RESUMEN

UNLABELLED: Two hallmarks of clear-cell renal cell carcinoma (ccRCC) are constitutive hypoxia-inducible factor (HIF) signaling and abundant intracellular lipid droplets (LD). However, regulation of lipid storage and its role in ccRCC are incompletely understood. Transcriptional profiling of primary ccRCC samples revealed that expression of the LD coat protein gene PLIN2 was elevated in tumors and correlated with HIF2α, but not HIF1α, activation. HIF2α-dependent PLIN2 expression promoted lipid storage, proliferation, and viability in xenograft tumors. Mechanistically, lipid storage maintained integrity of the endoplasmic reticulum (ER), which is functionally and physically associated with LDs. Specifically, PLIN2-dependent lipid storage suppressed cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Thus, in addition to promoting ccRCC proliferation and anabolic metabolism, HIF2α modulates lipid storage to sustain ER homeostasis, particularly under conditions of nutrient and oxygen limitation, thereby promoting tumor cell survival. SIGNIFICANCE: We demonstrate that HIF2α promotes lipid storage, ER homeostasis, and cell viability in ccRCC via upregulation of the LD coat protein PLIN2, revealing a novel function for the well-documented "clear-cell" phenotype and identifying ER stress as a targetable vulnerability created by HIF2α/PLIN2 suppression in this common renal malignancy.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Neoplasias Renales/metabolismo , Metabolismo de los Lípidos , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Estrés del Retículo Endoplásmico , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Oncogenes , Perilipina-2 , Biosíntesis de Proteínas , Carga Tumoral , Respuesta de Proteína Desplegada

RESUMEN

Therapeutic strategies designed to target cancer metabolism are an area of intense research. Antimetabolites, first used to treat patients in the early twentieth century, served as an early proof of concept for such therapies. We highlight strategies that attempt to improve on the anti-metabolite approach as well as new metabolic drug targets. Some of these targets have the advantage of a strong genetic anchor to drive patient selection (isocitrate dehydrogenase 1/2, Enolase 2). Additional approaches described here derive from hypothesis-driven and systems biology efforts designed to exploit tumor cell metabolic dependencies (fatty acid oxidation, nicotinamide adenine dinucleotide synthesis, glutamine biology).

Asunto(s)

Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Biomarcadores de Tumor/genética , Proteínas de Unión al ADN/genética , Ácidos Grasos/metabolismo , Glutamina/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Metotrexato/uso terapéutico , NAD/metabolismo , Neoplasias/genética , Fosfopiruvato Hidratasa/genética , Transducción de Señal , Proteínas Supresoras de Tumor/genética

RESUMEN

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

Asunto(s)

Hipoxia de la Célula , Fibroblastos/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Neoplasias/metabolismo , Proteínas/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica , Estrés del Retículo Endoplásmico , Endorribonucleasas/deficiencia , Endorribonucleasas/genética , Metabolismo Energético , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/biosíntesis , Lípidos/sangre , Lípidos/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Complejos Multiproteicos , Neoplasias/patología , Oxígeno/metabolismo , Oxígeno/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Suero , Serina-Treonina Quinasas TOR , Proteína 2 del Complejo de la Esclerosis Tuberosa , Microambiente Tumoral , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Respuesta de Proteína Desplegada

RESUMEN

We describe a novel technique for heme removal and replacement in the heme domain of P450BM-3 (BMP). The method was applied to obtain the aluminum-protoporphyrin IX (Al-PP) substituted derivative of BMP (Al-BMP). The overall yield of the purified Al-BMP was about 15% as related to the initial amount of the hemeprotein. Al-BMP possesses extensive fluorescence in the 550-650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM-3 (palmitic, arachidonic, and cis-parinaric acids) with affinities similar to those of the native enzyme (3-6 µM). However, the substrate-induced changes in fluorescence of Al-PP reveal the existence of a second, low-affinity substrate-binding site, which cannot be detected by the spin shift in the native, heme-containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al-BMP forms a complex with the flavoprotein domain of P450BM-3 labeled with 7-ethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum-substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.

Asunto(s)

Aluminio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Colorantes Fluorescentes/química , Hemo/química , Hemo/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , Especificidad por Sustrato

RESUMEN

MicroRNAs typically function at the level of posttranscriptional gene silencing within the cytoplasm; however, increasing evidence suggests that they may also function in nuclear, Argonaut-containing complexes, to directly repress target gene transcription. We have investigated the role of microRNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/ß, PERK, and ATF6, whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the proapoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter, where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 downregulation. Our data suggest a model in which PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment.

Asunto(s)

Regulación de la Expresión Génica , MicroARNs/genética , Factor de Transcripción CHOP/genética , eIF-2 Quinasa/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Estrés del Retículo Endoplásmico/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Metilación , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Células 3T3 NIH , Fosforilación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tapsigargina/farmacología , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , eIF-2 Quinasa/metabolismo

RESUMEN

The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.

Asunto(s)

Adipocitos/enzimología , Diferenciación Celular , eIF-2 Quinasa/metabolismo , Adipocitos/citología , Animales , Línea Celular , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Activación Enzimática , Metabolismo de los Lípidos , Ratones , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Mammary epithelial cells (MECs) detached from the extracellular matrix (ECM) produce deleterious reactive oxygen species (ROS) and induce autophagy to survive. The coordination of such opposing responses likely dictates whether epithelial cells survive ECM detachment or undergo anoikis. Here, we demonstrate that the endoplasmic reticulum kinase PERK facilitates survival of ECM-detached cells by concomitantly promoting autophagy, ATP production, and an antioxidant response. Loss-of-function studies show that ECM detachment activates a canonical PERK-eukaryotic translation initiation factor 2α (eIF2α)-ATF4-CHOP pathway that coordinately induces the autophagy regulators ATG6 and ATG8, sustains ATP levels, and reduces ROS levels to delay anoikis. Inducible activation of an Fv2E-ΔNPERK chimera by persistent activation of autophagy and reduction of ROS results in lumen-filled mammary epithelial acini. Finally, luminal P-PERK and LC3 levels are reduced in PERK-deficient mammary glands, whereas they are increased in human breast ductal carcinoma in situ (DCIS) versus normal breast tissues. We propose that the normal proautophagic and antioxidant PERK functions may be hijacked to promote the survival of ECM-detached tumor cells in DCIS lesions.

Asunto(s)

Autofagia/fisiología , Matriz Extracelular/metabolismo , Estrés Oxidativo/fisiología , eIF-2 Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Adhesión Celular , Línea Celular , Supervivencia Celular , Células Cultivadas , Embrión de Mamíferos/citología , Activación Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Interferencia de ARN , eIF-2 Quinasa/genética

RESUMEN

The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho-eIF2alpha were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2-eIF2alpha-ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti-tumour approaches.

Asunto(s)

Factor de Transcripción Activador 4/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Factor de Transcripción Activador 4/antagonistas & inhibidores , Aminoácidos/metabolismo , Animales , Proliferación Celular , Supervivencia Celular , Medios de Cultivo/química , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Humanos , Ratones

RESUMEN

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway.

Asunto(s)

Adipocitos/citología , Adipocitos/enzimología , Diferenciación Celular , Lipogénesis , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/enzimología , eIF-2 Quinasa/metabolismo , Animales , Células Cultivadas , Retículo Endoplásmico/enzimología , Activación Enzimática , Epitelio/enzimología , Epitelio/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Fibroblastos , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/metabolismo , Ratones , eIF-2 Quinasa/genética

RESUMEN

In this issue of Developmental Cell, two groups, Yamamoto et al. and Wu et al., describe the generation of mice with targeted deletion of the ATF6alpha gene. While ATF6alpha is nonessential for embryonic and postnatal development, deletion has a profound effect on the transcriptional program elicited by endoplasmic reticulum stress, revealing a broader than anticipated role for ATF6 in this signaling network.

Asunto(s)

Factor de Transcripción Activador 6/deficiencia , Factor de Transcripción Activador 6/metabolismo , Estrés Oxidativo , Factor de Transcripción Activador 6/química , Factor de Transcripción Activador 6/genética , Animales , Antígenos de Diferenciación/metabolismo , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Genes Reporteros , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1 , Estructura Terciaria de Proteína , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/metabolismo

RESUMEN

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Asunto(s)

Ciclo Celular/fisiología , Pliegue de Proteína , Ribosomas/química , Ribosomas/fisiología , Estrés Fisiológico/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/fisiología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Retículo Endoplásmico/metabolismo , Células HCT116 , Humanos , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/deficiencia

RESUMEN

The transcription factor NFkappaB (NFkappaB) is up-regulated in many cancer cells where it contributes to development of the pro-survival, anti-apoptotic state. The natural product curcumin is a known inhibitor of activation of NFkappaB. Enone analogues of curcumin were compared with curcumin for their abilities to inhibit the TNFalpha-induced activation of NFkappaB, using the Panomics' NFkappaB Reporter Stable Cell Line. The enones tested included curcumin analogues that retained the 7-carbon spacer between the aromatic rings, analogues with a 5-carbon spacer, and analogues with a 3-carbon spacer. Inhibitors of NFkappaB activation were identified in all three series, a number of which were more active than curcumin. Enone analogues in the series with the 5-carbon spacer were especially active, including members that contained heterocyclic rings. 1,5-Bis(3-pyridyl)-1,4-pentadien-3-one was the most active analogue, IC50 = 3.4 +/- 0.2 microM. The most active analogues retain the enone functionality, although some analogues devoid of the enone functionality exhibited activity. The activity of the analogues as inhibitors of the activation of NFkappaB did not correlate with their anti-oxidant activity. The data suggest that the abilities of curcumin and analogues to prevent the stress-induced activation of NFkappaB result from the inhibition of specific targets rather than from activity as anti-oxidants.

Asunto(s)

Curcumina/farmacología , Cetonas/farmacología , FN-kappa B/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Curcumina/síntesis química , Curcumina/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Cetonas/síntesis química , Cetonas/química , Estructura Molecular , FN-kappa B/metabolismo , Relación Estructura-Actividad Cuantitativa

RESUMEN

Vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8) are prominent pro-angiogenic and pro-metastatic proteins that represent negative prognostic factors in many types of cancer. Hypoxia is thought to be the primary environmental cause of VEGF and IL-8 expression in solid tumors. We hypothesized that a lack of nutrients other than oxygen could stimulate the expression of these factors and previously demonstrated that expression of VEGF and IL-8 is responsive to amino acid deprivation. In the present study, we examined the effect of glutamine availability on the expression of these factors as well as the role of transcription factors NFkappaB and activating protein-1 (AP-1) in the response of TSE human breast carcinoma cells to glutamine deprivation. VEGF and IL-8 secretion and mRNA levels were dramatically induced by glutamine deprivation. mRNA stabilization contributed to this response. Glutamine deprivation increased NFkappaB (p65/p50) and AP-1 (Fra-1/c-Jun+JunD) DNA-binding activities. Blocking NFkappaB and AP-1 activation with curcumin as well as expression of dominant inhibitors, inhibitor of nuclear factor-kappaB (IkappaB) super repressor (IkappaBM), and a mutant form of c-Fos (A-Fos) demonstrated that the activation of NFkappaB and AP-1 transcription factors was necessary for the induction of IL-8 expression but dispensable for the induction of VEGF expression. A macro-array containing 111 NFkappaB target genes identified a total of 17 that were up-regulated 2-fold or more in response to glutamine deprivation. These included growth regulated oncogene alpha (GROalpha/GRO1/CXCL1), another neutrophil chemoattractant implicated in tumor angiogenesis and metastasis.

Asunto(s)

Quimiocinas CXC/biosíntesis , Glutamina/deficiencia , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Interleucina-8/biosíntesis , FN-kappa B/fisiología , Factor de Transcripción AP-1/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiocina CXCL1 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Curcumina/farmacología , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Glutamina/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Interleucina-8/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.

Asunto(s)

Calcitriol/química , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/química , Ingeniería de Proteínas/métodos , Adrenodoxina/química , Sustitución de Aminoácidos , Animales , Sitios de Unión , Catálisis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Mutación , Unión Proteica , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ácido Retinoico 4-Hidroxilasa , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

BACKGROUND: The expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-kappaB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-kappaB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines. RESULTS: We found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153). CONCLUSION: These results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

Asunto(s)

Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Retículo Endoplásmico/metabolismo , Interleucina-8/biosíntesis , Neovascularización Patológica , Factores de Crecimiento Endotelial Vascular/biosíntesis , Aminoácidos/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Cobalto/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , ADN de Neoplasias/metabolismo , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Interleucina-8/metabolismo , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Factor de Transcripción CHOP , Factores de Transcripción/biosíntesis , Células Tumorales Cultivadas , Tunicamicina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Factores de Crecimiento Endotelial Vascular/metabolismo