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1.
Med Sci Monit ; 26: e918216, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32129321

RESUMO

BACKGROUND Chemoresistance is a primary hindrance for current cancer treatments. The influence of abnormal mitochondria in chemotherapy resistance is not well known. To explore the correlation between mitochondria and acquired chemoresistance, this work studied alterations in mitochondrial dynamics, biogenesis, and functions for paclitaxel-resistant cancer cell line A549/Taxol and its parental line A549. MATERIAL AND METHODS Mitochondrial morphology was observed by transmission electron microscopy and confocal microscopy. We measured the mitochondrial mass and mitochondrial membrane potential using fluorescent dyes. The glucose metabolic profile and ATP (adenosine triphosphate) content were determined by bioluminescent cell assays. Seahorse bio-energy analyzer XF24 was used to detect the mitochondrial respiratory function. The expressions of mitochondrial dynamics and biogenesis related genes were quantified using real-time polymerase chain reaction. RESULTS We observed fusion morphology of the mitochondrial network in A549/Taxol cells, with upregulation of fusion genes (Mfn1 and Mfn2) and downregulation of fission gene Fis1. In A549/Taxol cells, mitochondrial mass showed a significant decrease, while the mitochondrial biogenesis pathway was strongly activated. Despite the decreased mitochondrial membrane potential, the capability for mitochondrial respiration was not impaired in A549/Taxol cells. CONCLUSIONS Our study revealed a series changes of mitochondrial characteristics in paclitaxel-resistant cells. Mfn1 and Mfn2 and PGC-1alpha increased, while Fis1 expression and mitochondrial oxidative phosphorylation decreased in A549/Taxol cell lines. These changes to mitochondrial fusion, fission, and biological function contributed to the occurrence of paclitaxel resistance in tumor cells which induced paclitaxel resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Dinâmica Mitocondrial , Biogênese de Organelas , Paclitaxel/farmacologia , Células A549 , GTP Fosfo-Hidrolases/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
2.
Cell Physiol Biochem ; 54(2): 230-251, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32153152

RESUMO

BACKGROUND/AIMS: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). METHODS: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. RESULTS: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. CONCLUSION: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.


Assuntos
Aerossóis/química , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Material Particulado/toxicidade , Cálcio/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Material Particulado/química , Fumaça/efeitos adversos , Fatores de Tempo , Produtos do Tabaco/análise
3.
Cell Physiol Biochem ; 54(2): 161-179, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32045141

RESUMO

BACKGROUND/AIMS: We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. METHODS: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3'-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. RESULTS: In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. CONCLUSION: This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions.


Assuntos
Comunicação Celular/efeitos dos fármacos , Ranibizumab/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
4.
J Photochem Photobiol B ; 204: 111767, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32006893

RESUMO

Colon carcinoma is a recurring type of cancer that affects the intestine epithelial with a poor survival rate. It was already proven the anticancer property of hesperidin in various cancers but the bioavailability hesperidin is poor, which hinders the hesperidin usage. In this investigation we synthesized hesperidin loaded Zn2+@ SA/PCT nanocomposites and assessed its anticancer potential against colon cancer (HCT116) cells. Hesperidin loaded Zn2+@ SA/PCT nanocomposites were characterized using Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis. The drug releasing capacity and cytotoxic property was assessed via drug releasing assay, MTT assay with HCT116 cells. The anticancer potency of hesperidin nanocomposites were evaluated with TUNEL, DAPI staining, reactive oxygen species (ROS) generation assay and it is confirmed with flow cytometry analysis of MMP disruption in colon cancer (HCT116) cell line. Further the immunoblotting analysis of cysteine proteases Caspases 3, 9, PARP, proapoptotic protein Bax and antiapoptotic protein Bcl2 were performed. The results of FTIR, XRD and electroscopic analyses confirmed the synthesized hesperidin nanocomposites accomplish the properties of potent nanodrug and the MTT assay authentically confirmed that the synthesized hesperidin nanocomposite inhibited the HCT116 cell growth, and the results of fluorescent staining proved that the hesperidin nanocomposite induced the apoptotic mediated cell necrosis via promoting the expression of apoptotic proteins thereby induced the apoptosis in colon cancer (HCT116) cells. Hence, it was concluded that the, hesperidin loaded nanocomposites persuasively inhibited proliferation of colon carcinoma cell and induced apoptosis in in vitro condition.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Hesperidina/química , Nanocompostos/química , Alginatos/química , Antineoplásicos Fitogênicos/química , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Células HCT116 , Hesperidina/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pectinas/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zinco/química
5.
Life Sci ; 245: 117347, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981628

RESUMO

AIM: Oxidative stress plays an important role in myocardial ischemia-reperfusion injury. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was first identified in apoptosis induced by T cell receptor activation, and was shown to play a different role in different cell types and under different stimuli. The role and mechanism of PHLDA1 in oxidative stress-induced cardiomyocyte injury and cardiac ischemia-reperfusion were therefore determined. MAIN METHODS: Cell viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Mitochondrial membrane potential was measured using JC-1 test kit. Reactive oxygen species (ROS) production was detected using ROS kit. HE staining was used to detect histological morphology, 2,3,5-triphenyltetrazolium chloride staining to detect infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling staining to detect the apoptotic rate, and immunohistochemistry and western blot analysis to detect protein expression. The binding of PHLDA1 to Bcl-2 associated X (Bax) was detected by immunoprecipitation. KEY FINDINGS: The results indicated that PHLDA1 is highly expressed in oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. PHLDA1 overexpression in cardiomyocytes promoted oxidative stress-induced cardiomyocyte injury. At the same time, PHLDA1 knockdown improved oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. In addition, PHLDA1 binds to Bax and the interaction is enhanced under H2O2 stimulation. SIGNIFICANCE: The present results indicated that PHLDA1 interacts with Bax to participate in oxidative stress-induced cardiomyocyte injury and myocardial ischemia reperfusion injury.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Linhagem Celular , Citometria de Fluxo , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Masculino , Potencial da Membrana Mitocondrial , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
Life Sci ; 242: 117248, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899224

RESUMO

Diabetic nephropathy is the most common long-term complication of diabetes mellitus. The Methylglyoxal (MGO) production is mainly by metabolic pathways, such as lipolysis and glycolysis, its increases in the DM enhances oxidative stress and plays a crucial role in the diabetic nephrotic pathogenesis. Phosphocreatine (PCr) can improve lipopolysaccharide, ox-LDL-induced atherosclerosis, and alleviate vascular endothelial cell injury in diabetes. The aim of our present study is to examine the potential role of phosphocreatine (PCr) as a molecule protects against diabetes-induced Kidney Injury in-vitro and in-vivo through ERK/Nrf2/HO-1 signaling pathway. NRK-52E cells treatment with PCr obviously suppressed MGO-induced change of viability, apoptosis, coupled with decreased Bax/Bcl-2ratio, casapse-9 and caspase-3expressions. We determined the generation of reactive oxygen species (ROS) using membrane permeable fluorescent probe DCFH-DA as well as intracellular calcium by flow cytometry. ERK, Nrf2 and HO-1 expressions were determined by Western blot. PCr pretreatment significantly returned the oxidative stress enzymes to normal condition in-vitro and in-vivo. PCr pretreatment significantly reduced apoptosis, calcium and ROS production, induced by MGO, in NRK-52E cells. Moreover, pretreatment with PCr significantly inhibited cleaved caspase-3, cleaved caspase-9 and p-ERK expressions, while increased Nrf-2 and HO-1 expressions. Furthermore, PCr pretreatment significantly decreased p-ERK expression of MGO-induced injury in NRK-52E cells transfected with p-ERK cDNA. In conclusion, the renal protective effect of PCr in-vitro and in-vivo depends on suppressing apoptosis and ROS generation through ERK mediated Nrf-2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the diabetic nephropathy treatment.


Assuntos
Nefropatias Diabéticas/prevenção & controle , Heme Oxigenase (Desciclizante)/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fosfocreatina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/complicações , Citometria de Fluxo , Imunofluorescência , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
7.
Toxicol Lett ; 322: 87-97, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935479

RESUMO

1,2-Dichloroethane (1,2-DCE) is a widely used chlorinated organic toxicant, but little is known about the cerebellar dysfunction induced by excessive exposure to it. To uncover 1,2-DCE-induced neurotoxicity in cerebellar granular cells (CGCs), and to investigate the underlying mechanisms, we explored this, both in vitro and in vivo. Our findings showed significant cell viability inhibition in human CGCs (HCGCs) treated with 1,2-DCE. Flow cytometry and mitochondrial membrane potential analyses discovered an increase in apoptotic-mediated cell death in HCGCs after 1,2-DCE treatment. This HCGC apoptosis was involved in the increases of protein expression in Cytochrome c, Caspase-3, Bad, Bim, transformation related protein 53, Caspase-8, tumor necrosis factor-α, and Survivin. Quantitative real-time PCR (qPCR) and western blot confirmed the increases in Cytochrome c, Caspase-3, cleaved Caspase-3, and Bad in HCGCs after 1,2-DCE treatment. Bax inhibitor peptide V5 rescued 1,2-DCE-induced HCGC apoptosis. Furthermore, 80 CD-1 male mice were exposed to 1,2-DCE by inhalation at 0, 100, 350, and 700 mg/m3 for 6 h/day for 4 weeks. An open field test found abnormal neurobehavioral changes in the mice exposed to 1,2-DCE. Histopathological examination showed significantly shrunken and hypereosinophilic cytoplasm with nuclear pyknosis in mouse CGCs from the 700 mg/m3 1,2-DCE group. TdT-mediated dUTP nick-end labeling assay verified significant increases in apoptotic positive cells in the mouse CGCs after 1,2-DCE exposure. We confirmed the increases in the expressions of Cytochrome c, Caspase-3, cleaved Caspase-3 and Bad in the mice exposed to 1,2-DCE. These findings suggest that 1,2-DCE exposure can induce CGC apoptosis and cerebellar dysfunction, at least in part, through mitochondrial pathway.


Assuntos
Apoptose/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Dicloretos de Etileno/toxicidade , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/patologia , Cerebelo/fisiopatologia , Humanos , Locomoção/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Medição de Risco , Transdução de Sinais
8.
Pestic Biochem Physiol ; 163: 84-93, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31973874

RESUMO

Ivermectin (IVM) is a commercially well-known antiparasitic agent derived from the natural fermentation product avermectin. Originally used as a veterinary drug, IVM has been studied for its pharmacokinetic advantages, such as anticancer, antimigration, and antiproliferative effects, using several cell types. In the present study, we verified that IVM suppressed bovine mammary gland epithelial cell proliferation and induced the arrest of the cell cycle from the sub-G1 to the G2/M phase in these cells. Due to IVM treatment, the homeostasis of calcium ions, which play a crucial role in intracellular metabolism, deteriorated, leading to the loss of the mitochondrial membrane potential (MMP). To underpin these results, further studies using inhibitors of Ca2+ signaling were performed; combination treatment with IVM and these factors, including 2-APB, BAPTA-AM, or ruthenium red, inhibited the IVM-induced MMP disruption. Furthermore, following IVM treatment, the relationships among various cell signaling mediators were altered, and the balance between diverse cellular processes associated with cell survival or death was disturbed. In conclusion, we assessed the anti-survival effects of IVM on mammary gland epithelial cells; IVM may impede normal lactation in dairy cows.


Assuntos
Antiparasitários , Ivermectina , Animais , Apoptose , Bovinos , Células Epiteliais , Feminino , Potencial da Membrana Mitocondrial
9.
J Photochem Photobiol B ; 203: 111771, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31911399

RESUMO

Ultraviolet B (UVB) radiation triggers the activation of many reactive oxygen species (ROS)-sensitive signaling pathways, resulting in the induction of skin damage that can progress to premature skin aging with long-term exposure. Even after the cessation of UVB radiation, the activated photosensitizers can still cause cellular injury. Thus, the use of photoprotectors that inhibit or prevent intracellular ROS production during or after UV exposure is one alternative to counteract UV-induced oxidative damage. The present study investigated the photoprotective activity of protocatechuic acid (P0) and its alkyl esters ethyl protocatechuate (P2) and heptyl protocatechuate (P7) against UVB-induced damage in L929 fibroblasts by evaluating biomarkers of oxidative stress and photoaging. P0, P2 and P7 markedly increased cell viability after UVB exposure. This protective effect was related to the ability of these compounds to absorb UVB and restore cellular redox balance even 24 h after UVB exposure. P0, P2 and P7 also decreased oxidative damage to membrane lipids, mitochondrial membrane potential, and DNA. They also inhibited the nuclear translocation of NF-κB p65 and downregulated the expression of the photoaging-related proteins matrix metalloproteinases-1 and -9 and cyclooxygenase-2. As the lipophilicity of the P0 derivatives increased, their antioxidant potency increased, but more pronounced cytotoxic effects were also detected. In summary, P0 and P2 may be promising candidates for the prevention and treatment of UVB-induced skin photodamage and photoaging.


Assuntos
Senescência Celular/efeitos dos fármacos , Ésteres/química , Hidroxibenzoatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Raios Ultravioleta , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Linhagem Celular , Senescência Celular/efeitos da radiação , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/citologia , Hidroxibenzoatos/química , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , NADPH Oxidases/metabolismo , Oxirredução , Estresse Oxidativo/efeitos da radiação , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo
10.
J Photochem Photobiol B ; 203: 111778, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31931389

RESUMO

In the last decade, gold nanoparticles have emerged as promising agents for in vitro bio-sensing and in vivo cancer theranostics. However, different investigations have reported widely varying cytotoxicity and uptake efficiency of gold nanoparticles depending upon their size. Therefore, more extensive studies are needed to standardize these biological effects as a function of size on a particular cell line. In addition, to obtain robust confirmation on the correlation of a size to biological effect, thorough mechanistic study must also be performed. In this study, the size dependent biological activities of gold nanoparticles on osteosarcoma cells is investigated towards exploring their potential theranostic application in bone cancer, for which very scarce literature reports are available. Tris-assisted citrate based method was optimized to synthesize stable gold naoparticles of 40-60 nm sizes. Nanoparticles were characterized through UV-Vis spectroscopy, field emission scanning electron microscope (FESEM) and dynamic light scattering (DLS). Increasing concentrations of gold nanoparticles (AuNPs) of 46 nm size, enhanced the rate of reactive oxygen species (ROS)-induced apoptosis in MG63 cells by disrupting their mitochondrial membrane potential. Considerably higher cell death was observed for 46 and 60 nm AuNPs compared to 38 nm at all concentrations of 200, 400 and 800 ng/mL. Further, molecular signatures of cellular apoptosis under nanoparticle treatment were optically assessed through surface enhanced Raman scattering (SERS). A significant Raman enhancement in cancer cells under treatment of larger gold nanoparticles (46 and 60 nm) at fixed wavelength of 785 nm and laser power of 8.0 mW was evident. In corroboration with molecular biology techniques, SERS observation confirmed the size-dependent apoptotic phenomena in osteosarcoma cells under treatment of gold nanoparticles. Study demonstrates a facile, non-active targeting approach for detection of size-dependent AuNP-induced apoptosis in osteosarcoma cells through label-free SERS method.


Assuntos
Apoptose/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/toxicidade , Caspase 3/metabolismo , Linhagem Celular Tumoral , Difusão Dinâmica da Luz , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/química , Microscopia de Fluorescência , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral Raman
11.
Life Sci ; 244: 117322, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958419

RESUMO

AIMS: Mitochondrial dysfunction is an early prominent feature of Alzheimer's disease (AD). In the present study, we sought to investigate whether defective mitophagy is tightly related to amyloid-ß (Aß)-induced mitochondrial dysfunction. MAIN METHODS: Immunofluorescence, western blot and transmission electron microscopy were used to examine mitophagy. Mitochondrial membrane potential was assessed using the JC-1 dye. Mitochondrial ROS was detected using MitoSOX™ Red staining. KEY FINDINGS: Aß induced mitochondrial dysfunction in HEK293 cells. Moreover, Aß induced an increase in parkin translocation to mitochondria and led to a drastic reduction in cytosolic parkin. Furthermore, Aß-treated cells displayed a microtubule-associated protein 1 light chain 3 (LC3) punctate pattern and elevated mitochondrial LC3-II levels, suggesting the upregulation of mitophagy. Notably, Aß induced the accumulation of mitochondrial p62, which was associated with impaired mitophagy. In addition, Aß-treated cells exhibited fragmented or swollen mitochondria with severely decreased cristae. We then investigated whether overexpression of parkin could protect cells against Aß-induced mitochondrial dysfunction. Interestingly, parkin overexpression inhibited Aß-induced mitochondrial dysfunction. Besides, parkin overexpression increased cytosolic and mitochondrial parkin levels as well as mitochondrial LC3-II levels in Aß-treated cells. Additionally, parkin overexpression reversed the accumulation of p62 in mitochondria, indicating that parkin overexpression restored impaired mitophagy in Aß-treated cells. Importantly, parkin overexpression remarkably reversed Aß-induced mitochondrial fragmentation. SIGNIFICANCE: Our data demonstrate that overexpression of parkin ameliorates impaired mitophagy and promotes the removal of damaged mitochondria in Aß-treated cells, indicating that upregulation of parkin-mediated mitophagy may be a potential strategy for the therapy of AD.


Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Mitocôndrias/metabolismo , Doenças Mitocondriais/prevenção & controle , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/etiologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/genética
12.
Int J Occup Environ Med ; 11(1): 41-52, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905194

RESUMO

BACKGROUND: Arsenic, an environmental pollutant, is a carcinogenic metalloid and also an anticancer agent. OBJECTIVE: To evaluate the toxicity of arsenic nanoparticles in rat hepatocytes. METHODS: Freshly isolated rat hepatocytes were exposed to 0, 20, 40, and 100 µM of arsenic nanoparticles and its bulk counterpart. Their viability, reactive oxygen species level, glutathione depletion, mitochondrial and lysosomal damage, and apoptosis were evaluated. RESULTS: By all concentrations, lysosomal damage and apoptosis were clearly evident in hepatocytes exposed to arsenic nanoparticles. Evaluation of mitochondria and lysosomes revealed that lysosomes were highly damaged. CONCLUSION: Exposure to arsenic nanoparticles causes apoptosis and organelle impairment. The nanoparticles have potentially higher toxicity than the bulk arsenic. Lysosomes are highly affected. It seems that, instead of mitochondria, lysosomes are the first target organelles involved in the toxicity induced by arsenic nanoparticles.


Assuntos
Apoptose/efeitos dos fármacos , Arsênico/toxicidade , Hepatócitos/efeitos dos fármacos , Lisossomos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas/toxicidade , Animais , Células Cultivadas , Glutationa/metabolismo , Hepatócitos/citologia , Humanos , Masculino , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
13.
Environ Pollut ; 256: 113430, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31685329

RESUMO

Silver nanoparticles (AgNPs) are inevitably released into the environment owing to their widespread applications in industry and medicine. The potential of their toxicity has aroused a great concern. Previous studies have shown that AgNPs exposure in HepG2 cells is primarily related to the damage of mitochondria, which includes induction of mitochondrial swelling and increase of intracellular levels of reactive oxygen species (ROS), the collapse of mitochondrial membrane potential and induction of apoptosis through a mitochondrial pathway. In this study, the effects of AgNPs exposure in HepG2 cells on mitochondrial dynamics and biogenesis were investigated. AgNPs were found to induce mitochondrial morphological and structural alterations. The expressions of key proteins (Drp1, Fis1, OPA1, Mff, Mfn1, and Mfn2) related to mitochondrial fission/fusion event were changed. Especially the expression of fission-related protein 1 (p-Drp1) (Ser616) was significantly up-regulated, whereas the expression of mitochondrial biogenesis protein (PGC-1α) was reduced in AgNP-treated cells. Concomitantly, the expression of autophagy marker proteins (LC3B and p62) was increased. The results suggested that AgNPs could trigger cytotoxicity by targeting the mitochondria, resulting in the disruption of mitochondrial function, damage to the mitochondrial structure and morphology, interfering in mitochondrial dynamics and biogenesis. The mitochondria could be a critical target of AgNPs in cells. The functions of mitochondria could be used for assessing the cytotoxic effects associated with AgNPs in cells.


Assuntos
Nanopartículas Metálicas/toxicidade , Mitocôndrias/efeitos dos fármacos , Prata/toxicidade , Animais , Apoptose , Substâncias Perigosas , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade
14.
Toxicol Lett ; 319: 102-110, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706006

RESUMO

Crizotinib is a multi-target receptor tyrosine kinase inhibitor which is of great importance for the management of ALK-rearranged non-small cell lung cancer (NSCLC) patients. Serious erythroderma and toxic epidermal necrolysis have been reported associated with crizotinib treatment. The underlying mechanisms have not been examined. In this study, we tested the toxicity of crizotinib on immortal human keratinocytes (HaCaT) and human primary keratinocytes. We found that crizotinib directly cause cytotoxic on these two cells, which could be the explanation of the clinical characteristic of pathology. Apoptosis was observed and Z-VAD-FMK, a pan-caspase inhibitor can almost totally reverse the apoptosis induction effect of crizotinib. However, mitochondrial dysfunction and DNA damage were not involved in crizotinib-induced apoptosis, indicating the intrinsic apoptosis pathway have no connection with this cutaneous toxicity. Further studies showed that crizotinib significantly increased cleaved-caspase-8, a signaling protein of extrinsic apoptosis pathway, in a concentration and time-dependent manner. Moreover, we found the targets of crizotinib were not involved in HaCaT cells apoptosis. Collectively, our findings first report keratinocytes apoptosis is the key cause of crizotinib-induced cutaneous toxicity. We also reveal crizotinib induce apoptosis through the extrinsic apoptosis pathway due to detected up-regulated cleaved-caspase-8. Meanwhile, the apoptosis is independent of mitochondrial dysfunction, DNA damage and related drug targets inhibition.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Crizotinibe/toxicidade , Dermatite Esfoliativa/induzido quimicamente , Queratinócitos/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Dano ao DNA , Dermatite Esfoliativa/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Chem Biol Interact ; 315: 108850, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31634447

RESUMO

1,2,3-triazolium salts are poorly understood regarding their antileishmanial activity. Hence, as an effort to identify novel chemical scaffolds as antileishmanial agents, a series of 1,2,3-triazolium salts (TS) and corresponding 1,2,3-triazole (T) precursors including new epoxide derivatives were synthesized and assayed against Leishmania amazonensis promastigote and intracellular amastigote forms. Among them, the compound TS-6 exhibited promising activity on promastigotes (IC50 = 3.61 µM) and intracellular amastigotes (IC50 = 7.61 µM) of L. amazonensis, superior to miltefosine (IC50 > 10.0 µM), used as reference drug. In addition, TS-6 showed negligible cytotoxicity on murine peritoneal macrophages with a SI of about 10. Studies on the mode of action of TS-6 indicate mitochondrial dysfunction through an increase in 'total' and mitochondrial-ROS as well as depolarization of mitochondrial membrane potential of L. amazonensis promastigotes. In silico physicochemical studies indicate that the TS-6 could potentially be used as an oral drug.


Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Triazóis/farmacologia , Animais , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia
16.
Cell Prolif ; 53(1): e12718, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31721355

RESUMO

OBJECTIVES: We investigated the anti-cancer activity of pentamidine, an anti-protozoal cationic aromatic diamidine drug, in prostate cancer cells and aimed to provide valuable insights for improving the efficacy of prostate cancer treatment. MATERIALS AND METHODS: Prostate cancer cell lines and epithelial RWPE-1 cells were used in the study. Cell viability, wound-healing, transwell and apoptosis assays were examined to evaluate the influences of pentamidine in vitro. RNA-seq and qPCR were performed to analyse changes in gene transcription levels upon pentamidine treatment. Mitochondrial changes were assessed by measuring mitochondrial DNA content, morphology, membrane potential, cellular glucose uptake, ATP production and ROS generation. Nude mouse xenograft models were used to test anti-tumour effects of pentamidine in vivo. RESULTS: Pentamidine exerted profound inhibitory effects on proliferation, colony formation, migration and invasion of prostate cancer cells. In addition, the drug suppressed growth of xenograft tumours without exhibiting any obvious toxicity in nude mice. Mechanistically, pentamidine caused mitochondrial DNA content reduction and induced mitochondrial morphological changes, mitochondrial membrane potential dissipation, ATP level reduction, ROS production elevation and apoptosis in prostate cancer cells. CONCLUSIONS: Pentamidine can efficiently suppress prostate cancer progression and may serve as a novel mitochondria-targeted therapeutic agent for prostate cancer.


Assuntos
Proliferação de Células/efeitos dos fármacos , DNA Mitocondrial , DNA de Neoplasias , Mitocôndrias , Pentamidina/farmacologia , Neoplasias da Próstata , Animais , Proliferação de Células/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Nus , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Eur J Med Chem ; 186: 111851, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761381

RESUMO

Forty-eight analogues of CP-31398, an antitumor agent modulated the mutant p53 gene were synthesized and their cytotoxicities against four cancer cell lines with different p-53 status including bladder cell T24 (w-p53), gastric cell MGC-803 (m-p53), prostate cell DU145 (m-p53), prostate cell PC-3 (null-p53), lung cell A549 (w-p53) and normal liver cell line HL-7702 (w-p53) were examined. (E)-2-(4-Nitrostyryl)-4-(3-dimethylaminopropyl)-aminoquinazoline (10ah) was identified as the most potent compound in anti-proliferation against MGC-803 cells, with IC50 lowed to 1.73 µM, far potency than that of CP-31398. Molecular mechanism study revealed that 10ah and CP-31398 differ greatly in mechanism to exert their antitumor properties. 10ah could intercalate into DNA and resulted in significant DNA double-strand break. 10ah-treatment in MGC-803 cells increased the expression of p53, phosphorylated p53 (p-p53), CDK4, p21 to cause cell cycle arrest at G2/M phase, significantly up-regulated the levels of pro-apoptosis proteins Bak, Bax, Bim while down-regulated the anti-apoptosis proteins Bcl-2, Bcl-xL and the levels of cyclin B1, fluctuated the intracellular reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential, activated Caspase-9 and Caspase-3 to induce apoptosis. 10ah also displayed potent anticancer efficiency against MGC-803 xenograft tumors models, with tumor growth inhibition (TGI) up to 61.8% at 20 mg/kg without obvious toxicity.


Assuntos
Antineoplásicos/farmacologia , Quinazolinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/genética
18.
Eur J Med Chem ; 186: 111897, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761382

RESUMO

Diosgenin, a naturally occurring steroidal saponin, has been confirmed to possess potent anticancer properties. In the current work, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole (6a-6e and 7a-7e) or 1,3,4-thiadiazole (8a-8e and 9a-9e) moieties were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, A549, MCF-7 and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The results showed that compounds 8d and 9d exhibited significant cytotoxic activities against the HepG2 and A549 cells, being more potent than their parent compound diosgenin. Furthermore, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity compared with the 1,3,4-oxadiazole series against HepG2 and A549 cells, and the substitution of 3-pyridyl group at the C5 position of the 1,3,4-thiadiazole ring was the preferred option for these compounds to display significant cytotoxic activities. Compound 8d showed potent cytotoxic activity against A549 cell line (IC50 = 3.93 µM) and was 6.7-fold more potent than diosgenin (IC50 = 26.41 µM). Moreover, compound 8d displayed low toxicity against GES-1 cells (IC50 = 420.4 µM), showing specificity between normal and tumor cells. Further cellular mechanism studies in A549 cells indicated that compound 8d triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential, which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade. The above results indicated that compound 8d may be used as a promising skeleton for antitumor agents with improved efficacy.


Assuntos
Antineoplásicos/farmacologia , Diosgenina/farmacologia , Desenho de Drogas , Oxidiazóis/farmacologia , Tiadiazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diosgenina/síntese química , Diosgenina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Oxidiazóis/química , Relação Estrutura-Atividade , Tiadiazóis/química
19.
J Photochem Photobiol B ; 202: 111644, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31770706

RESUMO

Management of degenerative spine pathologies frequently leads to the need for bone growth. Rehmanniae Radix (RR), a Chinese herbal formulation was found to exhibit numerous therapeutic properties including its potent effect against cancer cell lines. However, the underlying mechanism through which the Zinc oxide nanoparticles (ZnONPs) synthesized from Rehmanniae Radix exerts its anti-cancer activity against osteosarcoma cell line MG-63 needs to be explored. Therefore, the study was performed to evaluate the anticancer, cytotoxicity and apoptotic effectiveness of ZnONPs from RR against MG-63 cells. Characterization studies such UV-vis spectroscopy, FTIR, TEM and XRD analysis were performed. Cytotoxicity assay, mitochondrial membrane potential (MMP), morphological examination of cells and formation of reactive oxygen species (ROS), and apoptosis inducing ability of RR were evaluated by various procedures. Western blot analysis of apoptotic markers such as Bax, caspase-3 and caspase-9 were also performed. RR was found to inhibit growth of MG-63 cells at increasing dose. AO/EB staining confirmed the apoptotic efficacy of ZnONPs induced by RR in MG-63 cells. ZnONPs was also found to initiate increased generation of ROS and decreased MMP. Decreased MMP has resulted in increased levels of apoptotic proteins Bax, caspase-3 and caspase-9 and induction of apoptosis was substantiated by western blot analysis. The outcomes of the work propose that ZnONPs from RR exhibits strong anticancer action and inducing apoptosis on MG-63 cells via stimulating increased generation of ROS. Thus, ZnONPs from RR might be used as a hopeful drug target against several types of cancer cell lines.


Assuntos
Caspase 3/metabolismo , Química Verde , Nanopartículas Metálicas/química , Óxido de Zinco/química , Proteína X Associada a bcl-2/metabolismo , Caspase 3/genética , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Rehmannia/química , Rehmannia/metabolismo , Proteína X Associada a bcl-2/genética
20.
J Photochem Photobiol B ; 202: 111718, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31790883

RESUMO

Metallic nanoparticles were extensively examined to explore their impending exploitations over pharmaceutical purposes. Current work attempting to explores the cytotoxic capacity of zinc oxide (ZnO) nanoparticles besides to human melanoma cell line (A375). Viability of cells was resoluted, and the promising cytotoxicity potential was exhibited by zinc oxide nanoparticles. Cellular adhesion and morphology was determined by propidium iodide assay. Characterization studies like UV-Spectroscopy, X-ray diffraction (XRD) investigation, transmission electron microscope (TEM), energy dispersive X-ray (EDX) Spec, and Fourier transform infrared (FT-IR) examination confirms the accessibility of measurement, form and volume. The mRNA expression of apoptotic genes like caspase 3, 8 and 9 was elevated followed by the exposure to ZnO nanoparticles and it was narrowly proved that ZnO nanoparticles stimulates the apoptotic cell necrosis at the transcriptional stage. Cardiospermum halicacabum down regulated the apoptotic gene expressions. Reactive oxygen species (ROS) accumulation was augmented at concentration reliant mode, that changed normalize numerous indicator pathways and manipulate the kinetic cellular actions. ZnO nanoparticle synthesized Cardiospermum halicacabum might persuades programmed cell necrosis via elevated ROS levels in cells. CH-ZnONPs was further stimulates the markers of apoptosis and aggravates necrosis of cancerous cells, toxicity to cells, and accretion of ROS. With sourced on above whole data, this might accomplished that CH-ZnONPs amalgamated Cardiospermum halicacabum appreciably possessed a toxicity to human melanoma cells (A375) via provoking the apoptotic cell necrosis, entailed feasible efficacy of CH-ZnONPs besides malignancy management.


Assuntos
Antineoplásicos/síntese química , Apoptose , Nanopartículas Metálicas/química , Sapindaceae/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Química Verde , Humanos , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Extratos Vegetais/química , Folhas de Planta/química , Folhas de Planta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sapindaceae/metabolismo , Óxido de Zinco/química
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