produced by certain species of Penicillium, Aspergillus and Byssochlamys, is mainly found in ripe apple and apple products. In our present study, a significant increase of the micronuclei frequency induced by PAT was found in human hepatoma HepG2 cells. To elucidate the role of glutathione (GSH) in the effect, the intracellular GSH level was modulated by pre-treatment with buthionine-(S, R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by pre-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PAT-induced micronuclei frequencies and that when the intracellular GSH content was elevated by NAC, the chromosome damage induced by PAT was significantly prevented in our test concentrations (0.19-0.75 microM). These results indicate that GSH play an important role in cellular defense against PAT-induced genotoxicity.