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No disponible
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Humanos , Masculino , Femenino , Persona de Mediana Edad , Anciano , Trasplante de Corazón/estadística & datos numéricos , Proteína Oncogénica v-akt/análisis , Serina-Treonina Quinasas TOR/análisis , Biomarcadores de Tumor/análisis , Terapia de Inmunosupresión/efectos adversosRESUMEN
Objetivo: Determinar la expresión proteica diferencial de los biomarcadores FGFR3, PI3K (subunidades PI3Kp110α, PI3KClassIII, PI3Kp85), AKT, p21Waf1/Cip1 y las ciclinas D1 y D3 en el cáncer de vejiga T1 versus tejido sano, así como estudiar su posible papel como marcadores de recidiva precoz. Material y método: Se trata de un estudio prospectivo en el que se utilizaron un total de 67 muestras de tejido (55 casos de tumores de vejiga T1 sometidos a resección transuretral y 12 casos de mucosa sana adyacente). Los niveles de expresión de las proteínas se evaluaron mediante Western blot, y las medias y los porcentajes fueron comparados utilizando el test «t» de Student y la prueba Chi cuadrado. El análisis de supervivencia se realizó mediante el método Kaplan-Meier y el test Log-rank. Resultados: Se detectó una mayor expresión proteica de FGFR3, PI3Kp110α, PI3KClassIII, ciclinas D1 y D3 y p21Waf1/Cip1 en tejido tumoral versus mucosa sana. Sin embargo, estas diferencias no fueron significativas para PI3Kp85 y AKT. Se observaron correlaciones estadísticamente significativas de PI3Kp110α, PI3KClassIII, PI3Kp85 y AKT con la recidiva temprana (p = 0,003, p = 0,045, p = 0,050 y p = 0,028 respectivamente), de ciclina D3 (p=0,001) con el tipo tumoral (primario versus recidivante), de FGFR3 (p = 0,035) con el tamaño tumoral y de ciclina D1 (p = 0,039) con la multifocalidad. El análisis de supervivencia seleccionó a FGFR3 (p = 0,024), PI3Kp110alfa (p = 0,014), PI3KClassIII (p = 0,042) y AKT (p= 0,008) como marcadores de supervivencia libre de recidiva precoz. Conclusiones: Existe un incremento de los niveles de expresión proteica en el tejido tumoral vesical, asimismo, la sobreexpresión de FGFR3, PI3Kp110α, PI3KClassIII y AKT se asocia con una mayor supervivencia libre de recidiva precoz en pacientes con tumores de vejiga T1
Objective: To determine the differential protein expression of biomarkers FGFR3, PI3K (subunits PI3Kp110α, PI3KClassIII, PI3Kp85), AKT, p21Waf1/Cip1 and cyclins D1 and D3 in T1 bladder cancer versus healthy tissue and to study their potential role as early recurrence markers. Material and method: This is a prospective study that employed a total of 67 tissue samples (55 cases of T1 bladder tumours that underwent transurethral resection and 12 cases of adjacent healthy mucosa). The protein expression levels were assessed using Western blot, and the means and percentages were compared using Student's t-test and the chi-squared test. The survival analysis was conducted using the Kaplan-Meier method and the log-rank test. Results: Greater protein expression was detected for FGFR3, PI3Kp110α, PI3KClassIII, cyclins D1 and D3 and p21Waf1/Cip1 in the tumour tissue than in the healthy mucosa. However, these differences were not significant for PI3Kp85 and AKT. We observed statistically significant correlations between early recurrence and PI3Kp110α, PI3KClassIII, PI3Kp85 and AKT (P = .003, P = .045, P = .050 and P = .028, respectively), between the tumour type (primary vs. recurrence) and cyclin D3 (P=.001), between the tumour size and FGFR3 (P = .035) and between multifocality and cyclin D1 (P = .039). The survival analysis selected FGFR3 (P = .024), PI3Kp110α (P = .014), PI3KClassIII (P = .042) and AKT (P = .008) as markers of early-recurrence-free survival. Conclusions:. There is an increase in protein expression levels in bladder tumour tissue. The overexpression of FGFR3, PI3Kp110α, PI3KClassIII and AKT is associated with increased early-recurrence-free survival for patients with T1 bladder tumours
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Humanos , Masculino , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Neoplasias de la Vejiga Urinaria/complicaciones , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/patología , Western Blotting/métodos , Recurrencia Local de Neoplasia/complicaciones , Pronóstico , Proteína Oncogénica v-akt/análisis , Ciclinas/análisis , Biomarcadores/análisis , Estudios Prospectivos , Estimación de Kaplan-MeierRESUMEN
Obesity is a risk factor for periodontitis, but the pathogenic mechanism involved is unclear. We studied the effects of insulin in periodontal tissues during the state of obesity-induced insulin resistance. Gingival samples were collected from fatty (ZF) and lean (ZL, control) Zucker rats. Endothelial nitric oxide synthase (eNOS) expression was decreased, and activities of protein kinase C (PKC) α, ß2, δ, and ϵ isoforms were significantly increased in the gingiva from ZF rats compared with those from ZL rats. Expression of oxidative stress markers (mRNA) and the p65 subunit of NF-κB was significantly increased in ZF rats. Immunohistochemistry revealed that NF-κB activation was also increased in the gingival endothelial cells from transgenic mice overexpressing NF-κB-dependent enhanced green fluorescent protein (GFP) and on a high-fat vs. normal chow diet. Analysis of the gingiva showed that insulin-induced phosphorylation of IRS-1, Akt, and eNOS was significantly decreased in ZF rats, but Erk1/2 activation was not affected. General PKC inhibitor and an anti-oxidant normalized the action of insulin on Akt and eNOS activation in the gingiva from ZF rats. This provided the first documentation of obesity-induced insulin resistance in the gingiva. Analysis of our data suggested that PKC activation and oxidative stress may selectively inhibit insulin-induced Akt and eNOS activation, causing endothelial dysfunction and inflammation.
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Gingivitis/etiología , Resistencia a la Insulina/fisiología , Obesidad/complicaciones , Vasculitis/etiología , Pérdida de Hueso Alveolar/etiología , Pérdida de Hueso Alveolar/metabolismo , Animales , Células Endoteliales/química , Endotelio Vascular/química , Gingivitis/metabolismo , Proteínas Sustrato del Receptor de Insulina/análisis , Masculino , Ratones , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo III/análisis , Proteína Oncogénica v-akt/análisis , Estrés Oxidativo/fisiología , Proteína Quinasa C beta/análisis , Proteína Quinasa C-alfa/análisis , Proteína Quinasa C-delta/análisis , Proteína Quinasa C-epsilon/análisis , Ratas , Ratas Zucker , Factor de Transcripción ReIA/análisis , Vasculitis/metabolismoRESUMEN
Previous studies have demonstrated that decreased bone mass results from either the impairment of osteoblastic insulin signaling or obesity. Our previous study revealed that 12-week high-fat-diet (HFD) consumption caused obesity as well as peripheral and brain insulin resistance. However, the osteoblastic insulin resistance induced by HFD has not been elucidated. Therefore, we hypothesized that 12-week HFD rats exhibited not only peripheral insulin resistance but also osteoblastic insulin resistance, which leads to decreased jawbone quality. We found that the jawbones of rats fed a 12-week HFD exhibited increased osteoporosis. The osteoblastic cells isolated from HFD-fed rats exhibited the impairment of osteoblastic insulin signaling as well as reduction of cell proliferation and survival. In conclusion, this study demonstrated that insulin resistance induced by 12-week HFD impaired osteoblastic insulin signaling, osteoblast proliferation, and osteoblast survival and resulted in osteoporosis in the jawbone.
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Densidad Ósea/fisiología , Resistencia a la Insulina/fisiología , Mandíbula/patología , Obesidad/fisiopatología , Osteoblastos/fisiología , Proceso Alveolar/patología , Animales , Apoptosis/fisiología , Glucemia/análisis , Proliferación Celular , Supervivencia Celular/fisiología , Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Insulina/sangre , Masculino , Obesidad/etiología , Proteína Oncogénica v-akt/análisis , Osteoblastos/metabolismo , Osteocalcina/sangre , Osteogénesis/fisiología , Osteoporosis/etiología , Osteoporosis/patología , Fosforilación , Distribución Aleatoria , Ratas , Ratas Wistar , Receptor de Insulina/análisis , Transducción de Señal/fisiología , Microtomografía por Rayos XRESUMEN
OBJECTIVES: The epidermal growth factor receptor (EGFR) is a validated target in head and neck squamous cell carcinoma (HNSCC). In recurrent and/or metastatic (R/M) HNSCC, resistance to anti-EGFR therapy inevitably occurs. Downstream activation of the PI3K/Akt/mTOR pathway is an established resistance mechanism. Concurrent mTOR blockade may improve efficacy of anti-EGFR therapy. MATERIALS AND METHODS: Erlotinib 150 mg daily and temsirolimus 15 mg weekly were administered to patients with platinum-refractory R/M HNSCC and ECOG performance status 0-2. The primary endpoint was progression-free survival (PFS). Correlative studies determined PIK3CA and HRAS mutation status; p16, EGFR, pS6K, pAkt and PTEN expression; and pre- and post-treatment plasma levels of 20 immunomodulatory cytokines. RESULTS: Twelve patients enrolled; six withdrew within 6 weeks due to toxicity or death, prompting early closure of the trial. Grade ≥ 3 toxicities included fatigue, diarrhea, gastrostomy tube infection, peritonitis, pneumonia, dyspnea, and HN edema. Median PFS was 1.9 months. Median overall survival was 4.0 months. Six/12 tumors were p16(+), 9/11 lacked measurable PTEN expression, and 1/12 harbored a PIK3CA mutation. On exploratory analysis, high baseline plasma VEGF and interferon-gamma levels marginally associated with tumor progression. CONCLUSIONS: The combination of erlotinib and temsirolimus was poorly tolerated. Low prevalence of PTEN expression and 8% incidence of PIK3CA mutations indicate biological relevance of this pathway in R/M disease. Investigation of more tolerable combinations of EGFR and PI3K/Akt/mTOR pathway inhibitors in selected HNSCC patients is warranted.
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Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Sirolimus/análogos & derivados , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Células Escamosas/secundario , Fosfatidilinositol 3-Quinasa Clase I , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Citocinas/análisis , Resistencia a Antineoplásicos , Clorhidrato de Erlotinib , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Proteína Oncogénica v-akt/análisis , Fosfohidrolasa PTEN/análisis , Fosfatidilinositol 3-Quinasas/análisis , Fosfatidilinositol 3-Quinasas/genética , Platino (Metal) , Proteínas Proto-Oncogénicas p21(ras)/análisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinazolinas/efectos adversos , Proteínas Quinasas S6 Ribosómicas/análisis , Sirolimus/administración & dosificación , Sirolimus/efectos adversos , Tasa de Supervivencia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Supresoras de Tumor/análisisRESUMEN
BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.
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Herpesvirus Humano 1/inmunología , Neutrófilos/inmunología , Estomatitis Herpética/inmunología , Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Femenino , Fluoresceínas , Colorantes Fluorescentes , Fluorometría , Glucógeno Sintasa Quinasa 3/análisis , Humanos , Sueros Inmunes/inmunología , Inmunoglobulina G/inmunología , Mediadores de Inflamación/análisis , Interleucina-8/análisis , Leucotrieno B4/análisis , Lipopolisacáridos/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Proteína Oncogénica v-akt/análisis , Fagosomas/virología , Porphyromonas gingivalis , Especies Reactivas de Oxígeno/análisis , Acoplamiento Viral , Adulto JovenRESUMEN
In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10µM SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen.
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Adenocarcinoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Oxígeno/farmacología , Ácido Salicílico/farmacología , Células CACO-2 , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/análisis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Oncogénica v-akt/análisis , Fosforilación/efectos de los fármacosRESUMEN
Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.
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Neoplasias Colorrectales/patología , Ácido Graso Sintasas/metabolismo , Receptores de Hialuranos/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación hacia Abajo/genética , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Quinasa 1 de Adhesión Focal/análisis , Técnicas de Silenciamiento del Gen , Humanos , Lipogénesis/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Ratones , Ratones Desnudos , Proteína Oncogénica v-akt/análisis , Paxillin/análisis , Proteínas Proto-Oncogénicas c-met/análisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Objectives: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oralleukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. Study design: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. Results: Kruskal-Wallis and Dunns post tests revealed a significant difference in TWIST and p-Akt immune expression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearsons correlation test. Conclusions: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multistep process of oral carcinogenesis since its early stages (AU)
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Humanos , Proteína 1 Relacionada con Twist/análisis , Proteína Oncogénica v-akt/análisis , Leucoplasia Bucal/diagnóstico , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas/patología , Hiperplasia Epitelial Focal/patología , Inmunohistoquímica/métodosRESUMEN
Multiple melanocytic markers are useful for differentiating between melanoma and nonmelanocytic lesions but generally do not distinguish melanoma from nevi and atypical melanocytic lesions. We sought to determine if several immunohistochemical markers recently described in the literature, including ezrin, KBA.62, p-Akt, CD166, and nestin, may be helpful in distinguishing these lesions. One hundred ten tissue microarray samples were scored for nestin and CD166 and 220 samples for ezrin, KBA.62, and p-Akt. We found that putative stem cell markers nestin and CD166 were both expressed in most melanomas (86% and 65% of samples, respectively), including desmoplastic melanoma, but were also expressed at similar levels in nevi (79% and 74%, respectively). In addition, these markers were not specific for melanocytic lesions. Ezrin was also expressed in both nevi and melanoma (81% each), including desmoplastic melanoma (75%), and in neural tumors. KBA.62 stained more cases of nevi versus melanoma (93% and 65%, respectively) and was positive in 53% of desmoplastic melanoma. However, it was also positive in several nonmelanocytic tumors. P-Akt expression was generally weak but was increased in nevi (75%) versus melanoma (43%), and was lost in desmoplastic melanomas (5%). Overall, only KBA.62 and p-Akt expression differed between melanoma and nevi, and none of these markers were completely specific for melanocytic tumors versus nonmelanocytic lesions.
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Biomarcadores de Tumor/análisis , Melanoma/metabolismo , Nevo/metabolismo , Neoplasias Cutáneas/metabolismo , Antígenos CD/análisis , Antígenos CD/biosíntesis , Moléculas de Adhesión Celular Neuronal/análisis , Moléculas de Adhesión Celular Neuronal/biosíntesis , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/biosíntesis , Diagnóstico Diferencial , Proteínas Fetales/análisis , Proteínas Fetales/biosíntesis , Humanos , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/análisis , Proteínas de Filamentos Intermediarios/biosíntesis , Antígenos Específicos del Melanoma/análisis , Antígenos Específicos del Melanoma/biosíntesis , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Nevo/diagnóstico , Proteína Oncogénica v-akt/análisis , Proteína Oncogénica v-akt/biosíntesis , Neoplasias Cutáneas/diagnóstico , Análisis de Matrices TisularesRESUMEN
PURPOSE: The clinical relevance of prolonged trastuzumab administration in combination therapy beyond progressive disease (PD) has been suggested. Here, we examined whether trastuzumab treatment is effective in combination after failing to show antitumor activity as monotherapy in HER2-positive human breast cancer xenograft models. METHODS: We established trastuzumab PD models with HER2-positive breast cancer xenograft models and compared the antitumor activity of trastuzumab in combination with a taxane versus monotherapy with a taxane in the models subsequent to tumor progression under trastuzumab monotherapy. RESULTS: We established trastuzumab PD model using the HER2-positive human breast cancer line MDA-MB-361 and KPL-4 in in vivo. In these models, trastuzumab at the same dose as the initial treatment showed no significant antitumor activity at 3 weeks after start of treatment. Re-inoculated tumor tissues showing PD regained sensitivity to trastuzumab. In the trastuzumab PD models, the HER2 status of the tumor tissues did not decrease. Also, the pAKT level continued to decrease, as with the initial treatment, and IGF-1R was not found to be up-regulated. Instead, differences were observed in the gene-expression profiles of the tumor tissues showing PD. Trastuzumab in combination with G-CSF, which is expected to enhance antibody-dependent cellular cytotoxicity (ADCC), showed significant antitumor activity, even though the single agents alone showed no antitumor activity in the PD model. In the MDA-MB-361 trastuzumab PD model, the combination of trastuzumab with paclitaxel showed significantly more potent antitumor activity compared with paclitaxel or docetaxel monotherapy. In the KPL-4 trastuzumab PD model as well, trastuzumab showed significant antitumor activity in combination with taxanes or capecitabine after PD had developed in response to trastuzumab monotherapy. CONCLUSION: We established in vivo trastuzumab PD models, in which trastuzumab monotherapy ceases to have antitumor activity during the treatment. The mechanisms of PD with trastuzumab are considered to involve both reversible changes in the gene expression profiles in tumor tissues and a decrease of ADCC activity in the host. Our present results demonstrated that trastuzumab showed antitumor activity in combination with taxanes or capecitabine even though it showed no antitumor activity as a monotherapy, suggesting a clinical relevance of treatment with trastuzumab as a combination therapy beyond PD.
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Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados , Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Factores Estimulantes de Colonias/uso terapéutico , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Oncogénica v-akt/análisis , Proteína Oncogénica v-akt/metabolismo , Receptor ErbB-2/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes , Taxoides/administración & dosificación , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To analyze the prognostic value of molecular biomarkers in curettages of endometrioid endometrial cancer pathologic FIGO stages 1 and 2. STUDY DESIGN: Population-based survival analysis in 258 patients of classical prognostic features and molecular biomarkers of cell cycle regulation, (anti)apoptosis, proliferation, squamous differentiation, and PTEN/Akt pathway. RESULTS: With 74 months median follow-up (range, 1-209), 24 (9.3%) patients had metastases develop. Pathologic FIGO stage 2B (6% of all cases) and age > 68 years had independent multivariate prognostic value. Many molecular biomarkers were prognostic, particularly cell-cycle regulators p16, p21, p27, p53, p63, and the antiapoptosis marker survivin (which mostly stains mitoses). The strong prognostic value of a multivariate model with survivin, p21, and p53 overshadowed all other prognosticators in pathologic FIGO 1 and 2A. CONCLUSION: In pathologic FIGO stage 1 and 2A endometrioid endometrial cancer curettages, combined biomarkers survivin, p21, and p53 expression patterns are prognostically stronger than classical feature combinations.
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Biomarcadores de Tumor/análisis , Carcinoma Endometrioide/química , Neoplasias Endometriales/química , Carcinoma Endometrioide/diagnóstico , Carcinoma Endometrioide/patología , Carcinoma Endometrioide/cirugía , Legrado , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/patología , Neoplasias Endometriales/cirugía , Femenino , Humanos , Inmunohistoquímica , Estadificación de Neoplasias , Proteína Oncogénica v-akt/análisis , Fosfohidrolasa PTEN/análisis , Valor Predictivo de las Pruebas , Análisis de Matrices TisularesRESUMEN
BACKGROUND: Ovarian tumors frequently express c-Kit and/or platelet-derived growth factor receptors (PDGFRs). Imatinib mesylate blocks the growth of ovarian cancer cells in vitro and may enhance the activity of chemotherapy. This study was conducted to determine the activity of imatinib in combination with docetaxel in patients with recurrent, platinum-resistant epithelial ovarian cancer (EOC). METHODS: Eligible patients had recurrent, platinum-resistant, or refractory EOC that expressed PDGFRalpha or c-kit, as determined by immunohistochemistry. Imatinib mesylate at a dose of 600 mg orally once daily was administered continuously with docetaxel at a dose of 30 mg/m(2) given intravenously once weekly in Weeks 1 through 4 of every 6-week cycle. The primary endpoint was objective response rate (ORR) as assessed by the Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: Thirty-four patients were screened for PDGFRalpha and c-kit expression to enroll 23 patients between December 2003 and October 2005. Four patients had c-kit-positive/PDGFR-negative tumors, 11 patients had PDGFR-positive/c-kit-negative tumors, and 8 patients had c-kit-positive/PDGFR-positive tumors. The median patient age was 56 years (range, 33-76 years). Patients had received a median of 3 prior treatments. The ORR was 21.7% and included 1 complete and 4 partial responses. An additional 3 patients had stable disease for more than 4 months. Expression of PDGFR, c-kit, phosphatase and tensin homolog (PTEN), and phosphorylated protein kinase B (Akt) did not predict response to therapy. The most common adverse events encountered were fatigue (83%), nausea (74%), diarrhea (61%), anorexia (52%), and edema (65%), and the majority of those events were graded as grade 1 or 2. CONCLUSIONS: The combination imatinib and docetaxel was tolerated in patients with heavily pretreated EOC that expressed c-kit or PDGFRalpha. Few patients had sustained responses or stable disease.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Taxoides/administración & dosificación , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Benzamidas , Docetaxel , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Femenino , Humanos , Mesilato de Imatinib , Persona de Mediana Edad , Proteína Oncogénica v-akt/análisis , Compuestos Organoplatinos/uso terapéutico , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/secundario , Proteínas Proto-Oncogénicas c-kit/análisis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/análisis , Recurrencia , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus-oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB(-) (colourless cytoplasm, high G6PDH activity) and BCB(+) (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB(+) and BCB(-) oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB(+) (33.1+/-3.1%) and BCB(-) (12.1+/-1.5%) oocytes. Moreover, BCB(+) oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP) and protein biosynthesis (RPS274A and mRNA for elongation factor 1alpha, EF1A). BCB(-) oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.
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Regulación del Desarrollo de la Expresión Génica , Glucosafosfato Deshidrogenasa/metabolismo , Oocitos/enzimología , Oogénesis/fisiología , Animales , Bovinos , Técnicas de Cultivo de Célula , Colorantes , Quinasas MAP Reguladas por Señal Extracelular/análisis , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Oncogénica v-akt/análisis , Proteína Oncogénica v-akt/metabolismo , Oocitos/metabolismo , Oogénesis/genética , Oxazinas , Fosforilación , Coloración y Etiquetado/métodosRESUMEN
OBJECTIVE: To evaluate roles of the Akt signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, expression of phosphorylated Akt (pAkt), PI3K, and PTEN was analyzed in ameloblastic tumors as well as in tooth germs. METHODS: 11 tooth germs, 40 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with antibodies against pAkt, PI3K, and PTEN. RESULTS: Immunoreactivity for pAkt, PI3K, and PTEN was detected predominantly in odontogenic epithelial cells near the basement membrane in tooth germs and ameloblastic tumors. The levels of immunoreactivity for pAkt and PI3K were slightly higher in ameloblastic tumors than in tooth germs. Plexiform ameloblastomas showed significantly higher expression of PI3K than follicular ameloblastomas, and PI3K immunoreactivity in ameloblastomas without cellular variation was significantly higher than that in acanthomatous ameloblastomas. The level of PTEN immunoreactivity was significantly lower in ameloblastomas than in tooth germs. CONCLUSION: Expression of pAkt, PI3K, and PTEN in tooth germs and ameloblastic tumors suggests that these signaling molecules regulate cell survival and growth in normal and neoplastic odontogenic tissues by mediating growth factor signals. Increased expression of pAkt and PI3K and decreased expression of PTEN in ameloblastic tumors may participate in oncogenesis of odontogenic epithelium by activating the Akt signaling pathway.
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Ameloblastoma/química , Neoplasias Maxilomandibulares/química , Proteínas de la Membrana/análisis , Proteína Oncogénica v-akt/análisis , Fosfohidrolasa PTEN/análisis , Ameloblastoma/cirugía , Humanos , Inmunohistoquímica , Neoplasias Maxilomandibulares/cirugía , Estadísticas no ParamétricasRESUMEN
Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-tuberous sclerosis complex (TSC), non-lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.
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Angiomiolipoma/patología , Biomarcadores de Tumor/análisis , Células Epitelioides , Neoplasias Renales/patología , Neoplasias de los Tejidos Conjuntivo y Blando/patología , Proteínas Quinasas/metabolismo , Angiomiolipoma/química , Angiomiolipoma/metabolismo , Western Blotting , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Renales/química , Neoplasias Renales/metabolismo , Neoplasias de los Tejidos Conjuntivo y Blando/química , Neoplasias de los Tejidos Conjuntivo y Blando/metabolismo , Proteína Oncogénica v-akt/análisis , Fosfohidrolasa PTEN/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/análisis , Transducción de Señal , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/análisis , Regulación hacia ArribaRESUMEN
OBJECTIVE: To determine the optimum rate of low-flow hypothermic cardiopulmonary bypass (LF), following circulatory arrest (DHCA) on brain oxygenation (bO(2)), extracellular dopamine (DA), phosphorylation of select neuroregulatory proteins responsible for neuronal injury, and survival following ischemic brain injury: CREB, Erk1/2, Akt, Bcl-2, and Bax. METHODS: The piglets were placed on cardiopulmonary bypass (CPB) and cooled to 18 degrees C. They were then subjected to 30 min of DHCA followed by 1h of LF at 20, 50, or 80 ml/(kg/min), rewarmed, separated from CPB, and maintained for 2h. The bO(2) was measured by quenching of phosphorescence; DA by microdialysis; phosphorylation of CREB, ERK1/2, Akt, Bcl-2, and Bax by Western blots. The results are means+/-SD for seven experiments. RESULTS: Pre-bypass bO(2) was 47.4+/-4.2 mmHg and decreased to 1.9+/-0.8 mmHg during DHCA. At the end of LF at 20, 50, and 80 ml/(kg/min), bO(2) was 11.8+/-1.6, 26+/-1.8, and 33.9+/-2.6 mmHg, respectively. The DA increased 510-fold relative to control (p<0.001) by 15 min of LF-20 with maximum increase occurring at 45 min. With LF-50, increase in DA was not statistically significant and no increase was observed when LF-80 was used. Bcl-2 immunoreactivity increased after LF-50 and LF-80 (140+/-14.5%, p<0.05 and 202+/-34%, p<0.05, respectively). Neither flow increased Bax immunoreactivity. The ratio of Bcl-2/Bax, pCREB, pAkt, pErk increased significantly with increasing the flow rate of LF. CONCLUSIONS: The protective effect of LF following DHCA on brain metabolism is dependent on the flow rate. Flow-dependent increase in pCREB, pErk1/2, pAkt, increase in Bcl-2/Bax, and decrease in DA indicated that to minimize DHCA-dependent neuronal injury, LF flow should be above 50 ml/(kg/min).
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Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Puente Cardiopulmonar/métodos , Paro Circulatorio Inducido por Hipotermia Profunda/métodos , Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/metabolismo , Circulación Cerebrovascular/fisiología , Cuerpo Estriado/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/análisis , Modelos Animales de Enfermedad , Dopamina/análisis , Dopaminérgicos/análisis , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteína Oncogénica v-akt/análisis , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Porcinos , Proteína X Asociada a bcl-2/análisisRESUMEN
Polycystic ovary syndrome (PCOS) manifests as chronic anovulation, ovarian hyperandrogenism, and follicular cysts, which are amplified by insulin as well as the inability of the hormone to stimulate glucose uptake in classic target tissues such as muscle and fat. In the present study, we evaluated the regulation of the insulin-signaling pathways by using immunoprecipitation and immunoblotting in whole extracts of ovaries from non-pregnant human chorionic gonadotropin (hCG)-treated rats, hyperinsulinemic-induced rats and hyperinsulinemic-induced rats, treated with hCG for 22 consecutive days. There were increased associations of insulin receptor substrate (IRS)-1 and IRS-2 with phosphatidylinositol (PI) 3-kinase, followed by enhanced protein kinase B (Akt) serine and threonine phosphorylation, in the ovaries of rats that were treated with hCG, either alone or with insulin. In contrast, the skeletal muscle demonstrated a reduced IRS-1/PI 3-kinase/Akt pathway in hyperinsulinemic-induced rats. These intracellular modifications were accompanied by follicular cysts, detected by optical microscopy, and increased androstenedione serum levels. In summary, our data show that chronic treatment with hCG or hCG plus insulin can induce changes in ovaries that simulate PCOS. In these situations, an increase in the insulin-induced IRS/PI 3-kinase/Akt pathway occurs in the ovary, suggesting that the activation of this pathway may have a role in the development of PCOS.
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Gonadotropina Coriónica/farmacología , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Animales , Quinasas MAP Reguladas por Señal Extracelular/análisis , Femenino , Immunoblotting/métodos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular/análisis , Proteína Oncogénica v-akt/análisis , Fosfoproteínas/análisis , Ratas , Ratas Wistar , Receptor de Insulina/análisisRESUMEN
Evidence suggests a sex difference in intrinsic physiological and diabetic myocardial contractile function related to antioxidant properties of female ovarian hormones. This study was designed to examine the effect of cardiac overexpression of antioxidant metallothionein on intrinsic and diabetic cardiomyocyte function. Weight-matched wild-type (FVB) and metallothionein transgenic mice of both sexes were made diabetic with streptozotocin (220 mg/kg). Contractile and intracellular Ca2+ properties were evaluated including peak shortening (PS), time to PS, time to 90% relengthening (TR90), maximal velocity of shortening or relengthening (+/- dL/dt), fura-2 fluorescence intensity change, and Ca2+ decay rate. Akt and transcription factor c-Jun levels were evaluated by Western blot. Myocytes from female FVB mice exhibited lower PS, +/- dL/dt, and fura-2 fluorescence intensity change, prolonged time to PS, TR90, and Ca2+ decay compared with male FVB mice. Interestingly, this sex difference was not present in metallothionein mice. Diabetes depressed PS, +/-dL/dt and caffeine-induced Ca2+ release, as well as prolonged TR90 and Ca2+ decay in male FVB mice, whereas it only reduced PS in female FVB mice. These diabetic dysfunctions were nullified by metallothionein in both sexes. Females displayed elevated Akt phosphorylation and reduced c-Jun phosphorylation. Diabetes dampened Akt phosphorylation in male FVB mice and enhanced c-Jun in both sexes. Diabetes-induced alterations in Akt phosphorylation and c-Jun were abolished by metallothionein. The sex difference in Akt phosphorylation but not c-Jun levels was reversed by metallothionein. These data indicate that antioxidant capacity plays an important role in sex differences in both intrinsic and diabetic cardiomyocyte contractile properties possibly related to phosphorylation of Akt and c-Jun.
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Diabetes Mellitus Experimental/fisiopatología , Metalotioneína/genética , Metalotioneína/fisiología , Miocitos Cardíacos/fisiología , Animales , Western Blotting , Calcio/análisis , Calcio/fisiología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Femenino , Regulación de la Expresión Génica , Glutatión/análisis , Masculino , Metalotioneína/análisis , Ratones , Ratones Transgénicos , Contracción Muscular/fisiología , Miocitos Cardíacos/química , Miocitos Cardíacos/patología , Proteína Oncogénica v-akt/análisis , Proteína Oncogénica v-akt/metabolismo , Estrés Oxidativo/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-jun/análisis , Proteínas Proto-Oncogénicas c-jun/metabolismo , Caracteres SexualesRESUMEN
Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-gamma (IFN-gamma). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-alpha) concentration. Lysates of cells were subjected to Western blot analyses of IKK-alpha, IKK-beta, p65 (RelA) subunit of nuclear factor kappaB (NF-kappaB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-gamma and on AM without IFN-gamma. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-gamma activation, and this phenomenon became apparent only after 48 hr. IFN-gamma-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-alpha during 72 hr culturing. In contrast, there was no NO and TNF-alpha production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-gamma-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-alpha, IKK-beta, p65 (RelA) subunit of NF-kappaB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-gamma-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-gamma activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-kappaB and Akt-FKHR signalling pathways.