RESUMEN
Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Fosfogluconato Deshidrogenasa/metabolismo , 6-Aminonicotinamida/química , 6-Aminonicotinamida/farmacología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Granzimas/genética , Granzimas/metabolismo , Humanos , Inmunoterapia , Listeria monocytogenes/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Vía de Pentosa Fosfato/efectos de los fármacos , Vía de Pentosa Fosfato/fisiología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Trasplante HeterólogoRESUMEN
BACKGROUND: The essential role of 6-phosphogluconate dehydrogenase (6PGD), the enzyme catalyzing the oxidative pentose phosphate pathway, in tumor growth and metabolism has garnered attention in recent years. In this work, we are the first to demonstrate that aberrant activation of 6PGD is a feature in renal cell carcinoma (RCC) and is critically involved in renal carcinogenesis and chemo- and immuno-resistance. MATERIALS AND METHODS: 6PGD expression and activity were systematically analyzed in normal and malignant renal cells and tissues. The roles of 6PGD and its downstream mechanism were investigated using gain-of-function and loss-of-function approaches. RESULTS: 6PGD expression and enzyme activity were increased in RCC cells and patients' samples. Activation of 6PGD via gain-of-function approach promoted growth of normal kidney but not RCC cells, and alleviated the efficacy of chemotherapeutic (e.g., 5-FU) and immunotherapeutic (e.g., IFN-α) agents. In contrast, 6PGD inhibition using siRNA knockdown and pharmacological inhibitor physcion augmented the inhibitory effects of 5-FU and IFN-α in RCC. Mechanistic studies demonstrated that 6PGD inhibition activated AMPK signaling, leading to ACC1 enzyme inhibition and reduction of lipid synthesis. In addition, 6PGD inhibition disrupted NADPH and NADH homeostasis in RCC cells as shown by the decreased level of NADPH and NADH, and suppressed SIRT-1 activity. AMPK inhibition by siRNA knockdown reversed the inhibitory effects of physcion, demonstrating that the effect of 6PGD inhibition is AMPK activation dependent. CONCLUSIONS: Our work provides preclinical evidence that 6PGD inhibition may represent a potential therapeutic strategy to augment the efficacy of RCC standard of care drugs.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Carcinoma de Células Renales/terapia , Reprogramación Celular/fisiología , Neoplasias Renales/terapia , Fosfogluconato Deshidrogenasa/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/genética , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Línea Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Fluorouracilo/uso terapéutico , Técnicas de Silenciamiento del Gen , Humanos , Inmunoterapia , Interferón-alfa/uso terapéutico , Riñón/patología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , NADP/fisiología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/genética , ARN Interferente Pequeño , Regulación hacia ArribaRESUMEN
The pentose phosphate pathway (PPP) plays a critical role in maintaining cellular redox homeostasis in tumor cells and macromolecule biosynthesis. Upregulation of the PPP has been shown in several types of tumor. However, how the PPP is regulated to confer selective growth advantages on drug resistant tumor cells is not well understood. Here we show a metabolic shift from tricarboxylic acid cycle (TCA) to PPP after a long period induction of Imatinib (IM). One of the rate-limiting enzymes of the PPP-phosphogluconate dehydrogenase (PGD), is dramatically upregulated in gastrointestinal stromal tumors (GISTs) and GIST cell lines resistant to Imatinib (IM) compared with sensitive controls. Functional studies revealed that the overexpression of PGD in resistant GIST cell lines promoted cell proliferation and suppressed cell apoptosis. Mechanistic analyses suggested that the protein level of hypoxia inducible factor-1α (HIF-1α) increased during long time stimulation of reactive oxygen species (ROS) produced by IM. Importantly, we further demonstrated that HIF-1α also had positive correlation with PGD, resulting in the change of metabolic pathway, and ultimately causing drug resistance in GIST. Our findings show that long term use of IM alters the metabolic phenotype of GIST through ROS and HIF-1α, and this may contribute to IM resistance. Our work offers preclinical proof of metabolic target as an effective strategy for the treatment of drug resistance in GIST.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Tumores del Estroma Gastrointestinal/enzimología , Tumores del Estroma Gastrointestinal/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mesilato de Imatinib/farmacología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Tumores del Estroma Gastrointestinal/patología , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Reprogrammed metabolism is key biochemical characteristic of malignant cells, which represents one of the emerging hallmarks of cancer. Currently, there is rising contemplation on oxidative pentose phosphate pathway (PPP) enzymes as potential therapeutic hits due to their affiliation with tumor metabolism. 6-Phosphogluconate dehydrogenase (6PGD), third oxidative decarboxylase of PPP, has received a great deal of attention during recent years due to its critical role in tumorigenesis and redox homeostasis. 6PGD has been reported to overexpress in number of cancer types and its hyperactivation is mediated through post-transcriptional and post-translational modifications by YTH domain family 2 (YTHDF2), Nrf2 (nuclear factor erythroid 2-related factor 2), EGFR (epidermal growth factor receptor) and via direct structural interactions with ME1 (malic enzyme 1). Upregulated expression of 6PGD provides metabolic as well as defensive advantage to cancer cells, thus, promoting their proliferative and metastatic potential. Moreover, enhanced 6PGD expression also performs key role in development of chemoresistance as well as radiation resistance in cancer. This review aims to discuss the historical timeline and cancer-specific role of 6PGD, pharmacological and genetic inhibitors of 6PGD and 6PGD as prognostic biomarker in order to explore its potential for therapeutic interventions. We anticipate that targeting this imperative supplier of NADPH might serve as tempting avenue to combat the deadly disease like cancer.
Asunto(s)
Carcinogénesis/genética , Resistencia a Antineoplásicos/genética , Neoplasias/genética , Vía de Pentosa Fosfato/genética , Fosfogluconato Deshidrogenasa/genética , Procesamiento Proteico-Postraduccional , Antineoplásicos/uso terapéutico , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , NADP/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Neoplasias/terapia , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tolerancia a Radiación/genética , Transducción de Señal , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The glucose metabolism in the pentose cycle is essential to the source of NADPH. Deficiency of these enzymes have been linked to depression and psychotic disorders. Depression is an increasingly prevalent mental disorder which may cause loss of labor. Antidepressant drugs are commonly employed in treatments of mood disorders and anxiety treatment. The purpose of this study is to investigate the effects of aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol on the activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD) enzymes purified from human erythrocytes. It was found that aripiprazole, mirtazapine, risperidone, escitalopram and haloperidol show effective inhibitor properties on purified G6PD and 6PGD enzymes. The IC50 values of these drugs were found in the range of 26.34 µM-5.78 mM for 6PGD and 16.26 µM-3.85 mM for G6PD. The Ki values of the drugs were found in the range of 30.21 ± 4.31 µM-4.51 ± 1.83 mM for 6PGD and 14.12 ± 3.48 µM-4.98 ± 1.14 mM for G6PD. Usage of drugs with significant biological effects may be a hazard in some conditions.
Asunto(s)
Antidepresivos/farmacología , Eritrocitos/efectos de los fármacos , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Aripiprazol/farmacología , Citalopram/farmacología , Eritrocitos/enzimología , Haloperidol/farmacología , Humanos , Mirtazapina/farmacologíaRESUMEN
In eukaryotes, adventitious oxidation of erythrose-4-phosphate, an intermediate of the pentose phosphate pathway (PPP), generates 4-phosphoerythronate (4PE), which inhibits 6-phosphogluconate dehydrogenase. 4PE is detoxified by metabolite-proofreading phosphatases such as yeast Pho13. Here, we report that a similar function is carried out in Bacillus subtilis by CpgA, a checkpoint protein known to be important for ribosome assembly, cell morphology and resistance to cell wall-targeting antibiotics. We find that ΔcpgA cells are intoxicated by glucose or other carbon sources that feed into the PPP, and that CpgA has high phosphatase activity with 4PE. Inhibition of 6-phosphogluconate dehydrogenase (GndA) leads to intoxication by 6-phosphogluconate, a potent inhibitor of phosphoglucose isomerase (PGI). The coordinated shutdown of PPP and glycolysis leads to metabolic gridlock. Overexpression of GndA, PGI, or yeast Pho13 suppresses glucose intoxication of ΔcpgA cells, but not cold sensitivity, a phenotype associated with ribosome assembly defects. Our results suggest that CpgA is a multifunctional protein, with genetically separable roles in ribosome assembly and metabolite proofreading.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/fisiología , Proteínas de Ciclo Celular/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Glucólisis , Modelos Moleculares , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Azúcares Ácidos/metabolismoRESUMEN
Better understanding of the molecular mechanism involved in hepatocellular carcinoma (HCC) progression is essential for the development of therapeutic strategies to overcome chemoresistance in HCC patients. In this work, we show that 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of the oxidative pentose phosphate pathway, is important for HCC growth and survival. Compared to normal liver tissues, we demonstrate that 6PGD expression is upregulated in HCC tissues. 6PGD overexpression increases 6PGD activity and promotes growth in normal liver cells. In contrast, targeting 6PGD using both genetic and pharmacological approaches inhibits HCC growth and survival. Combination of chemotherapeutic agents with 6PGD inhibition achieves greater efficacy in inhibiting HCC growth and survival than chemotherapeutic agent alone. We further show that inhibition of 6PGD activates AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase 1 (ACC1), and decreases level of NADPH/NAD + and NADH in HCC, leading to SIRT1 activity reduction and oxidative stress. Conversely, AMPK depletion significantly abolishes the effects of physcion (a selective small-molecule 6PGD inhibitor) in decreasing NADPH/NAD + ratio, growth and survival, confirming the role of AMPK as the relevant upstream activator with 6PGD inhibition in HCC cells. Our work is the first to demonstrate the upregulation of 6PGD and its critical involvement in growth and survival in HCC. Our findings suggest 6PGD as a promising therapeutic target to overcome chemoresistance in HCC.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Acetil-CoA Carboxilasa/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Humanos , NADP/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Sirtuina 1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: The oxidative pentose phosphate pathway (PPP) is essential for cancer metabolism and growth. However, the contribution of 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of PPP, to cervical cancer development remains largely unknown. METHODS: mRNA and protein levels of 6PGD were analyzed in cervical cancer cells and tissues derived from patients and compared to normal counterparts. Using cell culture system and xenograft mouse model, the functions of 6PGD in cervical cancer are determined and its molecular mechanism is analyzed. 6PGD inhibitor physcion and siRNA knockdown were used. RESULTS: In this work, we demonstrate that 6PGD is aberrantly upregulated and activated in cervical cancer cells and patient tissues compared to normal counterparts. Using different approaches and preclinical models, we show that 6PGD inhibition decreases growth and migration, and enhances chemosensitivity in cervical cancer. Mechanistically, inhibition of 6PGD activates AMP-activated protein kinase (AMPK) and decreases RhoA and Rac1 activities. AMPK depletion significantly reduces the effects of 6PGD inhibition in decreasing RhoA and Rac1 activities, growth and migration in cervical cancer cells. CONCLUSIONS: Our work is the first to demonstrate the aberrant expression of 6PGD and its predominant roles in cervical cancer cell growth and migration, via a AMPK-dependent activation. Our findings suggest 6PGD as a potential therapeutic target to enhance chemosensitivity in cervical cancer.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Biomarcadores de Tumor/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimioterapia Adyuvante , Emodina/análogos & derivados , Emodina/farmacología , Emodina/uso terapéutico , Femenino , Expresión Génica , Humanos , Ratones SCID , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/metabolismo , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human African trypanosomiasis, or sleeping sickness, is a lethal disease caused by the protozoan parasite Trypanosoma brucei. However, although many efforts have been made to understand the biochemistry of this parasite, drug development has led to treatments that are of limited efficiency and of great toxicity. To develop new drugs, new targets must be identified, and among the several metabolic processes of trypanosomes that have been proposed as drug targets, carbohydrate metabolism (glycolysis and the pentose phosphate pathway (PPP)) appears as a promising one. As far as the PPP is concerned, a limited number of studies are related to the glucose-6-phosphate dehydrogenase. In this work, we have focused on the activity of the second PPP enzyme (6-phospho-gluconolactonase (6PGL)) that transforms 6-phosphogluconolactone into 6-phosphogluconic acid. A lactam analog of the natural substrate has been synthesized, and binding of the ligand to 6PGL has been investigated by NMR titration. The ability of this ligand to inhibit 6PGL has also been demonstrated using ultraviolet experiments, and protein-inhibitor interactions have been investigated through docking calculations and molecular dynamics simulations. In addition, a marginal inhibition of the third enzyme of the PPP (6-phosphogluconate dehydrogenase) was also demonstrated. Our results thus open new prospects for targeting T. brucei.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Inhibidores Enzimáticos/farmacología , Lactamas/farmacología , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Trypanosoma brucei brucei/enzimología , Gluconatos/metabolismo , Glucólisis , Lactamas/síntesis química , Modelos Moleculares , Fosfogluconato Deshidrogenasa/metabolismo , Especificidad por SustratoRESUMEN
The enzyme 6-phosphogluconate dehydrogenase (6PGD) of the malaria parasite Plasmodium falciparum catalyzes the third step of the pentose phosphate pathway converting 6-phosphogluconate (6PG) to ribulose 5-phosphate. The NADPH produced by 6PGD is crucial for antioxidant defense and redox regulation, and ribose 5-phosphate is essential for DNA and RNA synthesis in the rapidly growing parasite. Thus, 6PGD represents an attractive antimalarial drug target. In this study, we present the X-ray structures of Pf6PGD in native form as well as in complex with 6PG or nicotinamide adenine dinucleotide phosphate (NADP+) at resolutions of 2.8, 1.9, and 2.9â¯Å, respectively. The overall structure of the protein is similar to structures of 6PGDs from other species; however, a flexible loop close to the active site rearranges upon binding of 6PG and likely regulates the conformation of the cofactor NADP+. Upon binding of 6PG, the active site loop adopts a closed conformation. In the absence of 6PG, the loop opens and NADP+ is bound in a waiting position, indicating that the cofactor and 6PG bind independently from each other. This sequential binding mechanism was supported by kinetic studies on the homodimeric wild-type Pf6PGD. Furthermore, the function of the Plasmodium-specific residue W104L mutant was characterized by site-directed mutagenesis. Notably, the activity of Pf6PGD was found to be post-translationally redox regulated via S-nitrosylation, and screening the Medicines for Malaria Venture Malaria Box identified several compounds with IC50s in the low micromolar range. Together with the three-dimensional structure of the protein, this is a promising starting point for further drug discovery approaches.
Asunto(s)
Antimaláricos/química , Inhibidores Enzimáticos/química , Fosfogluconato Deshidrogenasa/química , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Antimaláricos/farmacología , Sitios de Unión , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Fenómenos Mecánicos , Modelos Moleculares , Conformación Molecular , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Plasmodium falciparum/efectos de los fármacos , Unión Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N-benzoylindoles 9a--9d, which form the main framework of many natural indole derivatives such as indomethacin and N-benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N-benzoylindoles inhibited the enzymatic activity with IC50 in the range of 3.391505 µM for G6PD and 2.19-990 µM for 6PGD.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Eritrocitos/enzimología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Indoles/farmacología , Modelos Moleculares , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Unión Competitiva , Espectroscopía de Resonancia Magnética con Carbono-13 , Cromatografía de Afinidad , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/síntesis química , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glucosafosfato Deshidrogenasa/metabolismo , Indoles/síntesis química , Indoles/química , Indoles/metabolismo , Cinética , Estructura Molecular , NADP/química , NADP/metabolismo , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Ratas , Homología Estructural de Proteína , Temperatura de TransiciónRESUMEN
Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid malignancies and resistant to chemotherapy. Little is known on the underlying mechanisms of ATC resistance to chemotherapy. In our work, we identified that 6-phosphogluconate dehydrogenase (6PGD) is critically involved in the development of ATC resistance to doxorubicin. We found that 6PGD mRNA, protein and enzyme activity levels are significantly upregulated in ATC cells during the prolonged exposure to doxorubicin in a time-dependent manner. 6PGD inhibition by genetic and pharmacological approaches significantly inhibits growth and survival of ATC cells that are highly resistant to doxorubicin. Consistently, 6PGD inhibition also sensitizes ATC cells to doxorubicin treatment. Of note, we observed the decreased level of NADPH, NADH and enzymatic activity of sirtuin-1 in response to 6PGD inhibition in doxorubicin-resistant ATC cells. Lactate level was also reduced by 6PGD inhibition. All these indicate that 6PGD inhibition disrupts metabolic reprogramming in doxorubicin-resistant ATC cells. Our work demonstrates 6PGD activation-mediated resistance in response to doxorubicin and provides an alternative therapeutic strategy to overcome resistance to chemotherapy for ATC treatment. Our findings also highlight the importance of metabolic reprogramming in ATC chemoresistance.
Asunto(s)
Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Carcinoma Anaplásico de Tiroides/enzimología , Carcinoma Anaplásico de Tiroides/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Humanos , NADP/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Sirtuina 1/metabolismo , Carcinoma Anaplásico de Tiroides/patologíaRESUMEN
PURPOSE: 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of the oxidative pentose phosphate pathway, is involved in tumor growth and metabolism. Although high 6PGD activity has been shown to be associated with poor prognosis, its role and therapeutic value in breast cancer remain unknown. METHODS: The levels and roles of 6PGD were analyzed in breast cancer cells and their normal counterparts. The underlying mechanisms of 6PGD's roles are also analyzed. RESULTS: We found that 6PGD is aberrantly activated in breast cancer as shown by its increased transcriptional and translational levels as well as enzyme activity in breast cancer tissues and cell lines compared to normal counterparts. Although similar degree of enzyme activity inhibition was achieved in both breast cancer and normal breast cells, 6PGD inhibition by siRNA-mediated knockdown or pharmacological inhibitor physcion is more effective in inhibiting growth and survival in breast cancer than normal breast cells. Moreover, inhibiting 6PGD significantly sensitizes breast cancer response to chemotherapeutic agents in in vitro cell culture system and in vivo xenograft breast cancer model. We further show that 6PGD inhibition activates AMPK and its downstream substrate ACC1, leading to reduction of ACC1 activity and lipid biosynthesis. AMPK depletion significantly reverses the inhibitory effects of physcion in breast cancer cells, confirming that 6PGD inhibition targets breast cancer cell via AMPK activation. CONCLUSIONS: Our work provides experimental evidence on the association of 6PGD with poor prognosis in breast cancer and suggests that 6PGD inhibition may represent a potential therapeutic strategy to augment chemotherapy efficacy in breast cancer.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Activación Enzimática , Femenino , Humanos , RatonesRESUMEN
In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2',5'-ADP-Sepharose 4B affinity gel. The effects of Ast and Al3+ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al3+ inhibited the enzyme activity noncompetitively (IC50 values; 0.679 mM, Ki values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).
Asunto(s)
Compuestos de Aluminio , Cloruros , Inhibidores Enzimáticos , Eritrocitos/enzimología , Glucosafosfato Deshidrogenasa , Fosfogluconato Deshidrogenasa , Cloruro de Aluminio , Compuestos de Aluminio/química , Compuestos de Aluminio/farmacología , Animales , Cloruros/química , Cloruros/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/metabolismo , Ratas , Xantófilas/química , Xantófilas/farmacologíaRESUMEN
The oxidative pentose phosphate pathway (PPP) is crucial for cancer cell metabolism and tumor growth. We recently reported that targeting a key oxidative PPP enzyme, 6-phosphogluconate dehydrogenase (6PGD), using our novel small-molecule 6PGD inhibitors Physcion and its derivative S3, shows anticancer effects. Notably, humans with genetic deficiency of either 6PGD or another oxidative PPP enzyme, glucose-6-phosphate dehydrogenase, exhibit non-immune hemolytic anemia upon exposure to aspirin and various antimalarial drugs. Inspired by these clinical observations, we examined the anticancer potential of combined treatment with 6PGD inhibitors and antimalarial drugs. We found that stable knockdown of 6PGD sensitizes leukemia cells to antimalarial agent dihydroartemisinin (DHA). Combined treatment with DHA and Physcion activates AMP-activated protein kinase, leading to synergistic inhibition of human leukemia cell viability. Moreover, our combined therapy synergistically attenuates tumor growth in xenograft nude mice injected with human K562 leukemia cells and cell viability of primary leukemia cells from human patients, but shows minimal toxicity to normal hematopoietic cells in mice as well as red blood cells and mononucleocytes from healthy human donors. Our findings reveal the potential for combined therapy using optimized doses of Physcion and DHA as a novel antileukemia treatment without inducing hemolysis.
Asunto(s)
Antimaláricos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Emodina/análogos & derivados , Leucemia/tratamiento farmacológico , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Antimaláricos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Artemisininas/administración & dosificación , Artemisininas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Emodina/administración & dosificación , Emodina/farmacología , Femenino , Humanos , Células K562 , Leucemia/enzimología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.
Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/metabolismo , Células Germinativas de las Plantas/efectos de los fármacos , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Prostaglandina D2/antagonistas & inhibidores , Isoformas de Proteínas/antagonistas & inhibidores , Alelos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Clonación Molecular , Ciclopentanos/metabolismo , Citosol/metabolismo , ADN Bacteriano , ADN de Plantas/aislamiento & purificación , Germinación/efectos de los fármacos , Germinación/genética , Mutagénesis Sitio-Dirigida , Óxido Nítrico/metabolismo , Oxilipinas/metabolismo , Vía de Pentosa Fosfato , Peroxisomas/metabolismo , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/genética , Hojas de la Planta/metabolismo , Plastidios , Polen/efectos de los fármacos , Polen/crecimiento & desarrollo , Prostaglandinas D/antagonistas & inhibidores , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ProteínaRESUMEN
G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.
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Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutatión Reductasa/aislamiento & purificación , Pulmón/enzimología , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Reductasa/metabolismo , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.
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Digoxina/química , Dopamina/química , Inhibidores Enzimáticos/química , Furosemida/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutatión Reductasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Animales , Unión Competitiva , Pruebas de Enzimas , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/química , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Reductasa/química , Concentración de Iones de Hidrógeno , Cinética , Masculino , Peso Molecular , Miocardio/química , Miocardio/enzimología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/química , Unión Proteica , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato , TemperaturaRESUMEN
Polyphenols are the important compounds that have various bioactivities. They constitute vital active agents of not only daily diet but also natural medicines that are used traditionally. It is generally considered that they are safe because they are natural. In some conducted studies, different negative effects of these compounds were mentioned. Twelve phenolic compounds have been assayed to determine the effect of inhibition on glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzymes activity. For in vitro studies, the enzymes were purified from human erythrocytes using 2',5'-ADP Sepharose 4B affinity chromatography. Naringenin, caffeic acid, ellagic acid, ferulic acid, and sinapic acid against two enzymes, hesperidin and polydatin, only on G6PD activity and chrysin solely against 6PGD showed inhibitory effect. Chlorogenic acid, p-coumaric acid, and syringic acid did not exhibit an effect on the activity of the two enzymes.
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Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Polifenoles/farmacología , Inhibidores Enzimáticos/química , Eritrocitos/enzimología , Humanos , Polifenoles/químicaRESUMEN
The enzyme 6-phosphogluconate dehydrogenase is a potential drug target for the parasitic protozoan Trypanosoma brucei, the causative organism of human African trypanosomiasis. This enzyme has a polar active site to accommodate the phosphate, hydroxyl and carboxylate groups of the substrate, 6-phosphogluconate. A virtual fragment screen was undertaken of the enzyme to discover starting points for the development of inhibitors which are likely to have appropriate physicochemical properties for an orally bioavailable compound. A virtual screening library was developed, consisting of compounds with functional groups that could mimic the phosphate group of the substrate, but which have a higher pKa. Following docking, hits were clustered and appropriate compounds purchased and assayed against the enzyme. Three fragments were identified that had IC50 values in the low micromolar range and good ligand efficiencies. Based on these initial hits, analogues were procured and further active compounds were identified. Some of the fragments identified represent potential starting points for a medicinal chemistry programme to develop potent drug-like inhibitors of the enzyme.