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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445344

RESUMO

Thyroid hormones, including 3,5,3'-triiodothyronine (T3), cause a wide spectrum of genomic effects on cellular metabolism and bioenergetic regulation in various tissues. The non-genomic actions of T3 have been reported but are not yet completely understood. Acute T3 treatment significantly enhanced basal, maximal, ATP-linked, and proton-leak oxygen consumption rates (OCRs) of primary differentiated mouse brown adipocytes accompanied with increased protein abundances of uncoupling protein 1 (UCP1) and mitochondrial Ca2+ uniporter (MCU). T3 treatment depolarized the resting mitochondrial membrane potential (Ψm) but augmented oligomycin-induced hyperpolarization in brown adipocytes. Protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were activated by T3, leading to the inhibition of autophagic degradation. Rapamycin, as an mTOR inhibitor, blocked T3-induced autophagic suppression and UCP1 upregulation. T3 increases intracellular Ca2+ concentration ([Ca2+]i) in brown adipocytes. Most of the T3 effects, including mTOR activation, UCP1 upregulation, and OCR increase, were abrogated by intracellular Ca2+ chelation with BAPTA-AM. Calmodulin inhibition with W7 or knockdown of MCU dampened T3-induced mitochondrial activation. Furthermore, edelfosine, a phospholipase C (PLC) inhibitor, prevented T3 from acting on [Ca2+]i, UCP1 abundance, Ψm, and OCR. We suggest that short-term exposure of T3 induces UCP1 upregulation and mitochondrial activation due to PLC-mediated [Ca2+]i elevation in brown adipocytes.


Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Cálcio/metabolismo , Mitocôndrias/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Tecido Adiposo Marrom/metabolismo , Animais , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos
2.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445394

RESUMO

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.


Assuntos
Cálcio/metabolismo , Canabidiol/farmacologia , Ciclofilina D/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Ciclofilina D/química , Sinergismo Farmacológico , Homeostase/efeitos dos fármacos , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Conformação Proteica
3.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443472

RESUMO

Feruloylacetone (FER) is a natural degradant of curcumin after heating, which structurally reserves some functional groups of curcumin. It is not as widely discussed as its original counterpart has been previously; and in this study, its anticancer efficacy is investigated. This study focuses on the suppressive effect of FER on colon cancer, as the efficacious effect of curcumin on this typical cancer type has been well evidenced. In addition, demethoxy-feruloylacetone (DFER) was applied to compare the effect that might be brought on by the structural differences of the methoxy group. It was revealed that both FER and DFER inhibited the proliferation of HCT116 cells, possibly via suppression of the phosphorylated mTOR/STAT3 pathway. Notably, FER could significantly repress both the STAT3 phosphorylation and protein levels. Furthermore, both samples showed capability of arresting HCT116 cells at the G2/M phase via the activation of p53/p21 and the upregulation of cyclin-B. In addition, ROS elevation and changes in mitochondrial membrane potential were revealed, as indicated by p-atm elevation. The apoptotic rate rose to 36.9 and 32.2% after being treated by FER and DFER, respectively. In summary, both compounds exhibited an anticancer effect, and FER showed a greater proapoptotic effect, possibly due to the presence of the methoxy group on the aromatic ring.


Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Curcumina/farmacologia , Mitocôndrias/efeitos dos fármacos , Estirenos/farmacologia , Antineoplásicos/química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Curcumina/química , Curcumina/metabolismo , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/agonistas , Fase G2/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Fenol/química , Fenol/farmacologia , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Estirenos/química , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/agonistas
4.
Nat Commun ; 12(1): 4835, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376679

RESUMO

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ionóforos de Próton/farmacologia
5.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360883

RESUMO

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.


Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Suplementos Nutricionais , Mitofagia/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Valeratos/farmacologia , Adenocarcinoma/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo
6.
Molecules ; 26(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299490

RESUMO

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chalconas/farmacologia , Mitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Células A549 , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Feminino , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo
7.
Toxicology ; 459: 152859, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34273449

RESUMO

Vanadium dioxide nanoparticles (VO2 NPs) have been massively produced and widely applied due to their excellent metal-insulator transition property, making it extremely urgent to evaluate their safety, especially for low-dose long-term respiratory occupational exposure. Here, we report a comprehensive cytotoxicity and genotoxicity study on VO2 NPs to lung cell lines A549 and BEAS-2B following a long-term exposure. A commercial VO2 NP, S-VO2, was used to treat BEAS-2B (0.15-0.6 µg/mL) and A549 (0.3-1.2 µg/mL) cells for four exposure cycles, and each exposure cycle lasted for 4 consecutive days; then various bioassays were performed after each cycle. Significant proliferation inhibition was observed in both cell lines after long-term exposure of S-VO2 at low doses that did not cause apparent acute cytotoxicity; however, the genotoxicity of S-VO2, characterized by DNA damage and micronuclei, was only observed in A549 cells. These adverse effects of S-VO2 were exposure time-, dose- and cell-dependent, and closely related to the solubility of S-VO2. The oxidative stress in cells, i.e., enhanced reactive oxygen species (ROS) generation and suppressed reduced glutathione, was the main toxicity mechanism of S-VO2. The ROS-associated mitochondrial damage and DNA damage led to the genotoxicity, and cell proliferation retard, resulting in the cellular viability loss. Our results highlight the importance and urgent necessity of the investigation on the long-term toxicity of VO2 NPs.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Pulmão/patologia , Nanopartículas Metálicas/toxicidade , Mutagênicos/toxicidade , Óxidos/toxicidade , Compostos de Vanádio/toxicidade , Células A549 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Glutationa/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes para Micronúcleos , Estresse Oxidativo , Óxidos/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Compostos de Vanádio/farmacocinética
8.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199597

RESUMO

The disruption of iron homeostasis is an important factor in the loss of mitochondrial function in neural cells, leading to neurodegeneration. Here, we assessed the protective action of gossypitrin (Gos), a naturally occurring flavonoid, on iron-induced neuronal cell damage using mouse hippocampal HT-22 cells and mitochondria isolated from rat brains. Gos was able to rescue HT22 cells from the damage induced by 100 µM Fe(II)-citrate (EC50 8.6 µM). This protection was linked to the prevention of both iron-induced mitochondrial membrane potential dissipation and ATP depletion. In isolated mitochondria, Gos (50 µM) elicited an almost complete protection against iron-induced mitochondrial swelling, the loss of mitochondrial transmembrane potential and ATP depletion. Gos also prevented Fe(II)-citrate-induced mitochondrial lipid peroxidation with an IC50 value (12.45 µM) that was about nine time lower than that for the tert-butylhydroperoxide-induced oxidation. Furthermore, the flavonoid was effective in inhibiting the degradation of both 15 and 1.5 mM 2-deoxyribose. It also decreased Fe(II) concentration with time, while increasing O2 consumption rate, and impairing the reduction of Fe(III) by ascorbate. Gos-Fe(II) complexes were detected by UV-VIS and IR spectroscopies, with an apparent Gos-iron stoichiometry of 2:1. Results suggest that Gos does not generally act as a classical antioxidant, but it directly affects iron, by maintaining it in its ferric form after stimulating Fe(II) oxidation. Metal ions would therefore be unable to participate in a Fenton-type reaction and the lipid peroxidation propagation phase. Hence, Gos could be used to treat neuronal diseases associated with iron-induced oxidative stress and mitochondrial damage.


Assuntos
Flavonoides/farmacologia , Ferro/efeitos adversos , Mitocôndrias/metabolismo , Neurônios/citologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/efeitos adversos , Compostos Ferrosos/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos
9.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198834

RESUMO

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC50 and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.


Assuntos
Anidrase Carbônica IX/genética , Inibidores da Anidrase Carbônica/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Apoptose/efeitos dos fármacos , Anidrase Carbônica IX/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
10.
Chem Biol Interact ; 345: 109575, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34228970

RESUMO

In recent decades, interest has increased in the role of reactive oxygen species (ROS) in health and disease. The ROS are key causative factors in several hearing loss pathologies including ototoxicity, noise trauma, cochlear ageing and ischemic injury. In order to investigate ROS effects on inner ear cells and counteract them, we developed an in vitro model of oxidative stress by exposing the inner ear cell line OC-k3 to hydrogen peroxide (H2O2) at concentrations able to affect in vivo cellular components but allowing cell survival. The treatment with high concentrations (20 and 30 µM) resulted in reduction of cell viability, activation of apoptosis/necrosis and alteration of morphology, cell cycle progression and antioxidant defences. The ROS effects in inner ear cells are difficult to assess in vivo. Organocultures may provide preservation of tissue architecture but involve ethical issues and can be used only for a limited time. An in vitro model that could be commercially available and easy to handle is necessary to investigate inner ear oxidative stress and the ways to counteract it. The OC-k3 line is a suitable in vitro model to study ROS effects on inner ear cells because the observed cell alterations and damages were similar to those reported in studies investigating ROS effects of ototoxic drugs, noise trauma and cochlear ageing.


Assuntos
Orelha Interna/citologia , Peróxido de Hidrogênio/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
Am J Physiol Cell Physiol ; 321(1): C176-C186, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34106788

RESUMO

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.


Assuntos
Fibroblastos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Síndrome MELAS/genética , Mitocôndrias/efeitos dos fármacos , Sirolimo/farmacologia , Estudos de Casos e Controles , Pré-Escolar , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Lactente , Lisossomos/metabolismo , Síndrome MELAS/tratamento farmacológico , Síndrome MELAS/metabolismo , Síndrome MELAS/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Adulto Jovem
12.
Expert Opin Drug Metab Toxicol ; 17(7): 857-865, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34102941

RESUMO

BACKGROUND: Acrylamide (AA) is a water-soluble toxic chemical that is considered one of the most important food contaminants. Furthermore, AA is considered a major public health risk. METHODS: This study was designed to evaluate the effects of AA on cytotoxicity, oxidative damage and genotoxicity in human lymphocytes and also to evaluate the protective effects of the chrysin (CH). Lymphocytes after isolation from the blood were treated with AA (50 µM), AA (50 µM) plus CH (10, 25, 50 µM) and CH (50 µM), and parameters such as cell viability, mitochondrial and lysosomal damage, as well as oxidative damage to DNA were examined. RESULTS: The results showed that CH was able to reduce cytotoxicity, reactive oxygen species (ROS) levels, lipid peroxidation (LPO) level, collapse in mitochondrial membrane potential (MMP) and oxidative damage of DNA caused by AA in human lymphocytes. Also, co-treatment of the AA-exposed human lymphocytes with CH increases the glutathione (GSH) levels. CONCLUSION: Results suggest that CH (10, 25, 50 µM) shows a protective role in AA-induced cytotoxicity, oxidative stress, mitochondrial damage and DNA oxidative damage.


Assuntos
Acrilamida/toxicidade , Flavonoides/farmacologia , Linfócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
13.
Toxicology ; 458: 152838, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34153373

RESUMO

N,N-dimethylformamide (DMF) is an organic compound widely used in industrial production processes as a solvent with a low evaporation rate. Excessive exposure to DMF may lead to liver damage. Oxidative stress has been reported as one of the main causes of DMF-induced hepatotoxicity. Several doses of DMF (0, 1, 5, and 10 mM) were used to treat HL-7702 cells for a relatively long period to simulate the actual exposure pattern in occupational settings, and oxidative stress was induced. Previous studies illustrated that circular RNA (circRNA) plays a vital role in sustaining hepatocyte physiological function. To explore whether aberrant circRNA expression is involved in DMF-induced excessive ROS generation and hepatotoxicity, high-throughput transcriptional sequencing was performed to identify the altered circRNA expression profiles in HL-7702 liver cells after treatment with 0, 75, or 150 mM DMF for 48 h. We found that levels of induced oxidative stress were similar to those in the long-term exposure model. Among the altered circRNAs, one circRNA (hsa_circ_0005915) was significantly upregulated after DMF exposure, and it affected DMF-mediated oxidative stress in HL-7702 cells. Further experiments revealed that hsa_circ_0005915 downregulated the expression of nuclear factor erythoid-2-related factor 2 (NRF2) at the post-transcriptional level via promoting the ubiquitination and degradation of NRF2, which led to the increase of ROS accumulation. Further investigation demonstrated that the expression levels of NRF2-regulated antioxidative genes-heme oxygenase 1 (HO1) and NAD(P)H quinone dehydrogenase 1 (NQO1)-indeed declined after the overexpression of hsa_circ_0005915. In vivo study also indicated that DMF exposure can upregulate the expression of mmu_circ_0007941 (homologous circRNA of hsa_circ_0005915) and downregulated Nrf2 and Ho1 proteins. In summary, our results revealed that hsa_circ_0005915 plays an important role in promoting DMF-induced oxidative stress by inhibiting the transcriptional activity of the NRF2/ARE axis, which provides a potential molecular mechanism of DMF-mediated hepatotoxicity.


Assuntos
Dimetilformamida/toxicidade , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Circular/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , MicroRNAs , NAD(P)H Desidrogenase (Quinona)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
14.
Int J Biol Macromol ; 185: 40-48, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34144065

RESUMO

It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from Copaifera langsdorffii seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 µg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 µg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Fabaceae/química , Glucanos/farmacologia , Neoplasias Hepáticas/metabolismo , Vanadatos/química , Xilanos/farmacologia , Antineoplásicos/química , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucanos/química , Células Hep G2 , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Oxirredutases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Xilanos/química
15.
Ecotoxicol Environ Saf ; 221: 112450, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186417

RESUMO

Bisphenol A (BPA) is a widely distributed environmental endocrine disruptor. The accumulation of BPA has been proved that produce various toxic effects both on human and animals. However, the strategies to reduce the damage of BPA on the body and related mechanisms remain to be studied. Coenzyme Q10 (CoQ10), as a powerful antioxidant, is ubiquitous in many eukaryotic cells, which can improve the integrity of lysosomal membrane, lysosomal degradation function and promote autophagy. Here, we examined the ability of CoQ10 to alleviate oxidative stress and apoptosis in BPA-induced damages in C2C12 cells, and how to alleviate it. Our results showed that BPA treatment significantly reduced cell viability, increased the number of cell apoptosis and ROS production, decreased mitochondrial membrane potential, and inhibited the gene expression of mitochondria biogenesis. Moreover, we demonstrated that exposure to BPA increased expression levels of autophagy protein (LC3-II, p62), inhibited autophagy flux, and disrupted the acidic pH environment of lysosomes. Importantly, CoQ10 supplementation effectively restored these abnormalities caused by BPA. CoQ10 significantly decreased the apoptotic incidence and ROS levels, improved mitochondrial membrane potential. Moreover, CoQ10 improved lysosome function and enhanced autophagy flux. Taken together, our results indicate that CoQ10 supplementation is a feasible and effective way to promote the level of autophagy by improving lysosomal function, thereby reducing the apoptosis caused by BPA accumulation. This study aims to provide evidence for the role of CoQ10 in repairing BPA-induced cell damage in clinical practice.


Assuntos
Antioxidantes/toxicidade , Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Ubiquinona/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Ubiquinona/farmacologia
16.
Toxicol Appl Pharmacol ; 426: 115607, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34089742

RESUMO

Sertoli cells (SC) structurally support and transport nutrients to germ cells during spermatogenesis facilitated by an active cytoskeleton. Chemical perturbation of SC microtubule (MT) dynamics instability leads to premature germ cell exfoliation demonstrating that this process is essential for male fertility, yet the effects of MT damaging drugs on SC lipid metabolism have been less explored. The aim of this study was to advance our understanding of how adequate SC MT dynamicity is needed to finely tune lipid homeostasis. To elucidate the role of MT dynamics instability on the latter, we suppressed MT dynamicity by long-term exposures to 10 nM of nocodazole (NCZ) on TM4-SC cultures. Inhibition of MT dynamics instability affected the distribution of [3H] arachidonate on TM4-SC. Triacylglycerols (TAG) exhibited a higher proportion of the [3H] label, with significantly lower percentages in the mitochondrial phospholipid cardiolipin, and notably, also in phosphatidylethanolamine. A noteworthy and progressive accumulation of lipid droplets during the period of exposure to NCZ was accompanied by increased TAG levels but not cholesterol levels in TM4-SC. NCZ-exposed cells reduced their mitochondrial membrane potential and increased ROS production without triggering apoptosis, had a compromised autophagic flux, and lost their transferrin expression. Although SC morphology was preserved, the NCZ-exposed cells displayed alteration of the normal organization of microfilaments (f-actin) and intermediate filaments (vimentin). Our findings suggest that a preserved MT dynamicity is essential in the maintenance of lipid and fatty acids homeostasis in SC, and thus highlights a novel target in these cells for drugs that impair MT dynamicity.


Assuntos
Metabolismo dos Lipídeos , Microtúbulos/metabolismo , Células de Sertoli/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Homeostase/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Nocodazol/farmacologia , Células de Sertoli/efeitos dos fármacos , Moduladores de Tubulina/farmacologia
17.
Methods Mol Biol ; 2275: 227-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118041

RESUMO

Genetic mutations and defects in mitochondrial DNA (mtDNA) are associated with certain types of mitochondrial dysfunctions, ultimately resulting in the emergence of a variety of human diseases. To achieve an effective mitochondrial gene therapy, it will be necessary to deliver therapeutic agents to the innermost mitochondrial space (the mitochondrial matrix), which contains the mtDNA pool. We recently developed a MITO-Porter, a liposome-based nanocarrier that delivers cargo to mitochondria via a membrane-fusion mechanism. In this chapter, we discuss the methodology used to deliver bioactive molecules to the mitochondrial matrix using a Dual Function (DF)-MITO-Porter, a liposome-based nanocarrier that delivers it cargo by means of a stepwise process, and an evaluation of mtDNA levels and mitochondrial activities in living cells. We also discuss mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using the MITO-Porter system.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , RNA Antissenso/farmacologia , RNA Mitocondrial/genética , DNA Mitocondrial/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inativação Gênica , Células HeLa , Humanos , Lipossomos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Mutação , RNA Antissenso/química , RNA Mitocondrial/efeitos dos fármacos
18.
Ecotoxicol Environ Saf ; 221: 112425, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34146984

RESUMO

The frequent detection of (2,3-dibromopropyl) phosphate (TDBPP) in environment has led to a consistent risk to organisms. However, little is known about the toxicity of TDBPP exclusive for its carcinogen. Mitochondrion that tightly relates to adverse outcomes once deteriorated is referred as a target of environmental pollutants. Here, we investigated the role of mitochondrial abnormality in development of cellular pathobiology especially lipid deposition when response to TDBPP in mitochondria-rich hepatocyte (AML12) at the same order of magnitude as the environmental concentrations (10-6 mol/L or below) via multiplexed quantitative high content analytic system. The present study claimed TDBPP shifted mitochondria from fusion morphology to fission phenotype charactering by less mitochondrial networks, larger mitochondrial areas and shorter branch length at 10-7 mol/L or above. This dynamic imbalance was triggered by high levels of fis and drp1 genes when treated with TDBPP. The deformation caused by TDBPP reciprocally influenced biogenesis through PGC1α and electron transport chains via ectopic expression of genes encoding for mitochondria complex I and III subunits. Accordingly, we observed high mitoROS level and low mitochondria membrane potential. Consequently, cells contained those abnormal mitochondria were predisposed to accumulating lipids after exposure to TDBPP. Here we showed that TDBPP deteriorated mitochondrial morphology and function, which may induce lipid generation. As for a banned while still emerged contaminant, our study also claimed further exploration on the non-carcinogenic toxicity of TDBPP.


Assuntos
Retardadores de Chama/toxicidade , Hepatócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Organofosfatos/toxicidade , Animais , Linhagem Celular , Hepatócitos/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo
19.
Chem Biol Interact ; 345: 109553, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34126102

RESUMO

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Modelos Biológicos , Hipóxia Tumoral , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Desferroxamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacos
20.
Phytomedicine ; 88: 153605, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34107409

RESUMO

Osteoporosis is the process of bone loss, particular after menopause, when the production of estrogen in women is decreaing. Bioenergetic function is one of the critical roles in bone remodeling. Danggui Buxue Tang (DBT) is an herbal mixture containing Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), and which is consumed for "Qi-invigorating", i.e., stimulating energy metabolism, as a traditional Chinese medicine (TCM). However, the role of DBT in metabolism of osteoblast has not been examined. Here, we employed a metabolic flux to examine the mitochondrial functions of cultured osteoblast in the presence of herbal extracts, including DBT, ASR, AR, AR + ASR (single mixing of two herbal extracts), as well as DBT∆cal (a DBT extract depeleting calycosin), to examine their roles in osteoblastic metabolism, e.g. glycolysis and energy kinetics. By revealing the rates of oxygen consumption and extracellular acidification of mitochrondia, the DBT-treated osteoblasts were markedly strengthened with increases of maximal respiration, spare capacity, glycolysis capacity and glycolysis reserve, in comparing to other herbal extracts. In addition, the bioenergetic metabolism was modulated by DBT via the signaling of cellular Ca2+ and reactive oxgen species (ROS). Furthermore, DBT affected the morphology of mitochondria, as well as mitochondrial dynamic. Here, we propose that DBT can be regarded as benefit herbal extract in improving osteoblastic metabolism for bone disorders via central energy metabolism and mitochondrial bioenergetics.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Mitocôndrias/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Medicamentos de Ervas Chinesas/química , Metabolismo Energético/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoblastos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
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