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1.
Clin Cancer Res ; 16(23): 5734-49, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-20940278

RESUMEN

PURPOSE: We previously reported that small molecule X-linked inhibitor of apoptosis (XIAP) inhibitors synergize with soluble TRAIL to trigger apoptosis in pancreatic carcinoma cells. Because cancers may preferentially signal via 1 of the 2 agonistic TRAIL receptors, we investigated these receptors as a therapeutic target in pancreatic cancer in the present study. EXPERIMENTAL DESIGN: We examined TRAIL receptor expression and cytotoxicity of specific monoclonal antibodies to TRAIL-R1 (HGS-ETR1, mapatumumab) or TRAIL-R2 (HGS-ETR2, lexatumumab) and of TRAIL receptor selective mutants alone and in combination with small molecule XIAP inhibitors in pancreatic cancer cell lines, in primary specimens, and in a xenotransplant model in vivo. RESULTS: The majority of primary pancreatic carcinoma samples and all cell lines express one or both agonistic TRAIL receptors. Nine of 13 cell lines are more sensitive to mapatumumab-induced apoptosis, whereas lexatumumab requires cross-linking for maximal activity. Similarly, TRAIL-R1 selective mutants display higher cytotoxicity than TRAIL-R2 selective mutants. Small molecule XIAP inhibitors preferentially act in concert with mapatumumab to trigger caspase activation, caspase-dependent apoptosis, and suppress clonogenic survival. Also, primary cultured pancreatic carcinoma cells are more susceptible to mapatumumab than lexatumumab, which is significantly enhanced by a XIAP inhibitor. Importantly, combined treatment with mapatumumab and a XIAP inhibitor cooperates to suppress tumor growth in vivo. CONCLUSIONS: Mapatumumab exerts antitumor activity, especially in combination with XIAP inhibitors against most pancreatic carcinoma cell lines, whereas lexatumumab requires cross-linking for optimal cytotoxicity. These findings have important implications for the design of TRAIL-based protocols for pancreatic cancer.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma/patología , Neoplasias Pancreáticas/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Anciano , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/administración & dosificación , Carcinoma/metabolismo , Línea Celular Tumoral , Embrión de Pollo , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Pancreáticas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación
2.
J Clin Endocrinol Metab ; 95(6): 2836-40, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20382689

RESUMEN

OBJECTIVE: Leptin, a protein product of adipocytes, plays a critical role in the regulation of body weight, immune function, pubertal development, and fertility. So far, only three homozygous mutations in the leptin gene in a total of 13 individuals have been found leading to a phenotype of extreme obesity with marked hyperphagia and impaired immune function. DESIGN: Serum leptin was measured by ELISA. The leptin gene (OB) was sequenced in patient DNA. The effect of the identified novel mutation was assessed using HEK293 cells. RESULTS: We describe a 14-yr-old child of nonobese Austrian parents without known consanguinity. She had a body mass index of 31.5 kg/m(2) (+2.46 SD score) and undetectable leptin serum levels. Sequencing of the leptin gene revealed a hitherto unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein. RT-PCR, Western blot, and immunohistochemical analysis indicated that the mutant leptin was expressed in the patient's adipose tissue but retained within the cell. Using a heterologous cell system, we confirmed this finding and demonstrated that the side chain of Leu72 is crucial for intracellular leptin trafficking. Our patient showed signs of a hypogonadotropic hypogonadism. However, in contrast to the literature, she showed only mild obesity and a normal T cell responsiveness. CONCLUSIONS: These findings shed a new light on the clinical consequences of leptin deficiency. Congenital leptin deficiency should be considered possible in pediatric patients with mild obesity even if parents are lean and unrelated.


Asunto(s)
Hipogonadismo/genética , Leptina/genética , Mutación Missense/genética , Obesidad/genética , Linfocitos T/fisiología , Adiposidad/genética , Adolescente , Western Blotting , Calorimetría Indirecta , Proliferación Celular , Clonación Molecular , Frío , Citocinas/sangre , ADN Complementario/genética , Ingestión de Alimentos/fisiología , Ingestión de Energía , Metabolismo Energético , Femenino , Humanos , Inmunohistoquímica , Leptina/sangre , Leptina/química , Actividad Motora/fisiología , Obesidad/dietoterapia , Presión , Transfección
3.
Nat Med ; 15(6): 674-81, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19448635

RESUMEN

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification.


Asunto(s)
Ácidos , Densidad Ósea/fisiología , Calcio/metabolismo , Mucosa Gástrica/metabolismo , Homeostasis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enfermedades del Desarrollo Óseo/etiología , Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/metabolismo , Enfermedades del Desarrollo Óseo/patología , Calcio/farmacología , Homeostasis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Hipocalcemia/complicaciones , Hipocalcemia/genética , Hipocalcemia/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , ATPasas de Translocación de Protón Vacuolares/metabolismo
4.
Free Radic Biol Med ; 35(11): 1480-90, 2003 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-14642396

RESUMEN

After beta-carotene failed in certain clinical efficacy trials, there is evidence that the carotenoid might even be harmful, especially to smokers, when given in high dosages. These negative effects might be mediated in part also by carotenoid cleavage products (CPs) having a high reactivity towards biomolecules. The authors postulate that in certain tissues oxidative, nonenzymatic cleavage of carotenoids is carried out primarily by oxidants liberated by polymorphonuclear leukocytes (PML). In this study, we show that beta-carotene is degraded by stimulated PML in vitro. This gives the pathophysiological meaning to our further experiments in which beta-carotene degradation by hypochlorous acid and consecutive CP formation were investigated. While formation of apo-carotenals under these conditions has been studied before, this was not the case for short chain products. Performing gas chromatography mass spectrometry, we were able to identify for the first time 5,6-epoxi-beta-ionone, ionene, beta-cyclocitral, beta-ionone, dihydroactinidiolide, and 4-oxo-beta-ionone as CPs formed after degradation of beta-carotene mediated by hypochlorous acid. Our findings may be of biological relevance because beta-carotene CPs are highly reactive and, therefore, potentially toxic.


Asunto(s)
Ácido Hipocloroso/farmacología , Neutrófilos/metabolismo , Oxígeno/metabolismo , beta Caroteno/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Radicales Libres , Cromatografía de Gases y Espectrometría de Masas , Humanos , Ácido Hipocloroso/metabolismo , Metanol/química , Modelos Químicos
5.
J Biol Chem ; 277(47): 44791-800, 2002 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-12237314

RESUMEN

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.


Asunto(s)
Benzofuranos/química , Benzofuranos/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Quinasa I-kappa B , Proteínas I-kappa B/metabolismo , Leucemia de Células T/metabolismo , Ratones , Estructura Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Linfocitos T/citología , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología
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