RESUMEN
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
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Herpesvirus Humano 4/química , Ingeniería de Proteínas , Proteínas/farmacología , Proteínas Virales/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Biología Computacional , Cristalografía por Rayos X , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Herpesvirus Humano 4/fisiología , Xenoinjertos , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Trasplante de Neoplasias , Proteínas/química , Proteínas/metabolismo , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
Genetic polymorphisms in nuclear respiratory factor-1 (Nrf1), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß-cells. Expression of NRF1 target genes Tfam, Tfb1m and Tfb2m, and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi-genomic gene transcription in diabetes.
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PURPOSE: To investigate combined MRI and 18F-FDG PET for assessing breast tumor metabolism/perfusion mismatch and predicting pathological response and recurrence-free survival (RFS) in women treated for breast cancer. METHODS: Patients undergoing neoadjuvant chemotherapy (NAC) for locally-advanced breast cancer were imaged at three timepoints (pre, mid, and post-NAC), prior to surgery. Imaging included diffusion-weighted and dynamic contrast-enhanced (DCE-) MRI and quantitative 18F-FDG PET. Tumor imaging measures included apparent diffusion coefficient, peak percent enhancement (PE), peak signal enhancement ratio (SER), functional tumor volume, and washout volume on MRI and standardized uptake value (SUVmax), glucose delivery (K1) and FDG metabolic rate (MRFDG) on PET, with percentage changes from baseline calculated at mid- and post-NAC. Associations of imaging measures with pathological response (residual cancer burden [RCB] 0/I vs. II/III) and RFS were evaluated. RESULTS: Thirty-five patients with stage II/III invasive breast cancer were enrolled in the prospective study (median age: 43, range: 31-66 years, RCB 0/I: N = 11/35, 31%). Baseline imaging metrics were not significantly associated with pathologic response or RFS (p > 0.05). Greater mid-treatment decreases in peak PE, along with greater post-treatment decreases in several DCE-MRI and 18F-FDG PET measures were associated with RCB 0/I after NAC (p < 0.05). Additionally, greater mid- and post-treatment decreases in DCE-MRI (peak SER, washout volume) and 18F-FDG PET (K1) were predictive of prolonged RFS. Mid-treatment decreases in metabolism/perfusion ratios (MRFDG/peak PE, MRFDG/peak SER) were associated with improved RFS. CONCLUSION: Mid-treatment changes in both PET and MRI measures were predictive of RCB status and RFS following NAC. Specifically, our results indicate a complementary relationship between DCE-MRI and 18F-FDG PET metrics and potential value of metabolism/perfusion mismatch as a marker of patient outcome.
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Neoplasias de la Mama , Humanos , Femenino , Adulto , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/tratamiento farmacológico , Fluorodesoxiglucosa F18/uso terapéutico , Terapia Neoadyuvante/métodos , Radiofármacos/uso terapéutico , Estudios Prospectivos , Resultado del Tratamiento , Imagen por Resonancia Magnética/métodos , Tomografía de Emisión de Positrones/métodosRESUMEN
The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.
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Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Estrona/genética , Estrona/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Apoptosis/genética , División Celular/genética , Hematopoyesis/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The Fbw7 (F-box/WD repeat-containing protein 7) ubiquitin ligase targets multiple oncoproteins for degradation and is commonly mutated in cancers. Like other pleiotropic tumor suppressors, Fbw7's complex biology has impeded our understanding of how Fbw7 mutations promote tumorigenesis and hindered the development of targeted therapies. To address these needs, we employed a transfer learning approach to derive gene-expression signatures from The Cancer Gene Atlas datasets that predict Fbw7 mutational status across tumor types and identified the pathways enriched within these signatures. Genes involved in mitochondrial function were highly enriched in pan-cancer signatures that predict Fbw7 mutations. Studies in isogenic colorectal cancer cell lines that differed in Fbw7 mutational status confirmed that Fbw7 mutations increase mitochondrial gene expression. Surprisingly, Fbw7 mutations shifted cellular metabolism toward oxidative phosphorylation and caused context-specific metabolic vulnerabilities. Our approach revealed unexpected metabolic reprogramming and possible therapeutic targets in Fbw7-mutant cancers and provides a framework to study other complex, oncogenic mutations.
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Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Metaboloma , Mitocondrias/metabolismo , Mutación , Respiración de la Célula , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica , Humanos , Mitocondrias/patología , Fosforilación Oxidativa , Estrés Oxidativo , Fosforilación , Ubiquitina , UbiquitinaciónRESUMEN
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células Precursoras de Linfocitos B/fisiología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Glucólisis/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Fosforilación/efectos de los fármacos , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Proteína Reguladora Asociada a mTOR , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/deficiencia , Factores de Transcripción , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
AIM: Idelalisib (IDELA) treatment is associated with diarrhea/colitis (incidence of â¼15% grade ≥3). We performed a retrospective analysis of gastrointestinal biopsies from 29 patients treated with IDELA across nine clinical trials. METHODS: A central core laboratory performed histopathologic review, immunohistochemistry, and droplet digital PCR viral studies. These results were correlated with tissue immune profiling data and morphologic features per modified Geboes score. RESULTS: Out of 29 eligible patients with abdominal pain or diarrhea, 24 (82.8%) had reported adverse event terms of diarrhea and/or colitis. Infectious pathogens were detected in 9/29 samples. Most biopsies presented with mixed/inflammatory infiltrates and contained increased numbers of FOXP3+ cells versus normal controls. CONCLUSION: This study revealed evidence of T-cell dysregulation and a substantial infectious component in association with IDELA-related diarrhea/colitis.
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Colitis/inducido químicamente , Diarrea/inducido químicamente , Purinas/efectos adversos , Quinazolinonas/efectos adversos , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Biopsia , Colitis/tratamiento farmacológico , Colitis/patología , Colitis/virología , Colon/patología , Colon/virología , Infecciones por Citomegalovirus/patología , Diarrea/tratamiento farmacológico , Diarrea/patología , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Recto/patología , Recto/virologíaRESUMEN
Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I "red" slow twitch and type II "white" fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.
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Proteínas Portadoras/fisiología , Fibras Musculares de Contracción Rápida/patología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/patología , Fibras Musculares de Contracción Lenta/fisiología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Complejos Multiproteicos/metabolismo , Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Distrofia Muscular de Duchenne/genética , Mioglobina/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.
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Células Endoteliales/virología , Herpesvirus Humano 8/fisiología , Aerobiosis , Supervivencia Celular , Células Endoteliales/citología , Glucosa/biosíntesis , Ácido Láctico/biosíntesis , Consumo de Oxígeno , Internalización del VirusRESUMEN
Genetic polymorphisms in nuclear respiratory factor-1 ( NRF1 ), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß- cells. Expression of NRF1 target genes Tfam , T@1m and T@2m , and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi- genomic gene transcription in diabetes.
RESUMEN
Influx of calcium is an essential but insufficient signal in sustained nutrient-stimulated insulin secretion, and increased metabolic rate of the beta cell is also required. The aim of the study was to test the hypothesis that the reduced state of cytochrome c is a metabolic co-factor necessary for insulin secretion, over and above its participation in the ATP-generating function of electron transport/oxidative phosphorylation. We found that nutrient stimulation of insulin secretion by isolated rat islets was strongly correlated with reduced cytochrome c, and agents that acutely and specifically reduced cytochrome c led to increased insulin secretion, even in the face of decreased oxygen consumption and calcium influx. In contrast, neither sites 1 nor 4 of the electron transport chain were both necessary and essential for the stimulation of insulin secretion to occur. Importantly, stimulation of islets with glucose, α-ketoisocaproate, or glyceraldehyde resulted in the appearance of cytochrome c in the cytosol, suggesting a pathway for the regulation of exocytotic machinery by reduction of cytochrome c. The data suggest that the metabolic factor essential for sustained calcium-stimulated insulin secretion to occur is linked to reduction and translocation of cytochrome c.
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Citocromos c/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Mitochondria are principal actors in apoptosis as central hubs for diverse apoptotic signals. A new paper demonstrates the therapeutic potential of directly engaging these apoptotic pathways by identifying a mitochondrial toxin selective for tumor cells.
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Apoptosis/fisiología , Indoles/metabolismo , Mitocondrias/fisiología , Proteínas Mitocondriales/fisiología , Compuestos de Piridinio/metabolismo , Animales , Apoptosis/efectos de los fármacos , Indoles/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Compuestos de Piridinio/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Patients with extensive ulcerative colitis (UC) have an increased risk of colorectal cancer. Although UC patients generally undergo lifelong colonoscopic surveillance to detect dysplasia or cancer in the colon, detection of cancer in this manner is expensive and invasive. An objective biomarker of dysplasia would vastly improve the clinical management of cancer risk in UC patients. In the current study, accurate mass and time methods with ion intensity-based label-free proteomics are applied to profile individual rectal and colon samples from UC patients with dysplasia or cancer (UC progressors) compared to rectal samples from patients that are dysplasia/cancer free (UC nonprogressors) to identify a set of proteins in the rectum mucosa that differentiate the two groups. In addition to the identification of proteins in UC dysplastic colon tissue, we for the first time identified differentially expressed proteins in nondysplastic rectal tissue from UC progressors. This provides a candidate pool of biomarkers for dysplasia/cancer that could be detected in a random nondysplastic rectal biopsy. Mitochondrial proteins, cytoskeletal proteins, RAS superfamily, proteins relating to apoptosis and metabolism were important protein clusters differentially expressed in the nondysplastic and dysplastic tissues of UC progressors, suggesting their importance in the early stages of UC neoplastic progression. Among the differentially expressed proteins, immunohistochemistry analysis confirmed that TRAP1 displayed increased IHC staining in UC progressors, in both dysplastic and nondysplastic tissue, and CPS1 showed a statistically significant difference in IHC staining between the nonprogressor and progressor groups. Furthermore, rectal CPS1 staining could be used to predict dysplasia or cancer in the colon with 87% sensitivity and 45% specificity, demonstrating the feasibility of using surrogate biomarkers in rectal biopsies to predict dysplasia and/or cancer in the colon.
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Biomarcadores de Tumor/química , Colitis Ulcerosa/metabolismo , Progresión de la Enfermedad , Proteómica/métodos , Biomarcadores de Tumor/metabolismo , Colon/metabolismo , Humanos , Inmunohistoquímica , Procesos Neoplásicos , Proteínas/química , Proteínas/clasificación , Proteínas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Previous studies suggest regulatory T cells (Tregs) inhibit graft-versus-host disease (GVHD) in mouse and human hematopoietic cell transplant (HCT) recipients. As the gastrointestinal tract represents one of the most common and severe sites of GVHD-related tissue damage, we sought to determine whether a deficit in circulating or gastric mucosal Treg numbers correlates with the clinical onset of gastric GVHD. We used the marker FOXP3 to quantify Tregs in blood and in gastric antral biopsies in a cohort of 60 allogeneic HCT recipients undergoing endoscopy at a single center to evaluate symptoms suspicious for gastrointestinal GVHD. We show for the first time in the gastric mucosa and, contrary to existing reports, in the blood, that the percent of T cells expressing FOXP3 is at least as high in the presence as in the absence of GVHD involving the upper gut. There was no correlation of Treg frequency with the histologic or clinical severity of gastrointestinal GVHD. We conclude that Treg depletion is not a central feature in the pathogenesis of gastric GVHD in humans.
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Factores de Transcripción Forkhead , Mucosa Gástrica/inmunología , Enfermedad Injerto contra Huésped/sangre , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , Antro Pilórico/inmunología , Gastropatías/sangre , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Animales , Biopsia , Estudios de Cohortes , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Enfermedad Injerto contra Huésped/inmunología , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/inmunología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Antro Pilórico/metabolismo , Antro Pilórico/patología , Gastropatías/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Trasplante HomólogoRESUMEN
The Bcl-2 family of proteins includes pro- and anti-apoptotic factors acting at mitochondrial and microsomal membranes. An impressive body of published studies, using genetic and physical reconstitution experiments in model organisms and cell lines, supports a view of Bcl-2 proteins as the critical arbiters of apoptotic cell death decisions in most circumstances (excepting CD95 death receptor signaling in Type I cells). Evasion of apoptosis is one of the hallmarks of cancer [Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70], relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins is observed in many cancers [Manion MK, Hockenbery DM. Targeting BCL-2-related proteins in cancer therapy. Cancer Biol Ther. 2003;2:S105-14; Olejniczak ET, Van Sant C, Anderson MG, Wang G, Tahir SK, Sauter G, et al. Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains. Mol Cancer Res. 2007;5:331-9]. The rekindled interest in aerobic glycolysis as a cancer trait raises interesting questions as to how metabolic changes in cancer cells are integrated with other essential alterations in cancer, e.g. promotion of angiogenesis and unbridled growth signals. Apoptosis induced by multiple different signals involves loss of mitochondrial homeostasis, in particular, outer mitochondrial membrane integrity, releasing cytochrome c and other proteins from the intermembrane space. This integrative process, controlled by Bcl-2 family proteins, is also influenced by the metabolic state of the cell. In this review, we consider the role of reactive oxygen species, a metabolic by-product, in the mitochondrial pathway of apoptosis, and the relationships between Bcl-2 functions and oxidative stress.
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Apoptosis/fisiología , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Caspasas/metabolismo , Retículo Endoplásmico , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
OBJECTIVE: We investigated whether NADPH oxidase-dependent production of superoxide contributes to activation of NF-kappaB in endothelial cells by the saturated free fatty acid palmitate. METHODS AND RESULTS: After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 mumol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a >2-fold increase in superoxide levels, an effect associated with activation of NF-kappaB signaling as measured by phospho-IkappaBalpha, NF-kappaB activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of 3 different interventions-pretreatment with superoxide dismutase (SOD), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA-attenuated palmitate-mediated NF-kappaB signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitate-mediated superoxide production was not affected by overexpression of a phosphorylation mutant IkappaBalpha (NF-kappaB super repressor) that blocks cellular inflammation downstream of IKKbeta/NF-kappaB. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4(-/-) mice, compared to low-fat fed controls. CONCLUSIONS: These results suggest that NADPH oxidase-dependent superoxide production links palmitate-stimulated TLR4 activation to NF-kappaB signaling in endothelial cells.
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Células Endoteliales/enzimología , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Ácido Palmítico/metabolismo , Superóxidos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Aorta Torácica/enzimología , Aorta Torácica/inmunología , Proteína Morfogenética Ósea 4/metabolismo , Células Cultivadas , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Humanos , Proteínas I-kappa B/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Inhibidor NF-kappaB alfa , Compuestos Onio/farmacología , Fosforilación , Interferencia de ARN , ARN Mensajero/metabolismo , Transducción de Señal , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
OBJECTIVE: Clonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIPS) and FLIP long (FLIPL), which modulate TRAIL signals, showed disease-stage-dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIPL and FLIPS, on TRAIL and TNF-alpha-induced apoptosis in model leukemic cell lines. MATERIALS AND METHODS: Using lentiviral constructs, FLIPL and FLIPS, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-alpha, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed. RESULTS: Overexpression of FLIP reduced TRAIL and TNF-alpha-induced apoptosis in ML-1 cells. However, while FLIPL completely abrogated apoptosis, FLIPS allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIPS cells followed by apoptosis. Further, inhibition of nuclear factor-kappaB (NF-kappaB) activation in TNF-alpha-treated cells resulted in profound apoptosis in FLIPS, but not in FLIPL-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-kappaB had only minimal effects on TRAIL signaling. CONCLUSION: Thus, FLIPL and FLIPS exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-alpha. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms.
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Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática/genética , Regulación Leucémica de la Expresión Génica/genética , Humanos , Lentivirus , Leucemia Mieloide Aguda/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Factor de Necrosis Tumoral alfa/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Microbial metabolism of lignans from high-fiber plant foods produces bioactive enterolignans, such as enterolactone (ENL) and enterodiol (END). Enterolignan exposure influences cellular pathways important to cancer risk and is associated with reduced colon tumorigenesis in animal models and lower colorectal cancer risk in humans. OBJECTIVES: The aim of this study was to test the effects of a flaxseed lignan supplement (50 mg secoisolariciresinol diglucoside/d) compared with placebo on host gene expression in colon biopsies and exfoliated colonocyte RNA in feces and fecal microbial community composition, and to compare responses in relation to ENL excretion. METHODS: We conducted a 2-period randomized, crossover intervention in 42 healthy men and women (20-45 y). We used RNA-seq to measure differentially expressed (DE) genes in colonic mucosa and fecal exfoliated cells through the use of edgeR and functional analysis with Ingenuity Pathway Analysis. We used 16S ribosomal RNA gene (V1-V3) analysis to characterize the fecal microbiome, and measured END and ENL in 24-h urine samples by gas chromatography-mass spectrometry. RESULTS: We detected 32 DE genes (false discovery rate <0.05) in the exfoliome, but none in the mucosal biopsies, in response to 60 d of lignan supplement compared with placebo. Statistically significant associations were detected between ENL excretion and fecal microbiome measured at baseline and at the end of the intervention periods. Further, we detected DE genes in colonic mucosa and exfoliome between low- and high-ENL excreters. Analysis of biopsy samples indicated that several anti-inflammatory upstream regulators, including transforming growth factor ß and interleukin 10 receptor, were suppressed in low-ENL excreters. Complementary analyses in exfoliated cells also suggested that low-ENL excreters may be predisposed to proinflammatory cellular events due to upregulation of nuclear transcription factor κB and NOS2, and an inhibition of the peroxisome proliferator-activated receptor γ network. CONCLUSIONS: These results suggest that ENL or other activities of the associated gut microbial consortia may modulate response to a dietary lignan intervention. This has important implications for dietary recommendations and chemoprevention strategies. This study was registered at clinicaltrials.gov as NCT01619020.
Asunto(s)
Heces/microbiología , Lino/química , Perfilación de la Expresión Génica , Mucosa Intestinal/efectos de los fármacos , Lignanos/química , Extractos Vegetales/farmacología , Adulto , Colon/efectos de los fármacos , Estudios Cruzados , Método Doble Ciego , Femenino , Microbioma Gastrointestinal , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Masculino , Extractos Vegetales/químicaRESUMEN
Overexpression of Bcl-x(L) in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule Bcl-x(L) inhibitors. Based on knockout studies, nonneoplastic cells also require Bcl-x(L) survival functions, particularly when challenged with cytotoxic agents. We analyze the selective cytotoxicity of one Bcl-x(L) inhibitor, 2-methoxy antimycin A, toward cells with excess exogenous Bcl-x(L) in isogenic cell line pairs. This selectivity, characteristic of a gain-of-function mechanism, is not shared by other known Bcl-x(L) inhibitors, including BH3I-2, HA14-1, ABT-737, gossypol, or the stapled BH3 helical peptide SAHB-BID. We show that Bcl-x(L) overexpression induces a shift in energy metabolism from oxidative phosphorylation to glycolysis. Treatment with 2-methoxy antimycin A acutely reverses the metabolic effects of Bcl-x(L), causing mitochondrial hyperpolarization and a progressive increase in mitochondrial NAD(P)H. We identify an additional small-molecule Bcl-x(L) inhibitor, NSC 310343, establishing a class of Bcl-x(L) inhibitors with gain-of-function activity. In contrast to other Bcl-x(L) inhibitors, combining gain-of-function Bcl-x(L) inhibitors with a standard inducer of apoptosis, staurosporine, enhances selective cytotoxicity toward Bcl-x(L)-overexpressing cells. These results provide an example of the intersection of bioenergetic metabolism and Bcl-x(L) functions and suggest a metabolic basis for the gain-of-function mechanism of Bcl-x(L) inhibitors.
Asunto(s)
Antimicina A/análogos & derivados , Proteína bcl-X/antagonistas & inhibidores , Animales , Antimicina A/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Ácido Láctico/metabolismo , Ratones , Mitocondrias/metabolismo , NADP/metabolismo , Fenotipo , Ratas , Estaurosporina/farmacologíaRESUMEN
The Warburg effect is a well-known phenomenon in cancer, but the glutamine addiction in which cancer cells utilize glutamine as an alternative source of energy is less well known. Recent efforts have focused on preventing cancer cell proliferation associated with glutamine addiction by targeting glutaminase using the inhibitor BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). In the current study, an investigation of the BPTES induced changes in metabolism was made in two human breast cancer cell lines, MCF7 (an estrogen receptor dependent cell line) and MDA-MB231 (a triple negative cell line), relative to the non-cancerous cell line, MCF10A. NMR spectroscopy combined with a recently established smart-isotope tagging approach enabled quantitative analysis of 41 unique metabolites representing numerous metabolite classes including carbohydrates, amino acids, carboxylic acids and nucleotides. BPTES induced metabolism changes in the cancer cell lines were especially pronounced under hypoxic conditions with up to 1/3 of the metabolites altered significantly (p < 0.05) relative to untreated cells. The BPTES induced changes were more pronounced for MCF7 cells, with 14 metabolites altered significantly (p < 0.05) compared to seven for MDA-MB231. Analyses of the results indicate that BPTES affected numerous metabolic pathways including glycolysis, TCA cycle, nucleotide and amino acid metabolism in cancer. The distinct metabolic responses to BPTES treatment determined in the two breast cancer cell lines offer valuable metabolic information for the exploration of the therapeutic responses to breast cancer.