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The current 5-year survival rate of pancreatic cancer is about 12%, making it one of the deadliest malignancies. The rapid metastasis, acquired drug resistance, and poor patient prognosis necessitate better therapeutic strategies for pancreatic ductal adenocarcinoma (PDAC). Multiple studies show that combining chemotherapeutics for solid tumors has been successful. Targeting two distinct emerging hallmarks, such as non-mutational epigenetic changes by panobinostat (Pan) and delayed cell cycle progression by abemaciclib (Abe), inhibits pancreatic cancer growth. HDAC and CDK4/6 inhibitors are effective but are prone to drug resistance and failure as single agents. Therefore, we hypothesized that combining Abe and Pan could synergistically and lethally affect PDAC survival and proliferation. Multiple cell-based assays, enzymatic activity experiments, and flow cytometry experiments were performed to determine the effects of Abe, Pan, and their combination on PDAC cells and human dermal fibroblasts. Western blotting was used to determine the expression of cell cycle, epigenetic, and apoptosis markers. The Abe-Pan combination exhibited excellent efficacy and produced synergistic effects, altering the expression of cell cycle proteins and epigenetic markers. Pan, alone and in combination with Abe, caused apoptosis in pancreatic cancer cells. Abe-Pan co-treatment showed relative safety in normal human dermal fibroblasts. Our novel combination treatment of Abe and Pan shows synergistic effects on PDAC cells. The combination induces apoptosis, shows relative safety, and merits further investigation due to its therapeutic potential in the treatment of PDAC.
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AIMS: Insulin action is intertwined with changing levels of glucose and counter-regulatory hormone glucagon. While insulin lowers blood sugar level, glucagon raises it by promoting the breakdown of the stored glycogen in liver and releases glucose into the bloodstream. The hormones insulin and glucagon are key in the pathogenesis of type 2 diabetes (T2D). Insulin resistance is a primary predisposing factor for diabetes. Phosphorylation of insulin signaling molecules is altered in the insulin-resistant state. However, ubiquitin (Ub) modifications in insulin-resistant state are relatively understudied. To dissect the underlying mechanisms, we performed a proteomics study on hepatoma cells to study the regulation of ubiquitination by insulin and glucagon. MATERIALS AND METHODS: We performed western blotting, immunoprecipitations, and affinity pull down using tandem Ub binding entities (TUBE) reagents on hepatoma cells treated with insulin or glucagon. Next, we performed MS/MS analysis on Ub-linkage specific affinity pull down samples. Gene ontology analysis and protein-protein interaction network analysis was performed using DAVID GO and STRING db, respectively. KEY FINDINGS: The ubiquitination pattern of total Ub, K48-linked Ub, and K63-linked Ub was altered with the treatment of hormones insulin and glucagon. Ubiquitination in immunoprecipitated samples showed enrichment with total Ub and K48-linked Ub but not with K63-linked Ub. Ubiquitination by treatment with hormones mainly enriched key signaling pathways MAPK, Akt, oxidative stress etc. SIGNIFICANCE: Our study identified key altered proteins and signal transduction pathways which aids in understanding the mechanisms of hormonal action on ubiquitination and identify new therapeutic targets for T2D.
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Carcinoma Hepatocelular , Diabetes Mellitus Tipo 2 , Neoplasias Hepáticas , Humanos , Ubiquitina/metabolismo , Glucagon/metabolismo , Insulina/farmacologia , Insulina/metabolismo , Proteômica , Espectrometria de Massas em Tandem , UbiquitinaçãoRESUMO
PURPOSE: Glioblastoma multiforme (GBM) is a grade IV, highly proliferative, and malignant form of brain tumor with a 5-year survival rate at ~ 5%. Current treatment strategies for GBM include surgery, radiation, and chemotherapy. Major challenges in GBM management include difficulties in surgical resection due to brain's vital functions and GBM metastasis, development of resistance to temozolomide (TMZ), and protection of tumor by blood brain barrier (BBB). Therefore, we aimed to discover a novel therapeutic for GBM by targeting its metabolic reprogramming. METHOD: We screened metabolic inhibitors by their effects on GBM cell viability by MTT assay. We discovered an FDA-approved drug stiripentol (STP) in our screening of metabolic inhibitors in GBM cells. STP is used for Dravet syndrome (a rare epilepsy). We further tested efficacy of STP using proliferation assay, clonogenic assay, in vitro migration assay, cell cycle assay, apoptosis assay, and in U87 3D spheroids. We also tested the toxicity of STP, and combinations used in the study on normal human dermal fibroblasts. RESULTS: STP was effective in decreasing GBM cell viability, proliferation, clonogenic ability, and migration. Moreover, cell cycle changes were involved but robust apoptosis was absent in STP's anticancer effects. STP was effective in 3D spheroid models, and in TMZ-resistant cells. STP showed additive or synergistic effect with TMZ in different anticancer assays on GBM cells and was considerably less toxic in normal cells. CONCLUSION: Our results indicate that STP can be an effective GBM therapeutic that enhances the effects of TMZ on GBM cells. Importantly, STP reduced viability of TMZ-resistant cells. Our results warrant further studies in the mechanistic basis of STP's effects on GBM cells and the preclinical potential of STP in animal models.
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Neoplasias Encefálicas , Glioblastoma , Animais , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Anticonvulsivantes/farmacologia , Reposicionamento de Medicamentos , Linhagem Celular Tumoral , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Apoptose , Resistencia a Medicamentos Antineoplásicos , Antineoplásicos Alquilantes/uso terapêutico , Proliferação de Células , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. There is an urgent need for new targets to treat HCC due to limited treatment options and drug resistance. Many cancer cells are known to have high amount of glycogen than their tissue of origin and inhibition of glycogen catabolism induces cancer cell death by apoptosis. To further understand the role of glycogen in HCC and target it for pharmacotherapy, we studied metabolic adaptations and mitochondrial function in HepG2 cells after pharmacological inhibition of glycogen phosphorylase (GP) by CP-91149 (CP). GP inhibition increased the glycogen levels in HepG2 cells without affecting overall glucose uptake. Glycolytic capacity and importantly glycolytic reserve decreased significantly. Electron microscopy revealed that CP treatment altered mitochondrial morphology leading to mitochondrial swelling with less defined cristae. A concomitant decrease in mitochondrial oxygen consumption and mitochondria-linked ATP generation was observed. Metabolomics and enzyme activity / expression studies showed a decrease in the pentose phosphate pathway. In addition, CP treatment decreased the growth of HepG2 3D tumor spheroids in a dose- and time-dependent manner. Taken together, our study provides insights into metabolic alterations and mitochondrial dysfunction accompanying apoptosis in HepG2 cells upon GP inhibition. Our study can aid in the understanding of the mechanism and development of metabolic inhibitors to treat HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Carcinoma Hepatocelular/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismoRESUMO
This research deals with the development of asialoglycoprotein receptors (ASGPR) directed nanoliposomes incorporating a novel BRD4 (Bromodomain-containing protein 4) protein-targeted PROTAC (Proteolysis Targeting Chimera), ARV-825 (ARV) (GALARV), and to investigate the anticancer efficacy of GALARV for specific delivery in hepatocellular carcinoma. GALARV were prepared using the modified hydration method and characterized for their physicochemical properties as well as anticancer activity using 2D and 3D cell culture models. ARV and GALARV (93.83 ± 10.05 nm) showed significant in vitro cytotoxicity and apoptosis in hepatocellular carcinoma cells. GALARV also demonstrated a substantially higher intracellular concentration of ARV compared to non-targeted nanoliposomes (â¼3 fold) and ARV alone (â¼4.5 fold), showed good physical stability and negligible hemolysis. Immunoblotting results depicted substantial downregulation of target BRD4 protein, oncogenic c-Myc, apoptotic Bcl-2, and survivin proteins. Notably, GALARV treatment resulted in significant apoptosis and subsequent inhibition of the cell viability of 3D tumor spheroids of hepatocellular carcinoma. These results suggest that GALARV is a novel actively targeted PROTAC-based nanotherapeutic approach for hepatocellular carcinoma.
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A novel treatment strategy by co-targeting c-Myc and tumor stroma was explored in vemurafenib-resistant melanoma. BRD4 proteolysis targeting chimera (ARV-825) and nintedanib co-loaded PEGylated nanoliposomes (ARNIPL) were developed to incorporate a synergistic cytotoxic ratio. Both the molecules have extremely poor aqueous solubility. A modified hydration method with citric acid was used to improve the loading of both the molecules in liposomes. ARNIPL with mean particle size 111.1 ± 6.55 nm exhibited more than 90% encapsulation efficiency for both the drugs and was found to be physically stable for a month at 4 °C. Both the molecules and ARNIPL showed significantly higher cytotoxicity, apoptosis and down-regulation of target proteins BRD4 and c-Myc in vemurafenib-resistant cell line (A375R). Vasculogenic mimicry and clonogenic potential of A375R were significantly inhibited by ARNIPL. Tumor growth inhibition in 3D spheroids with reduction of TGF-ß1 was observed with ARNIPL treatment. Therefore, ARNIPL could be a promising therapeutic approach for the treatment of vemurafenib-resistant melanoma.
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Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease - a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.
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Doença de Lafora , Propanolaminas , Terapia Genética , Células HEK293 , Humanos , Doença de Lafora/genética , Doença de Lafora/terapia , Proteínas Tirosina Fosfatases não Receptoras/genéticaRESUMO
Gankyrin is an oncoprotein overexpressed in numerous cancer types and appears to play a key role in regulating cell proliferation, cell growth, and cell migration. These roles are largely due to gankyrin's protein-protein interaction with the 26S proteasome. We previously published a study exploring the aryl sulfonate ester of cjoc42 in an effort to enhance gankyrin binding and inhibit cancer cell proliferation. In order to further improve the gankyrin binding ability of the cjoc42 scaffold, an extensive SAR for the aryl-triazole moiety of cjoc42 was developed. Our cjoc42 derivatives exhibited enhanced gankyrin binding, as well as enhanced antiproliferative activity against Hep3B, HepG2, A549, and MDA-MB-231 cancer cell lines.
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Antineoplásicos/química , Benzenossulfonatos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Triazóis/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzenossulfonatos/metabolismo , Benzenossulfonatos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Dinâmica Molecular , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Relação Estrutura-Atividade , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Aim: To explore the anticancer activity of a novel BRD4 protein degrader ARV-825 (ARV) and its nanoformulation development (ARV-NP) for treatment of pancreatic cancer. Materials & methods: ARV-NP were prepared using nanoprecipitation method and characterized for their physicochemical properties and various anticancer cell culture assays. Results: ARV-NP (89.63 ± 16.39 nm) demonstrated good physical stability, negligible hemolysis and improved half-life of ARV. ARV-NP showed significant cytotoxicity, apoptosis and anticlonogenic effect in pancreatic cancer cells. Significant downregulation of target proteins BRD4, c-Myc, Bcl-2 and upregulation of apoptotic marker cleaved caspase-3 was observed. Most importantly, ARV-NP treatment significantly inhibited the cell viability of 3D tumor spheroids of pancreatic cancer. Conclusion: ARV-NP represents a novel therapeutic strategy for pancreatic cancer.
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Proteínas Nucleares , Neoplasias Pancreáticas , Humanos , Apoptose , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteólise , Fatores de Transcrição/metabolismoRESUMO
Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.
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Antineoplásicos/química , Benzenossulfonatos/química , Ésteres/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ácidos Sulfônicos/química , Triazóis/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzenossulfonatos/síntese química , Benzenossulfonatos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
Autophagy is an evolutionarily conserved cellular mechanism in eukaryotes that plays an important role in the maintenance of cellular homeostasis. The autophagy process maintains protein homeostasis by recycling damaged organelles and degrading many long-lived proteins in conjunction with the ubiquitin-proteasome system. Cytokines are low-molecular-weight secreted proteins that regulate a broad range of biological activities. For instance, pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce inflammation, autophagy, and apoptotic cell death. In this chapter, we discuss experimental techniques such as immunoblotting and fluorescence microscopy that can be utilized to measure autophagy in response to TNFα treatment.
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Autofagia , Bioensaio/métodos , Fator de Necrose Tumoral alfa/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , UbiquitinaRESUMO
BACKGROUND AND OBJECTIVE: Drug resistance and adverse effects are immense healthcare challenges in cancer therapy. Benzimidazole ring-based small molecules have been effective anticancer agents in drug development. In an effort to develop novel chemotherapeutics, we synthesized and assessed the anticancer and antibacterial activities of a small library of structurally unique benzimidazoles. METHODS: The benzimidazoles were derived from indole, N-alkyl indole, fatty acid, and alpha-amino acid scaffolds providing a panel of diverse structures. The compounds were tested in three different cancer cell lines for cytotoxicity: HepG2 (human hepatocellular carcinoma), HeLa (human cervical carcinoma), and A549 (human lung carcinoma). Mechanism of cell death induced by benzimidazoles was evaluated using fluorescent dye-based apoptosis-necrosis assay, immunoblotting for active caspases, topoisomerase-II activity assay, and cell cycle assay. RESULTS: Cell viability testing revealed that indole- and fatty acid-based benzimidazoles were most potent followed by the amino acid derivatives. Many compounds induced cytotoxicity in a concentration-dependent manner with cellular cytotoxicity (CC50) <20µM in the cell lines tested. Most compounds exhibited cytotoxicity via apoptosis through the intrinsic pathway. Inhibition of topoisomerase activity and cell cycle alterations were not the primary mechanisms of cytotoxicity. In addition, several compounds showed promising activity against S. aureus and S. epidermidis (Minimum Inhibitory Concentration (MIC) of as low as 0.04µmol/mL). CONCLUSION: The reported benzimidazole derivatives possess promising anticancer and antibacterial properties. Additionally, we discovered apoptosis to be the primary mechanism for cancer cell death induced by the tested benzimidazoles. Our findings suggest that further development of these scaffolds could provide drug leads towards new chemotherapeutics.
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Antibacterianos/síntese química , Antineoplásicos/síntese química , Benzimidazóis/síntese química , Células A549 , Aminoácidos/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , DNA Topoisomerases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/efeitos dos fármacos , Ácidos Graxos/química , Células HeLa , Células Hep G2 , Humanos , Indóis/química , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase/síntese química , Inibidores da Topoisomerase/farmacologiaRESUMO
The clinical outcomes of malignant melanoma have improved with the introduction of mitogen-activated protein kinase kinase (MEK) inhibitors. However, off-target toxicities of the MEK inhibitor trametinib (TMB) often result in dose interruption and discontinuation of therapy. The purpose of this study was to anchor a physically stable EphrinA1-mimicking peptide known as YSA (YSAYPDSVPMMS) on TMB-loaded PEGylated nanoliposomes (YTPLs), and evaluate them in BRAFV600E-mutated parent cells (lines A375 and SK-MEL-28) and vemurafenib-resistant cells lines (A375R and SK-MEL-28R) in melanoma. TMB-loaded PEGylated liposomes (TPL) functionalized with nickel-chelated phospholipids were prepared using a modified hydration method. The hydrodynamic diameter and zeta potential values of optimized YTPL were 91.20 ± 12.16 nm and -0.92 ± 3.27 mV, respectively. The drug release study showed TPL did not leak or burst release in 24 h. The hemolysis observed was negligible at therapeutic concentrations of TMB. A differential scanning calorimetry (DSC) study confirmed that TMB was retained in a solubilized state within lipid bilayers. YTPL showed higher intracellular uptake in parental cell lines compared to vemurafenib-resistant cell lines. Western blot analysis and a cytotoxicity study with the EphA2 inhibitor confirmed a reduction in EphA2 expression in resistant cell lines. Thus, EphA2 receptor-targeted nanoliposomes can be useful for metastatic melanoma-specific delivery of TMB.
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Nuclear glycogen was first documented in the early 1940s, but its role in cellular physiology remained elusive. In this study, we utilized pure nuclei preparations and stable isotope tracers to define the origin and metabolic fate of nuclear glycogen. Herein, we describe a key function for nuclear glycogen in epigenetic regulation through compartmentalized pyruvate production and histone acetylation. This pathway is altered in human non-small cell lung cancers, as surgical specimens accumulate glycogen in the nucleus. We demonstrate that the decreased abundance of malin, an E3 ubiquitin ligase, impaired nuclear glycogenolysis by preventing the nuclear translocation of glycogen phosphorylase and causing nuclear glycogen accumulation. Re-introduction of malin in lung cancer cells restored nuclear glycogenolysis, increased histone acetylation, and decreased growth of cancer cells transplanted into mice. This study uncovers a previously unknown role for glycogen metabolism in the nucleus and elucidates another mechanism by which cellular metabolites control epigenetic regulation.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Núcleo Celular/metabolismo , Glicogenólise/genética , Histonas/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Acetilação , Animais , Carbono/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Glicogênio/biossíntese , Glicogênio Fosforilase/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is one of the leading cancers in the world in incidence and mortality. Current pharmacotherapy of HCC is limited in the number and efficacy of anticancer agents. Metabolic reprogramming is a prominent feature of many cancers and has rekindled interest in targeting metabolic proteins for cancer therapy. Glycogen is a storage form of glucose, and the levels of glycogen have been found to correlate with biological processes in reprogrammed cancer cells. However, the contribution of glycogen metabolism to carcinogenesis, cancer cell growth, metastasis, and chemoresistance is poorly understood. Thus, we studied the processes involved in the inhibition of glycogen metabolism in HCC cells. Pharmacological inhibition of glycogen phosphorylase (GP), a rate-limiting enzyme in glycogen catabolism, by CP-91149 led to a decrease in HCC cell viability. GP inhibition induced cancer cell death through the intrinsic apoptotic pathway. Mitochondrial dysfunction and autophagic adaptations accompanied this apoptosis process whereas endoplasmic reticulum stress, necrosis, and necroptosis were not major components of the cell death. In addition, GP inhibition potentiated the effects of multikinase inhibitors sorafenib and regorafenib, which are key drugs in advanced-stage HCC therapy. Our study provides mechanistic insights into cell death by perturbation of glycogen metabolism and identifies GP inhibition as a potential HCC pharmacotherapy target.
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Amidas/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Glicogênio/metabolismo , Indóis/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Amidas/administração & dosagem , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Glicogênio Fosforilase/antagonistas & inibidores , Células Hep G2 , Humanos , Indóis/administração & dosagem , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Piridinas/administração & dosagem , Piridinas/farmacologia , Ratos , Sorafenibe/administração & dosagem , Sorafenibe/farmacologiaRESUMO
Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.
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Apoptose/efeitos dos fármacos , Glutationa/metabolismo , Mitocôndrias/efeitos dos fármacos , Neurônios/fisiologia , Apoptose/fisiologia , Azóis/farmacologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocromos c/metabolismo , Citosol/metabolismo , Ácido Etacrínico/farmacologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Isoindóis , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Compostos Organosselênicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Recent evidence suggests silicon dioxide micro- and nanoparticles induce cytotoxic effects on lung cells. Thus, there is an increasing concern regarding their potential health hazard. Nevertheless, the putative toxicity of nanoparticles in mammalian cells has not yet been systematically investigated. We previously noted that several metallic oxide nanoparticles exert differential cytotoxic effects on human neural and nonneural cells. Therefore, we hypothesized that silicon dioxide nanoparticles induce cytotoxicity in U87 cells by lowering their survival by decreasing cell survival signaling and disturbing mitochondrial function. To investigate this hypothesis, we determined the activities of the key mitochondrial enzymes, citrate synthase and malate dehydrogenase, in astrocytoma U87 cells treated with silicon dioxide nanoparticles. In addition, we studied the expression of the mitochondrial DNA-encoded proteins, cytochrome C oxidase II and nicotinamide adenine dinucleotide (NADPH) dehydrogenase subunit 6, and cell signaling pathway protein extracellular signal-regulated kinase (ERK) and phosphorylated ERK in treated U87 cells. The activated form of ERK controls cell growth, differentiation, and proliferation. In parallel, we determined survival of U87 cells after treating them with various concentrations of silicon dioxide nanoparticles. Our results indicated that treatment with silicon dioxide nanoparticles induced decreases in U87 cell survival in a dose-related manner. The activities of citrate synthase and malate dehydrogenase in treated U87 cells were increased, possibly due to an energetic compensation in surviving cells. However, the expression of mitochondrial DNA-encoded cytochrome C oxidase subunit II and NADH dehydrogenase subunit 6 and the cell signaling protein ERK and phosphorylated ERK were altered in the treated U87 cells, suggesting that silicon dioxide nanoparticles induced disruption of mitochondrial DNA-encoded protein expression, leading to decreased mitochondrial energy production and decreased cell survival/proliferation signaling. Thus, our results strongly suggest that the cytotoxicity of silicon dioxide nanoparticles in human neural cells implicates altered mitochondrial function and cell survival/proliferation signaling.
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Astrocitoma/metabolismo , Astrocitoma/patologia , Nanopartículas/toxicidade , Neurônios/efeitos dos fármacos , Dióxido de Silício/toxicidade , Astrocitoma/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA Mitocondrial/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nanomedicina , Nanopartículas/química , Neurônios/metabolismo , Neurônios/patologia , Dióxido de Silício/administração & dosagemRESUMO
Oxidative stress during development may predispose humans to neurodegenerative disorders in old age. Moreover, numerous ailments of brain disproportionately affect one of the genders. We therefore hypothesized that, activities of enzymes regenerating and utilizing glutathione (GSH) show sexual dimorphism and developmental differences in rat brain. To test this hypothesis, we collected cortex tissue from male and female Sprague-Dawley rats at post-natal day (PN) 5, PN 10, PN 20, PN 30, and PN 60. We measured tissue levels of NADP-linked isocitrate dehydrogenase (NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), and, glutathione reductase (GR) by UV spectrophotometry and determined glutathione peroxidase (GPx) expression therein by western blotting. Our results showed that sexual maturation had an impact on activities of enzymes that regenerate and utilize GSH and rat female cortex had more anti-oxidant capacity. Moreover, age-related decline in the activities of these key enzymes were observed. Reduced glutathione and NADPH protects the brain from oxidative stress. Thus, our results may have implications for neurodegenerative disorders like Parkinson's disease and developmental disorders of brain like autism in which oxidative stress plays a key role.
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Córtex Cerebral/enzimologia , Córtex Cerebral/crescimento & desenvolvimento , Glutationa/metabolismo , Caracteres Sexuais , Animais , Western Blotting , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Isocitrato Desidrogenase/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The use of titanium dioxide (TiO(2)) in various industrial applications (eg, production of paper, plastics, cosmetics, and paints) has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO(2) nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO(2) micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO) nanoparticles were the most effective, TiO(2) nanoparticles the second most effective, and magnesium oxide (MgO) nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO(2) micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.
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Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Teste de Materiais , Titânio/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Metais/toxicidade , Óxidos/toxicidadeRESUMO
Oxidative stress can induce mitochondrial dysfunction, mitochondrial DNA (mtDNA) depletion, and neurodegeneration, although the underlying mechanisms are poorly understood. The major mitochondrial antioxidant system that protects cells consists of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) and glutathione (GSH). To investigate the putative adaptive changes in antioxidant enzyme protein expression and targeting to mitochondria as mtDNA depletion occurs, we progressively depleted U87 astrocytoma cells of mtDNA by chronic treatment with ethidium bromide (EB, 50 ng/ml). Cellular MnSOD protein expression was markedly increased in a time-related manner while that of GPx showed time-related decreases. The mtDNA depletion also altered targeting or subcellular distribution of GPx, suggesting the importance of intact mtDNA in mitochondrial genome-nuclear genome signaling/communication. Cellular NADP(+)-ICDH activity also showed marked, time-related increases while their GSH content decreased. Thus, our findings suggest that interventions to elevate MnSOD, GPx, NADP(+)-ICDH, and GSH levels may protect brain cells from oxidative stress.