RESUMEN
BACKGROUND: Prostate cancer (PCa) remains the second leading cause of cancer-related death among men. Taxanes, such as docetaxel and cabazitaxel are utilized in standard treatment regimens for chemotherapy naïve castration-resistant PCa. However, tumors often develop resistance to taxane chemotherapeutics, highlighting a need to identify additional therapeutic targets. Fatty acid-binding protein 5 (FABP5) is an intracellular lipid carrier whose expression is upregulated in metastatic PCa and increases cell growth, invasion, and tumor formation. Here, we assessed whether FABP5 inhibitors synergize with semi-synthetic taxanes to induce cytotoxicity in vitro and attenuate tumor growth in vivo. METHODS: PC3, DU-145, and 22Rv1 PCa cells were incubated with FABP5 inhibitors Stony Brook fatty acid-binding protein inhibitor 102 (SBFI-102) or SBFI-103 in the presence or absence of docetaxel or cabazitaxel, and cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay. Cytotoxicity of SBFI-102 and SBFI-103 was also evaluated in noncancerous cells. For the in vivo studies, PC3 cells were subcutaneously implanted into BALB/c nude mice, which were subsequently treated with FABP5 inhibitors, docetaxel, or a combination of both. RESULTS: SBFI-102 and SBFI-103 produced cytotoxicity in the PCa cells. Coincubation of the PCa cells with FABP5 inhibitors and docetaxel or cabazitaxel produced synergistic cytotoxic effects in vitro. Treatment of mice with FABP5 inhibitors reduced tumor growth and a combination of FABP5 inhibitors with a submaximal dose of docetaxel reduced tumor growth to a larger extent than treatment with each drug alone. CONCLUSIONS: FABP5 inhibitors increase the cytotoxic and tumor-suppressive effects of taxanes in PCa cells. The ability of these drugs to synergize could permit more efficacious antitumor activity while allowing for dosages of docetaxel or cabazitaxel to be lowered, potentially decreasing taxane-resistance.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Docetaxel/farmacología , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Taxoides/farmacología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Docetaxel/administración & dosificación , Sinergismo Farmacológico , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células PC-3 , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Taxoides/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fatty acid-binding proteins (FABPs) are intracellular lipid carriers that regulate inflammation, and pharmacological inhibition of FABP5 reduces inflammation and pain. The mechanism(s) underlying the anti-inflammatory effects associated with FABP5 inhibition is poorly understood. Herein, we identify a novel mechanism through which FABP5 modulates inflammation. In mice, intraplantar injection of carrageenan induces acute inflammation that is accompanied by edema, enhanced pain sensitivity, and elevations in proinflammatory cytokines and prostaglandin E2 (PGE2). Inhibition of FABP5 reduced pain, edema, cytokine, and PGE2 levels. PGE2 is a major eicosanoid that enhances pain in the setting of inflammation, and we focused on the mechanism(s) through which FABP5 modulates PGE2 production. Cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1) are enzymes up-regulated at the site of inflammation and account for the bulk of PGE2 biosynthesis. Pharmacological or genetic FABP5 inhibition suppressed the induction of mPGES-1 but not COX-2 in carrageenan-injected paws, which occurred predominantly in macrophages. The cytokine interleukin 1ß (IL-1ß) is a major inducer of mPGES-1 during inflammation. Using A549 cells that express FABP5, IL-1ß stimulation up-regulated mPGES-1 expression, and mPGES-1 induction was attenuated in A549 cells bearing a knockdown of FABP5. IL-1ß up-regulates mPGES-1 via NF-κB, which activates the mPGES-1 promoter. Knockdown of FABP5 reduced the activation and nuclear translocation of NF-κB and attenuated mPGES-1 promoter activity. Deletion of NF-κB-binding sites within the mPGES-1 promoter abrogated the ability of FABP5 to inhibit mPGES-1 promoter activation. Collectively, these results position FABP5 as a novel regulator of mPGES-1 induction and PGE2 biosynthesis during inflammation.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Prostaglandina-E Sintasas/metabolismo , Células A549 , Animales , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células THP-1 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.
Asunto(s)
Analgésicos/uso terapéutico , Sistema Nervioso Central/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Dolor/tratamiento farmacológico , Nervios Periféricos/metabolismo , Analgésicos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Ciclobutanos/uso terapéutico , Ácidos Dicarboxílicos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Adyuvante de Freund/toxicidad , Ganglios Espinales/metabolismo , Hiperalgesia/etiología , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Dolor/complicaciones , Dolor/etiología , Umbral del Dolor/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción GenéticaRESUMEN
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is initiated and driven by the oncogenic fusion protein BCR-ABL, a constitutively active tyrosine kinase. Despite major advances in the treatment of this highly aggressive disease with potent inhibitors of the BCR-ABL kinase such as dasatinib, patients in remission frequently relapse due to persistent minimal residual disease possibly supported, at least in part, by salutary cytokine-driven signaling within the hematopoietic microenvironment. Using a mouse model of Ph+ ALL that accurately mimics the genetics, clinical behavior, and therapeutic response of the human disease, we show that a combination of 2 agents approved by the US Food and Drug Administration (dasatinib and ruxolitinib, which inhibit BCR-ABL and Janus kinases, respectively), significantly extends survival by targeting parallel signaling pathways. Although the BCR-ABL kinase cancels the cytokine requirement of immature leukemic B cells, dasatinib therapy restores cytokine dependency and sensitizes leukemic cells to ruxolitinib. As predicted, ruxolitinib alone had no significant antileukemic effect in this model, but it prevented relapse when administered with dasatinib. The combination of dasatinib, ruxolitinib, and the corticosteroid dexamethasone yielded more durable remissions, in some cases after completion of therapy, avoiding the potential toxicity of other cytotoxic chemotherapeutic agents.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasas Janus/antagonistas & inhibidores , Recurrencia Local de Neoplasia/tratamiento farmacológico , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Factor 1 de Ribosilacion-ADP/fisiología , Animales , Western Blotting , Dasatinib , Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Interleucina-7/genética , Interleucina-7/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Nitrilos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Tasa de Supervivencia , Tiazoles/administración & dosificación , Células Tumorales CultivadasRESUMEN
Prostate cancer (PCa) is defined by dysregulated lipid signaling and is characterized by upregulation of lipid metabolism-related genes including fatty acid binding protein 5 (FABP5), fatty acid synthase (FASN), and monoacylglycerol lipase (MAGL). FASN and MAGL are enzymes that generate cellular fatty acid pools while FABP5 is an intracellular chaperone that delivers fatty acids to nuclear receptors to enhance PCa metastasis. Since FABP5, FASN, and MAGL have been independently implicated in PCa progression, we hypothesized that FABP5 represents a central mechanism linking cytosolic lipid metabolism to pro-metastatic nuclear receptor signaling. Here, we show that the abilities of FASN and MAGL to promote nuclear receptor activation and PCa metastasis are critically dependent upon co-expression of FABP5 in vitro and in vivo. Our findings position FABP5 as a key driver of lipid-mediated metastasis and suggest that disruption of lipid signaling via FABP5 inhibition may constitute a new avenue to treat metastatic PCa.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Células PC-3 , Neoplasias de la Próstata/patologíaRESUMEN
The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.
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Dronabinol/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Animales , Sitios de Unión , Biotransformación , Células Cultivadas , Cristalografía por Rayos X , Dronabinol/administración & dosificación , Proteínas de Unión a Ácidos Grasos/química , Femenino , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos MolecularesRESUMEN
Fatty acid binding proteins (FABPs) serve as critical modulators of endocannabinoid signaling by facilitating the intracellular transport of anandamide and whose inhibition potentiates anandamide signaling. Our previous work has identified a novel small-molecule FABP inhibitor, α-truxillic acid 1-naphthyl monoester (SB-FI-26, 3) that has shown efficacy as an antinociceptive and anti-inflammatory agent in rodent models. In the present work, we have performed an extensive SAR study on a series of 3-analogs as novel FABP inhibitors based on computer-aided inhibitor drug design and docking analysis, chemical synthesis and biological evaluations. The prediction of binding affinity of these analogs to target FABP3, 5 and 7 isoforms was performed using the AutoDock 4.2 program, using the recently determined co-crystal structures of 3 with FABP5 and FABP7. The compounds with high docking scores were synthesized and evaluated for their activities using a fluorescence displacement assay against FABP3, 5 and 7. During lead optimization, compound 3l emerged as a promising compound with the Ki value of 0.21⯵M for FABP 5, 4-fold more potent than 3 (Ki, 0.81⯵M). Nine compounds exhibit similar or better binding affinity than 3, including compounds 4b (Ki, 0.55⯵M) and 4e (Ki, 0.68⯵M). Twelve compounds are selective for FABP5 and 7 with >10⯵M Ki values for FABP3, indicating a safe profile to avoid potential cardiotoxicity concerns. Compounds 4f, 4j and 4k showed excellent selectivity for FABP5 and would serve as other new lead compounds. Compound 3a possessed high affinity and high selectivity for FABP7. Compounds with moderate to high affinity for FABP5 displayed antinociceptive effects in mice while compounds with low FABP5 affinity lacked in vivo efficacy. In vivo pain model studies in mice revealed that exceeding hydrophobicity significantly affects the efficacy. Thus, among the compounds with high affinity to FABP5 in vitro, the compounds with moderate hydrophobicity were identified as promising new lead compounds for the next round of optimization, including compounds 4b and 4j. For select cases, computational analysis of the observed SAR, especially the selectivity of new inhibitors to particular FABP isoforms, by comparing docking poses, interaction map, and docking energy scores has provided useful insights.
Asunto(s)
Analgésicos/farmacología , Ciclobutanos/farmacología , Ésteres/farmacología , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Analgésicos/síntesis química , Analgésicos/química , Animales , Diseño Asistido por Computadora , Ciclobutanos/síntesis química , Ciclobutanos/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ésteres/síntesis química , Ésteres/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
BACKGROUND: Fatty acid amide hydrolase 2 (FAAH2) is a hydrolase that mediates the degradation of endocannabinoids in man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatric conditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activity have not previously been described. We report a male with autistic features with an onset before the age of 2 years who subsequently developed additional features including anxiety, pseudoseizures, ataxia, supranuclear gaze palsy, and isolated learning disabilities but was otherwise cognitively intact as an adult. METHODS AND RESULTS: Whole exome sequencing identified a rare missense mutation in FAAH2, hg19: g.57475100G > T (c.1372G > T) resulting in an amino acid change (p.Ala458Ser), which was Sanger confirmed as maternally inherited and absent in his healthy brother. Alterations in lipid metabolism with abnormalities of the whole blood acyl carnitine profile were found. Biochemical and molecular modeling studies confirmed that the p.Ala458Ser mutation results in partial inactivation of FAAH2. Studies in patient derived fibroblasts confirmed a defect in FAAH2 activity resulting in altered levels of endocannabinoid metabolites. CONCLUSIONS: We propose that genetic alterations in FAAH2 activity contribute to neurologic and psychiatric disorders in humans.
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Amidohidrolasas/metabolismo , Ansiedad/patología , Enfermedades del Sistema Nervioso Central/patología , Depresión/patología , Adulto , Amidohidrolasas/genética , Ansiedad/genética , Enfermedades del Sistema Nervioso Central/genética , Clonación Molecular , Depresión/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutación Missense , Conformación ProteicaRESUMEN
UNLABELLED: We evaluated the ability of the PET imaging agent (89)Zr-trastuzumab to delineate HER2-positive gastric cancer and to monitor the pharmacodynamic effects of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor afatinib. METHODS: Using (89)Zr-trastuzumab, (18)F-FDG, or 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT PET), we imaged HER2-positive NCI-N87 and HER2-negative MKN74 gastric cancer xenografts in mice. Next, we examined the pharmacodynamic effects of afatinib in NCI-N87 xenografts using (89)Zr-trastuzumab and (18)F-FDG PET and comparing imaging results to changes in tumor size and in protein expression as monitored by Western blot and histologic studies. RESULTS: Although (18)F-FDG uptake in NCI-N87 tumors did not change, a decrease in (89)Zr-trastuzumab uptake was observed in the afatinib-treated versus control groups (3.0 ± 0.0 percentage injected dose per gram (%ID/g) vs. 21.0 ± 3.4 %ID/g, respectively; P < 0.05). (89)Zr-trastuzumab PET results corresponded with tumor reduction, apoptosis, and downregulation of HER2 observed on treatment with afatinib. Downregulation of total HER2, phosphorylated (p)-HER2, and p-EGFR occurred within 24 h of the first dose of afatinib, with a sustained effect over 21 d of treatment. CONCLUSION: Afatinib demonstrated antitumor activity in HER2-positive gastric cancer in vivo. (89)Zr-trastuzumab PET specifically delineated HER2-positive gastric cancer and can be used to measure the pharmacodynamic effects of afatinib.