RESUMEN
Intervertebral disc degeneration (IDD) is an important pathological basis for degenerative spinal diseases and is involved in mitophagy dysfunction. However, the molecular mechanisms underlying mitophagy regulation in IDD remain unclear. This study aimed to clarify the role of DJ-1 in regulating mitophagy during IDD pathogenesis. Here, we showed that the mitochondrial localization of DJ-1 in nucleus pulposus cells (NPCs) first increased and then decreased in response to oxidative stress. Subsequently, loss- and gain-of-function experiments revealed that overexpression of DJ-1 in NPCs inhibited oxidative stress-induced mitochondrial dysfunction and mitochondria-dependent apoptosis, whereas knockdown of DJ-1 had the opposite effect. Mechanistically, mitochondrial translocation of DJ-1 promoted the recruitment of hexokinase 2 (HK2) to damaged mitochondria by activating Akt and subsequently Parkin-dependent mitophagy to inhibit oxidative stress-induced apoptosis in NPCs. However, silencing Parkin, reducing mitochondrial recruitment of HK2, or inhibiting Akt activation suppressed DJ-1-mediated mitophagy. Furthermore, overexpression of DJ-1 ameliorated IDD in rats through HK2-mediated mitophagy. Taken together, these findings indicate that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NPCs and could be a therapeutic target for IDD.
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Degeneración del Disco Intervertebral , Mitofagia , Proteína Desglicasa DJ-1 , Animales , Ratas , Apoptosis , Hexoquinasa/genética , Hexoquinasa/farmacología , Hexoquinasa/uso terapéutico , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/metabolismo , Mitofagia/genética , Mitofagia/fisiología , Proteínas Proto-Oncogénicas c-akt , Ubiquitina-Proteína Ligasas/genética , Proteína Desglicasa DJ-1/metabolismoRESUMEN
Neuropathic pain is a complex pain condition accompanied by prominent neuroinflammation involving activation of both central and peripheral immune cells. Metabolic switch to glycolysis is an important feature of activated immune cells. Hexokinase 2 (HK2), a key glycolytic enzyme enriched in microglia, has recently been shown important in regulating microglial functions. Whether and how HK2 is involved in neuropathic pain-related neuroinflammation remains unknown. Using a HK2-tdTomato reporter line, we found that HK2 was prominently elevated in spinal microglia. Pharmacological inhibition of HK2 effectively alleviated nerve injury-induced acute mechanical pain. However, selective ablation of Hk2 in microglia reduced microgliosis in the spinal dorsal horn (SDH) with little analgesic effects. Further analyses showed that nerve injury also significantly induced HK2 expression in dorsal root ganglion (DRG) macrophages. Deletion of Hk2 in myeloid cells, including both DRG macrophages and spinal microglia, led to the alleviation of mechanical pain during the first week after injury, along with attenuated microgliosis in the ipsilateral SDH, macrophage proliferation in DRGs, and suppressed inflammatory responses in DRGs. These data suggest that HK2 plays an important role in regulating neuropathic pain-related immune cell responses at acute phase and that HK2 contributes to neuropathic pain onset primarily through peripheral monocytes and DRG macrophages rather than spinal microglia.
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Neuralgia , Traumatismos de los Nervios Periféricos , Humanos , Microglía/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Enfermedades Neuroinflamatorias , Hiperalgesia/metabolismo , Macrófagos/metabolismo , Neuralgia/metabolismo , Ganglios Espinales/metabolismo , Médula Espinal/metabolismo , Traumatismos de los Nervios Periféricos/metabolismoRESUMEN
Evidence has demonstrated that oxidative stress plays a crucial role in regulating cellular glucose metabolism. In previous studies, wheat germ peptide (WGP) was found to effectively mitigate oxidative stress induced by high glucose. Based on the information provided, we hypothesized that WGP could exhibit antihyperglycemic and anti-insulin-resistant effects in cells. The insulin-resistant cell model was established by insulin stimulation. The glucose consumption, glycogen content, and the activities of hexokinase and pyruvate kinase following WGP treatment were measured. The protein expression of SOCS3, phosphorylated insulin receptor substrate-1 (p-IRS1), IRS1, phosphorylated protein kinase B (p-Akt), Akt, glucose transporter 2 (GLUT2), phosphorylated GSK 3ß, GSK 3ß, FOXO1, G6P, and phosphoenolpyruvate carboxykinase were assessed by western blot analysis. Our results demonstrated that WGP treatment increased cellular glucose consumption and glycogen synthesis and enhanced hexokinase and pyruvate kinase activities. Additionally, WGP treatment was observed to cause a significant reduction in the expression of SOCS3, FOXO1, G6P, and phosphoenolpyruvate carboxykinase, as well as in the ratio of p-IRS1/IRS1. Conversely, the expression of GLUT2 and the ratios of p-Akt/Akt and p-GSK3ß/GSK3ß were upregulated by WGP. These findings suggested that WGP can activate the SOCS3/IRS1/Akt signaling pathway, thus promoting the phosphorylation of GSK-3ß and increasing the expression of FOXO1 and GLUT2, which contribute to enhancing glycogen synthesis, inhibiting gluconeogenesis, and promoting glucose transport in insulin-resistant HepG2 cells.
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Resistencia a la Insulina , Proteínas Proto-Oncogénicas c-akt , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/farmacología , Triticum , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Piruvato Quinasa/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/farmacología , Hepatocitos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Glucógeno/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismoRESUMEN
BACKGROUND: Therapies for cholangiocarcinoma are largely limited and ineffective. Herein, we examined the role of the FGF and VEGF pathways in regulating lymphangiogenesis and PD-L1 expression in intrahepatic cholangiocarcinoma (iCCA). METHODS: The lymphangiogenic functions of FGF and VEGF were evaluated in lymphatic endothelial cells (LECs) and iCCA xenograft mouse models. The relationship between VEGF and hexokinase 2 (HK2) was validated in LECs by western blot, immunofluorescence, ChIP and luciferase reporter assays. The efficacy of the combination therapy was assessed in LECs and xenograft models. Microarray analysis was used to evaluate the pathological relationships of FGFR1 and VEGFR3 with HK2 in human lymphatic vessels. RESULTS: FGF promoted lymphangiogenesis through c-MYC-dependent modulation of HK2 expression. VEGFC also upregulated HK2 expression. Mechanistically, VEGFC phosphorylated components of the PI3K/Akt/mTOR axis to upregulate HIF-1α expression at the translational level, and HIF-1α then bound to the HK2 promoter region to activate its transcription. More importantly, dual FGFR and VEGFR inhibition with infigratinib and SAR131675 almost completely inhibited lymphangiogenesis, and significantly suppressed iCCA tumor growth and progression by reducing PD-L1 expression in LECs. CONCLUSIONS: Dual FGFR and VEGFR inhibition inhibits lymphangiogenesis through suppression of c-MYC-dependent and HIF-1α-mediated HK2 expression, respectively. HK2 downregulation decreased glycolytic activity and further attenuated PD-L1 expression. Our findings suggest that dual FGFR and VEGFR blockade is an effective novel combination strategy to inhibit lymphangiogenesis and improve immunocompetence in iCCA.
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Colangiocarcinoma , Linfangiogénesis , Humanos , Ratones , Animales , Antígeno B7-H1/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/patologíaRESUMEN
BACKGROUND: Copper is a micronutrient vital to several cellular energy metabolic processes and drives erythropoiesis. However, it disrupts cellular biological activities and causes oxidative damage when in excess of cellular needs. This study investigated the effects of copper toxicity on erythrocyte energy metabolism in male Wistar rats. METHODS: Ten Wistar rats (150-170 g) were randomly divided into 2 groups: control (given 0.1 ml distilled water) and copper toxic (given 100 mg/kg copper sulphate). Rats were orally treated for 30 days. Blood, collected retro-orbitally after sodium thiopentone anaesthesia (50 mg/kg i.p.) into fluoride oxalate and EDTA bottles, was subjected to blood lactate assay and extraction of red blood cell respectively. Red blood cell nitric oxide (RBC NO), glutathione (RBC GSH), adenosine triphosphate (RBC ATP) levels, RBC hexokinase, glucose-6-phosphate (RBC G6P), glucose-6-phosphate dehydrogenase (RBC G6PDH), and lactate dehydrogenase (RBC LDH) activity was estimated spectrophotometrically. Values (Mean±SEM, n = 5) were compared by Student's unpaired T-test at p < 0.05. RESULTS AND CONCLUSION: Copper toxicity significantly increased RBC hexokinase (23.41 ± 2.80 µM), G6P (0.48 ± 0.03 µM), G6PDH (71.03 ± 4.76nmol/min/ml) activities, ATP (624.70 ± 57.36 µmol/gHb) and GSH (3.08 ± 0.37 µM) level compared to control (15.28 ± 1.37 µM, 0.35 ± 0.02 µM, 330.30 ± 49.58 µmol/gHb, 54.41 ± 3.01nmol/min/ml and 2.05 ± 0.14 µM respectively, p < 0.05). Also, RBC LDH activity (145.00 ± 19.88mU/ml), NO (3.45 ± 0.25 µM) and blood lactate (31.64 ± 0.91 mg/dl) level were lowered significantly compared to control (467.90 ± 94.23mU/ml, 4.48 ± 0.18 µM and 36.12 ± 1.06 mg/dl respectively). This study shows that copper toxicity increases erythrocyte glycolytic rate and glutathione production. This increase could be connected to a compensatory mechanism for cellular hypoxia and increased free radical generation.
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Cobre , Oxibato de Sodio , Masculino , Ratas , Animales , Ratas Wistar , Cobre/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Oxibato de Sodio/metabolismo , Oxibato de Sodio/farmacología , Eritrocitos/metabolismo , Adenosina Trifosfato/metabolismo , Glutatión/metabolismo , Lactatos/metabolismo , Lactatos/farmacología , Equilibrio HidroelectrolíticoRESUMEN
BACKGROUND: Sestrins (SESN1-3) act as proximal sensors in leucine-induced activation of the protein kinase mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), a key regulator of cell growth and metabolism. OBJECTIVE: In the present study, the hypothesis that SESNs also mediate glucose-induced activation of mTORC1 was tested. METHODS: Rats underwent overnight fasting, and in the morning, either saline or a glucose solution (4 gâ kg-1 BW/10 mLâ kg-1) was administered by oral gavage; mTORC1 activation in the tibialis anterior muscle was assessed. To further assess the mechanism through which glucose promotes mTORC1 activation, wild-type (WT) HEK293T and HEK293T cells lacking either all 3 SESNs (SESNTKO) or hexokinase 2 (HK2KO) were deprived of glucose, followed by glucose addback, and mTORC1 activation was assessed. In addition, glucose-induced changes in the association of the SESNs with components of the GAP activity toward the Rags (GATOR2) complex and with hexokinase 2 (HK2) were assessed by co-immunoprecipitation. One- and two-way ANOVA with Tukey post hoc comparisons were used. RESULTS: Glucose administration to fasted rats promoted mTORC1 activation. Similarly, glucose readdition (GluAB) to the medium of glucose-deprived WT cells also promoted mTORC1 activation. By contrast, SESNTKO cells demonstrated attenuated mTORC1 activation following GluAB compared with WT cells. Interestingly, HK2 associated with all 3 SESNs in a glucose-dependent manner, i.e., HK2 abundance in SESN immunoprecipitates was high in cells deprived of glucose and decreased in response to GluAB. Moreover, similar to SESNTKO cells, the sensitivity of mTORC1 to GluAB was attenuated in HK2KO cells compared with WT cells. CONCLUSIONS: The results of this study demonstrate that the SESNs and HK2 play important roles in glucose-induced mTORC1 activation in HEK293T cells. However, unlike leucine-induced mTORC1 activation, the effect was independent of the changes in SESN-GATOR2 interaction, and instead, it was associated with alterations in the association of SESNs with HK2.
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Transducción de Señal , Serina-Treonina Quinasas TOR , Ratas , Animales , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células HEK293 , Serina-Treonina Quinasas TOR/metabolismo , Leucina/farmacología , Sestrinas/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Glucosa/farmacologíaRESUMEN
BACKGROUND: Silencing specific genes in pests using RNA interference (RNAi) technology is a promising new pest-control strategy. The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important citrus pest because it transmits Candidatus Liberibacter asiaticus, which causes huanglongbing. Chitin is essential for insect development, and enzymes in this pathway are attractive targets for pest control. RESULTS: The hexokinase gene DcHK was characterized from D. citri to impair proper growth and chitin synthesis through RNAi. The transcription of DcHK was more highly developed in third-instar nymphs, adults and the Malpighian tube. The RNAi needed for D. citri is dose-dependent, with 600 ng µl-1 dsDcHK sufficient to knockdown endogenous DcHK expression. The messenger RNA (mRNA) level was lowest at 36 h after dosing, and there were significant effects on the relative levels of mRNA in the chitin synthesis pathway (DcTre, DcG6PI, DcGNAT, DcGFAT, DcPGM, DcUAP and DcCHS), leading to mortality, reduced body weight and abnormal or lethal phenotypes. CONCLUSION: RNAi can be triggered by orally delivered double-stranded RNA in D. citri. These results can provide support for HK genes as a new potential target for citrus psyllid control. © 2022 Society of Chemical Industry.
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Citrus , Hemípteros , Animales , Quitina , Citrus/genética , Hexoquinasa/genética , Hexoquinasa/farmacología , Fenotipo , Enfermedades de las Plantas , Interferencia de ARN , ARN MensajeroRESUMEN
The aryl hydrocarbon receptor (AHR) regulates the expression of numerous genes in response to activation by agonists including xenobiotics. Although it is well appreciated that environmental signals and cell intrinsic features may modulate this transcriptional response, how it is mechanistically achieved remains poorly understood. We show that hexokinase 2 (HK2) a metabolic enzyme fuelling cancer cell growth, is a transcriptional target of AHR as well as a modulator of its activity. Expression of HK2 is positively regulated by AHR upon exposure to agonists both in human cells and in mice lung tissues. Conversely, over-expression of HK2 regulates the abundance of many proteins involved in the regulation of AHR signalling and these changes are linked with altered AHR expression levels and transcriptional activity. HK2 expression also shows a negative correlation with AHR promoter methylation in tumours, and these tumours with high HK2 expression and low AHR methylation are associated with a worse overall survival in patients. In sum, our study provides novel insights into how AHR signalling is regulated which may help our understanding of the context-specific effects of this pathway and may have implications in cancer.
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Hexoquinasa , Receptores de Hidrocarburo de Aril , Animales , Hexoquinasa/genética , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Humanos , Ratones , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , XenobióticosRESUMEN
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. In this study, we induced a young-adult PCOS rat model by oral administration of letrozole combined with a high-fat diet and then treated with mogroside V (MV) to evaluate the protective effects of MV on endocrine and follicle development in young-adult PCOS rats. MV (600 mg/kg/day) administration not only significantly reduced the body weight and ovary weight, but also attenuated the disrupted estrous cycle and decreased the level of testosterone. MV restored the follicular development, especially by increasing the number of corpus luteum and the thickness of the granular layer in young-adult POCS rats. Moreover, metabolomics showed that MV markedly increased the levels of D-Glucose 6-phosphate, lactate and GTP, while decreased the level of pyruvate. Bioinformatic analysis revealed that MV recovered multiple metabolism-related processes including gluconeogenesis, glycolysis and glucose metabolic process. Further real-time quantitative PCR analysis showed that MV upregulated the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Western blotting and immunohistochemistry analysis showed that MV restored the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Collectively, these findings indicated that MV could effectively improve the ovarian microenvironment by upregulating the expression of LDHA, HK2 and PKM2 in granulosa cells and enhancing lactate and energy production, which may contribute to follicle development and ovulation of young-adult PCOS rats.
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Síndrome del Ovario Poliquístico , Animales , Dieta Alta en Grasa/efectos adversos , Femenino , Glucólisis , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Humanos , Lactato Deshidrogenasa 5 , Ácido Láctico/efectos adversos , Letrozol , Ovulación , Síndrome del Ovario Poliquístico/inducido químicamente , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Síndrome del Ovario Poliquístico/metabolismo , Piruvato Quinasa/metabolismo , Piruvato Quinasa/farmacología , Ratas , Triterpenos , Microambiente TumoralRESUMEN
Rodent studies highlight enhancement of glucose tolerance and insulin sensitivity as potential clinically relevant effects of chronic beta2 -agonist treatment. However, the doses administered to rodents are not comparable with the therapeutic doses used for humans. Thus, we investigated the physiological effects of prolonged beta2 -agonist treatment at inhaled doses resembling those used in respiratory diseases on insulin-stimulated whole-body glucose disposal and putative mechanisms in skeletal muscle and adipose tissue of healthy men. Utilizing a randomized placebo-controlled parallel-group design, we assigned 21 healthy men to 4 weeks daily inhalation of terbutaline (TER; 4 mg × day-1 , n = 13) or placebo (PLA, n = 8). Before and after treatments, we assessed subjects' whole-body insulin-stimulated glucose disposal and body composition, and collected vastus lateralis muscle and abdominal adipose tissue biopsies. Glucose infusion rate increased by 27% (95% CI: 80 to 238 mg × min-1 , P = 0.001) in TER, whereas no significant changes occurred in PLA (95% CI: -37 to 195 mg × min-1 , P = 0.154). GLUT4 content in muscle or adipose tissue did not change, nor did hexokinase II content or markers of mitochondrial volume in muscle. Change in lean mass was associated with change in glucose infusion rate in TER (r = 0.59, P = 0.03). Beta2 -agonist treatment in close-to-therapeutic doses may augment whole-body insulin-stimulated glucose disposal in healthy young men and part of the change is likely to be explained by muscle hypertrophy. These findings highlight the therapeutic potential of beta2 -agonists for improving insulin sensitivity. KEY POINTS: While studies in rodents have highlighted beta2 -agonists as a means to augment insulin sensitivity, these studies utilized beta2 -agonists at doses inapplicable to humans. Herein we show that a 4-week treatment period with daily therapeutic inhalation of beta2 -agonist increases insulin-stimulated whole-body glucose disposal in young healthy lean men. This effect was associated with an increase of lean mass but not with changes in GLUT4 and hexokinase II or basal glycogen content in skeletal muscle nor GLUT4 content in abdominal adipose tissue. These findings suggest that the enhanced insulin-stimulated whole-body glucose disposal induced by a period of beta2 -agonist treatment in humans, at least in part, is attributed to muscle hypertrophy. Our observations extend findings in rodents and highlight the therapeutic potential of beta2 -agonists to enhance the capacity for glucose disposal and whole-body insulin sensitivity, providing important knowledge with potential application in insulin resistance.
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Glucosa , Resistencia a la Insulina , Agonistas de Receptores Adrenérgicos beta 2 , Glucosa/farmacología , Hexoquinasa/farmacología , Humanos , Hipertrofia , Insulina/farmacología , Músculo Esquelético , Poliésteres/farmacologíaRESUMEN
Psychosis is a state of altered thoughts which often accompanies schizophrenia. It was suggested that changes in energetic metabolism accompany psychosis and post-psychosis states. Here, we use the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 to experimentally induce psychosis-like behavior in rats. We addressed an effect of single and repeated (5×) MK-801 application (0.3â¯mg/kg; i.p.) on the energy metabolism in homogenates and crude mitochondrial fraction (CMF) of the striatum (STR), prefrontal cortex (PFC), and the hippocampus (HIP) of the adult male Wistar rat (nâ¯=â¯39). In each brain region, we assessed activity of glycolytic (hexokinase (HK) and lactate dehydrogenase (LDH)) and Krebs cycle enzymes (citrate synthase (CS) and malate dehydrogenase (MDH)) 2â¯h and 3 days (3d) after the last MK-801 application together with relative respiratory rates assessment in tissue homogenate. In STR, a single MK-801 application led to a decrease in the LDH (pâ¯=â¯0.0035) and the increase of the MDH (pâ¯=â¯0.0043) activities following 3d. Therein, repeated MK-801 doses evoked increased LDH (pâ¯=â¯0.0204) and CS (pâ¯=â¯0.0019) activities in the homogenate 2â¯h and increased HK (pâ¯=â¯0.0007) 3d after the last application. Elevated HK activity within CMF was observed after 3d (pâ¯=â¯0.0054). In PFC, repeated MK-801 application decreased HK activity in the homogenate 3d after the final application (pâ¯=â¯0.0234). Correspondingly, PFC HK activity in CMF of repeated administration samples dropped (pâ¯=â¯0.003). In HIP, repeated MK-801 administration led to increased respiration of SDH (pâ¯=â¯0.0475) only 2â¯h after the last application and decreased CS activity (pâ¯=â¯0.0160) was observed 3d after the last application. Our results indicate a progressive metabolic dysregulation of glycolytic and Krebs cycle enzymes following repeated inhibition of NMDA receptors activity in a region-specific manner. Energetic alterations may form a basis for persisting cognitive problems during and following a psychosis in schizophrenia patients.
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Maleato de Dizocilpina , N-Metilaspartato , Animales , Citrato (si)-Sintasa/metabolismo , Citrato (si)-Sintasa/farmacología , Ciclo del Ácido Cítrico , Maleato de Dizocilpina/farmacología , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Hipocampo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , N-Metilaspartato/farmacología , Corteza Prefrontal , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
BACKGROUND: Most cancers favor glycolytic-based glucose metabolism. Hexokinase-2 (HK2), the first glycolytic rate-limiting enzyme, shows limited expression in normal adult tissues but is overexpressed in many tumor tissues, including ovarian cancer. HK2 has been shown to be correlated with the progression and chemoresistance of ovarian cancer and could be a therapeutic target. However, the systemic toxicity of HK2 inhibitors has limited their clinical use. Since follicle-stimulating hormone (FSH) receptor (FSHR) is overexpressed in ovarian cancer but not in nonovarian healthy tissues, we designed FSHR-mediated nanocarriers for HK2 shRNA delivery to increase tumor specificity and decrease toxicity. RESULTS: HK2 shRNA was encapsulated in a polyethylene glycol-polyethylenimine copolymer modified with the FSH ß 33-53 or retro-inverso FSH ß 33-53 peptide. The nanoparticle complex with FSH peptides modification effectively depleted HK2 expression and facilitated a shift towards oxidative glucose metabolism, with evidence of increased oxygen consumption rates, decreased extracellular acidification rates, and decreased extracellular lactate and glucose consumption in A2780 ovarian cancer cells and cisplatin-resistant A2780CP counterpart cells. Consequently, cell proliferation, invasion and migration were significantly inhibited, and tumor growth was suppressed even in cisplatin-resistant ovarian cancer. No obvious systemic toxicity was observed in mice. Moreover, the nanoparticle complex modified with retro-inverso FSH peptides exhibited the strongest antitumor effects and effectively improved cisplatin sensitivity by regulating cisplatin transport proteins and increasing apoptosis through the mitochondrial pathway. CONCLUSIONS: These results established HK2 as an effective therapeutic target even for cisplatin-resistant ovarian cancer and suggested a promising targeted therapeutic approach.
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Antineoplásicos/farmacología , Glucosa/metabolismo , Hexoquinasa/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Receptores de HFE/efectos de los fármacos , Receptores de HFE/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hexoquinasa/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Nanopartículas , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Polietileneimina/metabolismo , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Beta-toxin produced by Clostridium perfringens is a key virulence factor of fatal hemorrhagic enterocolitis and enterotoxemia. This toxin belongs to a family of ß-pore-forming toxins (PFTs). We reported recently that the ATP-gated P2X7 receptor interacts with beta-toxin. The ATP-release channel pannexin 1 (Panx1) is an important contributor to P2X7 receptor signaling. Hence, we investigated the involvement of Panx1 in beta-toxin-caused cell death. METHODS: We examined the effect of Panx1 in beta-toxin-induced cell death utilizing selective antagonists, knockdown of Panx1, and binding using dot-blot analysis. Localization of Panx1 and the P2X7 receptor after toxin treatment was determined by immunofluorescence staining. RESULTS: Selective Panx1 antagonists (carbenoxolone [CBX], probenecid, and Panx1 inhibitory peptide) prevented beta-toxin-caused cell death in THP-1 cells. CBX did not block the binding of the toxin to cells. Small interfering knockdown of Panx1 blocked beta-toxin-mediated cell death through inhibiting the oligomer formation of the toxin. Beta-toxin triggered a transient ATP release from THP-1 cells, but this early ATP release was blocked by CBX. ATP scavengers (apyrase and hexokinase) inhibited beta-toxin-induced cytotoxicity. Furthermore, co-administration of ATP with beta-toxin enhanced the binding and cytotoxicity of the toxin. CONCLUSIONS: Based on our results, Panx1 activation is achieved through the interaction of beta-toxin with the P2X7 receptor. Then, ATP released by the Panx1 channel opening promotes oligomer formation of the toxin, leading to cell death. GENERAL SIGNIFICANCE: Pannexin 1 is a novel candidate therapeutic target for beta-toxin-mediated disease.
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Toxinas Bacterianas/toxicidad , Conexinas/fisiología , Proteínas del Tejido Nervioso/fisiología , Adenosina Trifosfato/metabolismo , Apirasa/farmacología , Carbenoxolona/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Hexoquinasa/farmacología , Humanos , Receptores Purinérgicos P2X7/fisiologíaRESUMEN
BACKGROUND: Peritoneal dialysis (PD) is used as an alternative to hemodialysis in end-stage renal disease (ESRD). Icodextrin has been used as a hyperosmotic agent in PD. The aim of the study was to assess two different point-of-care testing (POCT) glucose strips, affected and not affected by icodextrin, with serum glucose concentrations of the patients using and not using icodextrin. METHODS: Fifty-two chronic ambulatory peritoneal dialysis (CAPD) patients using icodextrin (Extraneal®) and 20 CAPD patients using another hyperosmotic fluid (Dianeal®) were included in the study. Duplicate capillary and serum glucose concentrations were measured with two different POCT glucose strips and central laboratory hexokinase method. Assay principles of glucose strips were based on glucose dehydrogenase-pyrroloquinoline quinone (GDH-PQQ) and a mutant variant of GDH (Mut Q-GDH). The results of both strips were compared with those of hexokinase method. RESULTS: Regression equations between POCT and hexokinase methods in icodextrin group were y = 2.55x + 1.12 mmol/l and y = 1.057x + 0.16 mmol/l for the GDH-PQQ and Mut Q-GDH methods, respectively. The mean difference between the results of hexokinase and those of GDH-PQQ and Mut Q-GDH in icodextrin group was 3.41 ± 1.56 and 0.72 ± 0.64 mmol/l, respectively. However, the mean differences were found much lower in the control group; 0.64 mmol/l for GDH-PQQ and 0.52 mmol/l for Mut Q-GDH. CONCLUSION: Compared to GDH-PQQ, glucose strips of Mut Q-GDH correlated better with hexokinase method in PD patients using icodextrin.
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Glucemia/efectos de los fármacos , Glucanos/farmacología , Glucosa/farmacología , Soluciones para Hemodiálisis/farmacología , Diálisis Peritoneal/métodos , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/terapia , Adulto , Anciano , Anciano de 80 o más Años , Glucemia/análisis , Femenino , Glucosa Deshidrogenasas/metabolismo , Pruebas Hematológicas , Hexoquinasa/farmacología , Humanos , Icodextrina , Masculino , Persona de Mediana EdadRESUMEN
Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPßS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.
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Receptores Purinérgicos P2Y1/metabolismo , Retina/metabolismo , Fase S/fisiología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apirasa/farmacología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Relojes Circadianos/fisiología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Hexoquinasa/farmacología , Fotoperiodo , Antagonistas del Receptor Purinérgico P2Y/farmacología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Purinérgicos P2Y1/genética , Retina/citología , Retina/enzimología , Fase S/efectos de los fármacos , Transducción de Señal , Tionucleótidos/farmacología , Pez CebraRESUMEN
The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). This elevation of [Ca(2+)](i) and activation of Ca(2+)-dependent Cl(-) channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca(2+)](i) in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na(+) and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca(2+) signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca(2+) entry was either inhibited or stimulated by Zn(2+) depending on the extracellular Na(+) concentration. In Na(+)-free environment, Zn(2+) and other divalent cations elicited a biphasic Ca(2+) signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca(2+) entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.
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Calcio/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Líquido Extracelular/efectos de los fármacos , Zinc/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Apirasa/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Transformada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Estrenos/farmacología , Líquido Extracelular/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Hexoquinasa/farmacología , Humanos , Lactonas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Pirrolidinonas/farmacología , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Sesquiterpenos/farmacología , Sodio/metabolismo , Suramina/farmacología , Transfección/métodos , Zinc/metabolismoRESUMEN
Ciliary beat frequency (CBF) is a key factor in the defense of the airways, and ATP can stimulate CBF by increasing intracellular calcium concentration ([Ca2+]i). However, the regulatory effects of ATP have been mainly studied in cultured or isolated epithelial cells from the large cartilaginous airways. The aim of this study was to evaluate the regulation of CBF in small airways of lung slices that are representative of in vivo tissue. Mice lungs were inflated with agarose and cut into thin slices with a vibratome. CBF in the small bronchioles was observed with differential interference contrast microscopy and quantified using high-speed digital imaging (at 240 images s(-1)). We found that the in situ organization of the ciliated cells was well preserved and that their CBF was high. We verified the fidelity of our recording system by analyzing rapid changes in CBF in response to temperature. However, we found that ATP had no effect on CBF, despite the fact that the [Ca2+]i, measured with confocal fluorescence imaging, was increased. Ionomycin and purinergic or beta-adrenergic agonists also failed to increase CBF. Similar results were obtained in outgrowths of cells cultured from lung slices. By contrast, ATP increased the slower CBF of outgrowths of ciliated cells cultured from tracheal rings. Therefore, we conclude that CBF in intrapulmonary airways of mice is maintained at a maximum rate and cannot be further increased by agonist stimulation. These conditions would ensure that mucociliary clearance is constantly active to provide continuous airway protection.
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Cilios/fisiología , Pulmón/anatomía & histología , Pulmón/citología , Adenosina Desaminasa/farmacología , Adenosina Trifosfato/farmacología , Animales , Apirasa/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Hexoquinasa/farmacología , Técnicas In Vitro , Ionomicina/farmacología , Isoproterenol/farmacología , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Temperatura , Tráquea/efectos de los fármacosRESUMEN
Transformed cells are highly glycolytic and overexpress hexokinase II (HXK II). HXK II is capable of binding to the mitochondria through an interaction with the voltage-dependent anion channel (VDAC), an abundant outer mitochondrial membrane protein. The binding of HXK II to mitochondria has been shown to protect against loss of cell viability. Akt activation inhibits apoptosis partly by promoting the binding of HXK II to the mitochondria, but the mechanism through which Akt accomplishes this has not been characterized. The present report shows that Akt mediates the binding of HXK II to the mitochondria by negatively regulating the activity of glycogen synthase kinase 3beta (GSK3beta). On inhibition of Akt, GSK3beta is activated and phosphorylates VDAC. HXK II is unable to bind VDAC phosphorylated by GSK3beta and dissociates from the mitochondria. Inhibition of Akt potentiates chemotherapy-induced cytotoxicity, an effect that is dependent on GSK3beta activation and its attendant ability to disrupt the binding of HXK II to the mitochondria. Moreover, agents that can force the detachment of HXK II from mitochondria in the absence of Akt inhibition or GSK3beta activation promoted a synergistic increase in cell killing when used in conjunction with chemotherapeutic drugs. Such findings indicate that interference with the binding of HXK II to mitochondria may be a practicable modality by which to potentiate the efficacy of conventional chemotherapeutic agents.
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Canales de Calcio/metabolismo , Doxorrubicina/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Hexoquinasa/metabolismo , Mitocondrias/metabolismo , Paclitaxel/farmacología , Sinergismo Farmacológico , Activación Enzimática , Glucógeno Sintasa Quinasa 3 beta , Células HeLa , Hexoquinasa/antagonistas & inhibidores , Hexoquinasa/farmacología , Humanos , Fragmentos de Péptidos/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Substance P (SP) triggers responses in astrocytoma cells, which are considered important for proliferation and neuroimmunomodulatory activity. In this study, we compared the effects of SP with those of the novel tachykinin Hemokinin-1 (HK-1) in the human astrocytoma cell line U-251 MG. We show that U-251 MG cells express high levels of Neurokinin-1 (NK-1) receptors. The binding affinities of 125I-SP and 125I-mHK-1 to these receptors were in a similar, subnanomolar range. HK-1 and SP stimulated Ca2+ mobilization and induced increased cytokine mRNA expression. A specific NK-1 receptor antagonist blocked the observed effects. We conclude that there are no qualitative differences in SP and HK-1-evoked responses, suggesting that both peptides act through NK-1 receptors in U-251 MG cells. Moreover, we show TAC4 mRNA expression in gliomas, indicating a possible involvement of HK-1 in glioma biology.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hexoquinasa/farmacología , Precursores de Proteínas/farmacología , Sustancia P/farmacología , Taquicininas/farmacología , Animales , Astrocitoma , Northern Blotting/métodos , Calcio/metabolismo , Recuento de Células/métodos , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Hexoquinasa/farmacocinética , Humanos , Inmunohistoquímica/métodos , Isótopos de Yodo/farmacocinética , Ratones , Unión Proteica/efectos de los fármacos , Precursores de Proteínas/farmacocinética , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante/métodos , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sustancia P/farmacocinética , Taquicininas/farmacocinética , Factores de TiempoRESUMEN
In thymocytes, dexamethasone initiates cytochrome c-dependent processing of caspase-9 and the activation of caspase-3 to trigger apoptotic damage. Using murine thymocytes or a thymocyte cell line WEHI 7.1, we show that this pathway is inhibited by dominant-negative caspase-9, the anti-apoptotic protein Bcl-2, or by blocking components of the mitochondrial permeability transition pore complex (PTPC). We use DIDS (dithiocyanatostilbene-2,2-disulfonic acid), a pharmacological modifier of VDAC (voltage-dependent anion channel) function or ectopic expression of hexokinase-II, to examine the role of the VDAC--a mitochondrial outer membrane protein--in this apoptotic pathway. This approach implicated the VDAC in dexamethasone-mediated cytochrome c release, processing of caspase-9 and caspase-3, the loss of mitochondrial transmembrane potential (Deltapsim), nuclear damage and cell lysis. Inhibiting the adenine nucleotide transporter (ANT), a protein on the mitochondrial inner membrane, also blocks dexamethasone-induced apoptosis, but the ANT regulates caspase-3 processing and nuclear damage but not the mitochondrial efflux of cytochrome c. Collectively, the data identify two separable, but connected events in dexamethasone-induced mitochondrial damage in thymocytes. The first event is an increase in permeability of the mitochondrial outer membrane leading to VDAC-regulated efflux of cytochrome c and initial processing of caspase-9 followed by ANT-dependent caspase-3 processing and apoptotic damage to cells.