RESUMEN

Human aortic endothelial cells (HAEC) exposed to 50 microg/ml oxidized L-A-phosphatidylcholine B-arachidonoyl-gamma-palmitoyl (ox-PAPC) for 6h increased in interleukin-8 mRNA and protein levels. Preincubation of HAEC with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) inhibitor, (20 microM), significantly inhibited ox-PAPC-stimulated interleukin-8 mRNA and protein levels. Mevalonate (200 microM) reversed the inhibition of ox-PAPC-stimulated mRNA and protein levels by lovastatin, indicating the inhibitory effect of lovastatin was due to inhibition of mevalonate synthesis. Addition of the geranylgeraniol (GGOL, 10 microM) but not farnesol (FOL, 10 microM), reversed the inhibitory effect of lovastatin on interleukin-8 mRNA and protein levels stimulated by ox-PAPC, indicating that lovastatin exerted its effect by inhibiting stores of geranylgeranyl pyrophosphate (GGPP) which are necessary for geranylgeranylation of proteins. These results suggest a new mechanism for lovastatin in preventing atherosclerosis by inhibiting the inflammatory response that takes place in the vascular wall.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Interleucina-8/genética , Lovastatina/farmacología , Fosfatidilcolinas/farmacología , Fosfatos de Poliisoprenilo/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/efectos de los fármacos , Aorta/citología , Células Cultivadas , Endotelio Vascular/citología , Humanos

RESUMEN

Recurrent prostate cancer (PC) is usually treated with androgen deprivation therapy, which, despite initial success, eventually fails due to the development of androgen-independent PC. Androgen deprivation stimulates a significant increase in the phosphorylation (activation) of Akt, a serine/threonine kinase, which regulates cell growth and survival. Hence, we asked whether the increase in Akt phosphorylation contributes to the development of androgen independence. Akt regulates transcriptional activity of the androgen receptor (AR), and our data show that Akt-stimulated AR transcriptional activity is dependent on androgen-binding to the AR. PC proliferation has both androgen-sensitive and insensitive components. The androgen sensitive component is Akt-dependent, while the androgen-insensitive is not. However, Akt-induced cell survival is largely AR independent, suggesting that the cell stimulates Akt phosphorylation when subjected to androgen deprivation as an alternate pathway to maintain survival.

Asunto(s)

Proliferación Celular , Neoplasias Hormono-Dependientes/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos/farmacología , Andrógenos/metabolismo , Anilidas/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Masculino , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Nitrilos/farmacología , Fosforilación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Transducción de Señal , Compuestos de Tosilo/farmacología , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

Prostate cancer is initially dependent on androgens for growth; hence, recurrent prostate is treated with androgen ablation which may result in progression to androgen independence characterized by a resistance to such therapy. Androgens bind to and activate the androgen receptor (AR), a member of the nuclear steroid receptor family of transcription factors, which regulates prostate cancer cell proliferation and survival in androgen-independent, as well as -dependent, tumors. Another pathway regulating proliferation and survival is the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Here we analyze reports in the literature indicating that these two pathways cooperate to regulate prostate tumor development and progression. Studies show that AR transcriptional activity and expression are regulated by Akt. In addition, androgens regulate the Akt pathway by both genomic and non-genomic effects. This explains why prostate tumors subjected to androgen ablation experience an increase in Akt phosphorylation, and suggest that the tumor compensates for the loss of one pathway with another. Different modes of interaction between the two pathways, including direct interaction, or regulation via downstream intermediates, such as the wnt/GSK-3beta/beta-catenin pathway, NF-kappaB, and the FOXO family of transcription factors, will be discussed. In addition, we will discuss the role of Akt in the interaction of the AR with upstream regulators of Akt phosphorylation, such as receptor tyrosine kinases of the EGF and IGF-1 receptor families and the tumor suppressor PTEN.

Asunto(s)

Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cross-Talk , Receptores Androgénicos/metabolismo , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/enzimología

RESUMEN

PURPOSE: A considerable fraction of patients who undergo radical prostatectomy as treatment for primary prostate cancer experience biochemical recurrence detected by elevated serum levels of prostate-specific antigen. In this study, we investigate whether loss of expression of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the phosphorylated form of the cell survival protein Akt (pAkt) predicts biochemical recurrence. EXPERIMENTAL DESIGN: Expression of PTEN and pAkt was detected by immunohistochemistry in paraffin-embedded prostate cancer tissue obtained from men undergoing radical prostatectomy. Outcome was determined by 60-month follow-up determining serum prostate-specific antigen levels. RESULTS: By itself, PTEN was not a good predictor of biochemical recurrence; however, in combination with pAkt, it was a better predictor of the risk of biochemical recurrence compared with pAkt alone. Ninety percent of all cases with high pAkt and negative PTEN were recurrent whereas 88.2% of those with low pAkt and positive PTEN were nonrecurrent. In addition, high Gleason scores resulted in reduced protection from decreased pAkt and increased PTEN. By univariate logistic regression, pAkt alone gives an area under the receiver-operator characteristic curve of 0.82 whereas the area under the receiver-operator characteristic curve for the combination of PTEN, pAkt, and Gleason based on a stepwise selection model is 0.89, indicating excellent discrimination. CONCLUSIONS: Our results indicate that loss of PTEN expression, together with increased Akt phosphorylation and Gleason score, is of significant predictive value for determining, at the time of prostatectomy, the risk of biochemical recurrence.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Fosfohidrolasa PTEN/biosíntesis , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Recurrencia , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia Celular , Activación Enzimática , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Riesgo

RESUMEN

Epidermal growth factor receptor (EGFR) is widely expressed in a number of solid tumors including colorectal cancers. Overexpression of this receptor is one means by which a cell can achieve positive signals for survival and proliferation; another effective means is by constitutive activation of EGFR. We have elucidated the role of constitutive EGFR signaling in malignant progression by stably transfecting colon cancer cells with a human transforming growth factor-alpha cDNA (a ligand for EGFR) under repressible control by tetracycline. We show that constitutive expression of transforming growth factor-alpha and its subsequent constitutive activation of EGFR allows for cancer cell survival in response to environmental stress in vitro and in vivo as well. The reversal of constitutive EGFR activation results in the loss of downstream mitogen-activated protein kinase and Akt activation, and a reduction in xenograft size that is associated with decreased proliferation and increased apoptosis. We used CI-1033, a small molecule antagonist of EGFR, to dissect an activation pathway that shows the ability of ERBb2 to activate Akt, but not Erk in the face of EGFR antagonism. This novel escape mechanism is a possible explanation of why anti-EGFR therapies have shown disappointing results in clinical trials.

Asunto(s)

Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Morfolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Procesos de Crecimiento Celular , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador alfa/antagonistas & inhibidores , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

PURPOSE: To assess the feasibility of administering erlotinib, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, in combination with paclitaxel and carboplatin, and to identify pharmacokinetic interactions, evaluate downstream effects of EGFR inhibition on surrogate tissues, and seek preliminary evidence for clinical activity. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated continuously with erlotinib at doses of 100, 125, and 150 mg/d orally along with fixed i.v. doses of paclitaxel 225 mg/m(2) and carboplatin AUC 6 mg x min/mL, both on day 1 every 3 weeks. RESULTS: Twenty evaluable patients were treated with 136 courses of erlotinib, paclitaxel, and carboplatin. Myelosuppression, skin rash, and diarrhea were the principal toxicities. Dose limiting diarrhea occurred in 1 of 6 patients at the 100 mg erlotinib dose level, whereas 0 of 9 evaluable patients at the 125 mg erlotinib dose level experienced dose limiting toxicity and 3 of 5 evaluable patients at 150 mg erlotinib experienced dose limiting skin rash and neutropenic sepsis. There was no evidence of pharmacokinetic interactions between paclitaxel and erlotinib; however, total carboplatin exposure trended higher in the presence of erlotinib. No consistent downstream effects on EGFR inhibition were found in skin. Durable objective responses were observed in non-small-cell lung and head and neck cancers. CONCLUSIONS: A dose level of erlotinib 125 mg combined with paclitaxel 225 mg/m(2) and carboplatin AUC 6 mg.min/mL is recommended for disease-directed studies. This phase I trial was followed by a randomized phase III study in non-small-cell lung cancer using a similar regimen.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Quinazolinas/farmacocinética , Quinazolinas/uso terapéutico , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carboplatino/administración & dosificación , Carboplatino/efectos adversos , Carboplatino/farmacocinética , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Clorhidrato de Erlotinib , Femenino , Enfermedades Hematológicas/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/patología , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversos , Paclitaxel/farmacocinética , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/efectos adversos , Piel/efectos de los fármacos , Piel/metabolismo

RESUMEN

BACKGROUND: Temsirolimus (CCI-779) is a small-molecule inhibitor of the mammalian target of rapamycin (mTOR) and represents a rational therapeutic target against glioblastoma multiforme (GBM). METHODS: Recurrent GBM patients with < or = 1 chemotherapy regimen for progressive disease were eligible. Temsirolimus was administered in a 250-mg intravenous dose weekly. RESULTS: Sixty-five patients were treated. The incidence of grade 3 or higher nonhematologic toxicity was 51%, and consisted mostly of hypercholesterolemia (11%), hypertriglyceridemia (8%), and hyperglycemia (8%). Grade 3 hematologic toxicity was observed in 11% of patients. Temsirolimus peak concentration (Cmax), and sirolimus Cmax and area under the concentration-time curve were decreased in patients receiving p450 enzyme-inducing anticonvulsants (EIACs) by 73%, 47%, and 50%, respectively, but were still within the therapeutic range of preclinical models. Twenty patients (36%) had evidence of improvement in neuroimaging, consisting of decrease in T2 signal abnormality +/- decrease in T1 gadolinium enhancement, on stable or reduced steroid doses. Progression-free survival at 6 months was 7.8% and median overall survival was 4.4 months. Median time to progression (TTP) for all patients was 2.3 months and was significantly longer for responders (5.4 months) versus nonresponders (1.9 months). Development of grade 2 or higher hyperlipidemia in the first two treatment cycles was associated with a higher percentage of radiographic response (71% v 31%; P = .04). Significant correlation was observed between radiographic improvement and high levels of phosphorylated p70s6 kinase in baseline tumor samples (P = .04). CONCLUSION: Temsirolimus is well tolerated in recurrent GBM patients. Despite the effect of EIACs on temsirolimus metabolism, therapeutic levels were achieved. Radiographic improvement was observed in 36% of temsirolimus-treated patients, and was associated with significantly longer TTP. High levels of phosphorylated p70s6 kinase in baseline tumor samples appear to predict a patient population more likely to derive benefit from treatment. These findings should be validated in other studies of mTOR inhibitors.

Asunto(s)

Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/efectos de los fármacos , Sirolimus/análogos & derivados , Adulto , Anciano , Antineoplásicos Alquilantes/efectos adversos , Neoplasias Encefálicas/patología , Supervivencia sin Enfermedad , Femenino , Genes erbB-1/fisiología , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Pronóstico , Inhibidores de Proteínas Quinasas/efectos adversos , Terapia Recuperativa , Sirolimus/efectos adversos , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR , Resultado del Tratamiento , Proteínas Supresoras de Tumor/genética

RESUMEN

In a previous report, we showed that increased activation of Akt, a downstream effector of phosphoinositide 3-kinase (PI3K) together with decreased activation of extracellular-signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, predicted poor clinical outcome in prostate cancer (Kreisberg et al. 2004 Cancer Research 64 5232-5236). We now show that Akt activation, but not ERK activation, is correlated with proliferation in human prostate tumors as estimated by the expression of the cell proliferation antigen Ki67. We verified these results in vitro, using the androgen-dependent prostate cancer cell line LNCaP and its androgen-independent clone C4-2 as models of prostate cancer of good and poor clinical outcome, respectively. C4-2 cells expressed higher Akt activation, lower ERK activation and increased proliferation compared with LNCaP cells, similar to cases of poor clinical outcome. The PI3K inhibitor LY294002, but not the MAPK/ERK kinase inhibitor PD98059, induced growth arrest in both cell lines. Transient transfection with constitutively active Akt increased proliferation while dominant negative Akt decreased it, thus showing that Akt plays an important role in prostate cancer proliferation. Akt regulates the expression and activation of the androgen receptor. Androgen receptor inhibition with Casodex induced growth arrest in LNCaP cells, but not in C4-2 cells. Another PI3K downstream effector, p70 S6 kinase, requires prior phosphorylation by mammalian target of rapamycin (mTOR) for complete activation. Activation of p70 S6 kinase was higher in C4-2 compared with LNCaP cells. Rapamycin, an mTOR inhibitor, had a growth-inhibitory effect in C4-2 cells, but not in LNCaP cells. Our data suggest a shift from a Casodex-sensitive proliferation pathway in LNCaP cells to a rapamycin-sensitive pathway in C4-2 cells.

Asunto(s)

Andrógenos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Antagonistas de Andrógenos/farmacología , Antagonistas de Receptores Androgénicos , Anilidas/farmacología , Antibióticos Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Antígeno Ki-67/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/secundario , Nitrilos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/secundario , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Receptores Androgénicos/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Compuestos de Tosilo , Células Tumorales Cultivadas

RESUMEN

We previously showed, by immunohistochemistry with phospho-specific antibodies, increased phosphorylation (activation) of Akt (Ser(473)) [phosphorylated Akt (pAkt)] in high-Gleason grade prostate cancer (Malik SN, et al., Clin Cancer Res 2002;8:1168-71). Elevation of pAkt was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr(202)/Tyr(204)) [phosphorylated ERK (pERK)], indicative of inactivation. In this report, we determined whether increased pAkt and decreased pERK predicted clinical outcome. Prostate-specific antigen (PSA) failure (detectable and rising PSA) versus PSA non-failure (undetectable PSA 5 years after prostatectomy) was used as a surrogate for clinical outcome. Prostate tumors from cases of PSA failure versus non-failure were stained for pAkt and pERK. A significant increase in mean pAkt staining (P < 0.001) in the PSA failures versus non-failures was seen based on the Wilcoxon signed ranks test [222.18 +/- 33.9 (n = 37) versus 108.79 +/- 104.57 (n = 16)]. Using the best-fitting multiple logistic regression equation, a 100-point increase in pAkt staining resulted in a 160% increase in the odds of being a PSA failure. There was decreased staining for pERK in PSA failures versus non-failures: a 100-point decrease resulted in an 80% increase in the odds of being a PSA failure. Each of these effects assumed the other biomarker was held constant. The area under the receiver-operating characteristic curve for these two biomarkers predicting PSA failure was 0.84, indicating excellent discrimination between PSA failure and non-failure cases. These data indicate that increased pAkt, alone or together with decreased pERK, is an important predictor of probability of PSA failure. However, pERK alone was not a significant predictor of PSA failure.

Asunto(s)

Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Valor Predictivo de las Pruebas , Antígeno Prostático Específico/metabolismo , Prostatectomía , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt

RESUMEN

PURPOSE: To assess the feasibility of administering oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the Bcl-2 mRNA, with docetaxel to patients with hormone-refractory prostate cancer; to characterize the pertinent pharmacokinetic parameters, Bcl-2 protein inhibition in peripheral blood mononuclear cell(s) (PBMC) and tumor; and to seek preliminary evidence of antitumor activity. EXPERIMENTAL DESIGN: Patients were treated with increasing doses of oblimersen sodium administered by continuous i.v. infusion on days 1 to 6 and docetaxel administered i.v. over 1 h on day 6 every 3 weeks. Plasma was sampled to characterize the pharmacokinetic parameters of both oblimersen and docetaxel, and Bcl-2 protein expression was measured from paired collections of PBMCs pretreatment and post-treatment. RESULTS: Twenty patients received 124 courses of the oblimersen and docetaxel combination at doses ranging from 5 to 7 mg/kg/day oblimersen and 60 to 100 mg/m(2) docetaxel. The rate of severe fatigue accompanied by severe neutropenia was unacceptably high at doses exceeding 7 mg/kg/day oblimersen and 75 mg/m(2) docetaxel. Nausea, vomiting, and fever were common, but rarely severe. Oblimersen mean steady-state concentrations were 3.44 +/- 1.31 and 5.32 +/- 2.34 at the 5- and 7-mg/kg dose levels, respectively. Prostate-specific antigen responses were observed in 7 of 12 taxane-naïve patients, but in taxane-refractory patients no responses were observed. Preliminary evaluation of Bcl-2 expression in diagnostic tumor specimens was not predictive of response to this therapy. CONCLUSIONS: The recommended Phase II doses for oblimersen and docetaxel on this schedule are 7 mg/kg/day continuous i.v. infusion days 1 to 6, and 75 mg/m(2) i.v. day 6, respectively, once every 3 weeks. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition in PBMC and tumor tissue, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination.

Asunto(s)

Antineoplásicos/farmacocinética , Oligonucleótidos Antisentido/farmacocinética , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Taxoides/farmacocinética , Tionucleótidos/farmacocinética , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Área Bajo la Curva , Biopsia , Docetaxel , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Humanos , Inmunohistoquímica , Infusiones Intravenosas , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Fosforilación , Antígeno Prostático Específico/biosíntesis , ARN Mensajero/metabolismo , Taxoides/administración & dosificación , Taxoides/efectos adversos , Taxoides/uso terapéutico , Tionucleótidos/administración & dosificación , Tionucleótidos/efectos adversos , Factores de Tiempo

RESUMEN

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, Gö6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.

Asunto(s)

Arginina Vasopresina/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Fármacos Renales/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación/efectos de los fármacos , Ratas

RESUMEN

Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), has often been implicated in prostate cancer. Studies in prostate tumor cell lines revealed that Akt activation is probably important for the progression of prostate cancer to an androgen-independent state. Investigations of human prostate cancer tissues show that although there is neither Akt gene amplification nor enhanced protein expression in prostate cancer compared to normal tissue, poorly differentiated tumors exhibit increased expression of a phosphorylated (activated) form of Akt compared to normal tissue, prostatic intraepithelial neoplasia (PIN) or well-differentiated prostate cancer. Akt phosphorylation is accompanied by the inactivation of ERK, a member of the mitogen activated protein kinase (MAPK) family. In this article, we postulate that Akt promotes androgen-independent survival of prostate tumor cells by modulating the expression and activation of the androgen receptor (AR).

Asunto(s)

Andrógenos/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Animales , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores Androgénicos/fisiología , Transducción de Señal , Proteínas Supresoras de Tumor/fisiología , Proteínas ras/fisiología

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) cell lines, MIA PaCa-2, and UK Pan-1, were used to investigate the role of ErbB2 in PDAC oncogenesis. Both these cell lines exhibit exogenous growth factor-independent proliferation that was attributed to the production of autocrine growth factors and/or overexpression of growth factor receptors. The exogenous growth factor-independent phenotype displayed by these PDAC cell lines was dependent on ErbB2 kinase activity since treatment of cells with tyrphostin AG879 prevented serum-free media (SFM) induction of cell proliferation. We determined that ErbB2 kinase contributed to aberrant cell cycle regulation in PDAC through the induction of cyclin D1 levels and the suppression of p21(Cip1) and p27(Kip1). Inhibition of ErbB2 kinase led to cell cycle arrest marked by an increased association of p27(Kip1) with cdk2 and reduced levels of phosphorylated pRb. We further observed constitutive STAT3 activation in the PDAC cell lines and an increase in STAT3 activation upon stimulating quiescent cells with SFM. Inhibitors of ErbB2 kinase blocked STAT3 activation, whereas inhibition of EGFR kinase led to a slight reduction of STAT3 activation. STAT3 was coimmunoprecipitated with ErbB2. SFM stimulation caused an increase in the association of ErbB2 and STAT3, which was blocked by inhibition of ErbB2 kinase. Expression of a STAT3 dominant negative prevented SFM-stimulated cell proliferation of MIA PaCa-2 cells, suggesting that activation of STAT3 by ErbB2 is required for a growth factor-independent phenotype of these cells. Consistent with this observation in PDAC cell lines, we found that most PDAC tumor specimens (10 of 11) showed constitutive activation of STAT3 and that ErbB2 was readily detected in most of these tumors (nine of 11). We believe that these findings indicate a novel mechanism of oncogenesis in PDAC and may suggest future therapeutic strategies in the treatment of PDAC.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Neoplasias Pancreáticas/patología , Receptor ErbB-2/fisiología , Transactivadores/fisiología , Quinasas CDC2-CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclina D1/biosíntesis , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Receptores ErbB/fisiología , Sustancias de Crecimiento/fisiología , Humanos , Fosforilación , Receptor ErbB-2/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción STAT3 , Proteínas Supresoras de Tumor/metabolismo , Tirosina/metabolismo

RESUMEN

BACKGROUND: OSI-774 is an inhibitor of the epidermal growth factor receptor tyrosine kinase (EGFR-TK) currently in clinical development. In preclinical models, the antitumor activity of OSI-774 was directly related to its ability to inhibit the EGFR-TK. On the basis of these data, we hypothesized that inhibition of the EGFR-TK will be required for this agent to be effective in the clinic. This study evaluated the pharmacodynamic effects of OSI-774 in normal skin tissues collected from patients treated with the agent in a Phase I study. METHODS: Patients with advanced cancer who were treated in a Phase I study of OSI-774 underwent a biopsy of normal skin epidermis at baseline and after the last dose of drug in the first course of treatment. The expression and activation of the EGFR, downstream signaling extracytoplasmatic-regulated kinase (Erk), and cell cycle regulator p27 were determined in paraffin-embedded skin tissues using an immunohistochemical method (IHC). The IHC data were analyzed using both a semiquantitative scoring system and an automatic absorbance quantitative IHC method. The number of cells with nuclear staining of p27 per 500 cells was determined. Plasma samples were collected to quantitate OSI-774 plasma concentrations. RESULTS: A total of 56 skin specimens was collected from 28 patients treated with OSI-774 at doses ranging from 25 to 200 mg/day. There was a significant decrease in phospho-EGFR (Tyr 1173) expression as determined semiquantitatively with OSI-774 treatment [2.75 +/- 0.51 (mean +/- SD) pretreatment versus 2.36 +/- 0.76 after treatment, pair comparison P = 0.01]. The quantitative ratio [(phopho-EGFR/EGFR) x 100] of phospho-EGFR (Tyr1173) decreased from 64.16 +/- 36.58 pretreatment to 48.87 +/- 35.37 post-treatment (pair comparison, P = 0.02). No significant differences were observed in phospho-Erk (Thr202/Tyr204) expression. The mean number of cells with nuclear staining for p27 increased from 185 +/- 101 (mean +/- SD) pretreatment to 253 +/- 111 post-treatment (pair comparison P = 0.02). A total of 12 (42.8%), 7 (25%), and 14 (50%) patients had >25% variation in the ratio of phospho-EGFR (Tyr1173), phospho-Erk (Thr202/Tyr204), and p27 expression, respectively. Only changes in p27 expression were related to the administered dose of OSI-774. CONCLUSIONS: OSI-774 exerted pharmacodynamic effects in skin tissues of 30-50% of patients treated with the agent. Up-regulation of p27, which is a downstream effect of EGFR inhibition, was dose related. Although there was a significant decrement in phospho-EGFR (Tyr1173), it was not related to the administered dose of OSI-774. On the basis of these findings and the relatively simple and reliable method to measure p27 expression, this biomarker appears to be the most promising and is being evaluated in Phase II studies as a predictor of clinical outcome.

Asunto(s)

Antineoplásicos/farmacocinética , Receptores ErbB/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Quinazolinas/farmacocinética , Piel/efectos de los fármacos , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Humanos , Inmunohistoquímica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Piel/metabolismo , Tirosina/metabolismo , Regulación hacia Arriba

RESUMEN

TGF-beta1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-beta1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-beta1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle alpha-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-beta1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-beta1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-beta1-stimulated alpha1(I) collagen mRNA but did not inhibit TGF-beta-stimulated alpha1(IV) mRNA expression. A similar decrease in alpha1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-beta1-stimulated alpha1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-beta receptor number, or receptor affinity. Thus, TGF-beta1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.

Asunto(s)

Colágeno/metabolismo , Citoesqueleto/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Actinas/metabolismo , Northern Blotting , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Citocalasina D/farmacología , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Mesangio Glomerular/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Metaloproteinasas de la Matriz/metabolismo , Fosforilación , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Tiazoles/farmacología , Tiazolidinas , Factores de Tiempo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Factor de Crecimiento Transformador beta1

RESUMEN

Tubulin, the main component of microtubules, is a major target for antitumor drugs such as vinblastine. We have recently discovered that the betaII isotype of tubulin is present in the nuclei of cultured rat kidney mesangial cells, smooth-muscle-like cells present in the renal glomerular mesangium (Walss C, Kreisberg JI, Ludueña RF: Cell Motil Cytoskeleton 42: 274-284, 1999). Here, we have investigated the effect of vinblastine on nuclear betaII-tubulin in these cells. We have found that, at concentrations of 15 nM and higher, vinblastine caused a reversible loss of betaII-tubulin from the nucleus. Our results raise the possibility that nuclear betaII-tubulin constitutes a population of tubulin that could be a novel target for antitumor drugs such as vinblastine.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Núcleo Celular/efectos de los fármacos , Mesangio Glomerular/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Vinblastina/farmacología , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley

Asunto(s)

Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Técnicas para Inmunoenzimas , Proteínas de Neoplasias/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Antineoplásicos/uso terapéutico , Biopsia , Inhibidores Enzimáticos/uso terapéutico , Receptores ErbB/metabolismo , Hematoxilina , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Adhesión en Parafina , Fosforilación/efectos de los fármacos , Manejo de Especímenes , Coloración y Etiquetado

RESUMEN

We previously showed that RhoA played an important role in the proliferation of murine We prostate cancer (TRAMP) cells (P. M. Ghosh et al., Oncogene, 18: 4120-4130, 1999). Untransfected TRAMP cells as well as those expressing constitutively active RhoA (Q63L) mutant protein (Q63L cells) were highly proliferative. In contrast, TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein (T19N cells) were slow growing. In this study, we showed, in addition, that T19N cells displayed reduced rates of apoptotic cell death in response to serum deprivation, compared with TRAMP and Q63L cells, and we studied the mechanisms of the effects of RhoA on TRAMP cell proliferation and apoptosis. Both proliferation and apoptosis of TRAMP and Q63L cells were dependent on the activation of phosphatidylinositol 3-kinase (PI3K). The ubiquitous mitogen-activated Ser/Thr kinase, p70S6 kinase, a downstream effector of PI3K, was overexpressed in TRAMP and Q63L cells. Another PI3K effector, the cell survival protein Akt, displayed increased activity in T19N cells, which did not express active RhoA, compared with TRAMP and Q63L cells. The atypical protein kinase C (PKC) isoform PKCzeta, which is downstream of PI3K, was activated in cells expressing active RhoA. In addition, expression of constitutively activated PKCzeta in TRAMP cells enhanced proliferation and p70S6 kinase phosphorylation, whereas the inhibition of PKCzeta activation resulted in activation of Akt and enhanced cell survival. Thus, the effects of RhoA on TRAMP cell proliferation and apoptosis may be mediated by PKCzeta.

Asunto(s)

Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas , Proteína de Unión al GTP rhoA/fisiología , Animales , Apoptosis/fisiología , División Celular/fisiología , Supervivencia Celular/fisiología , Activación Enzimática , Masculino , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas/biosíntesis , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/biosíntesis

RESUMEN

TGFbeta overexpression in human cancer cells has been shown to promote tumor progression. In the present study, we sought to determine whether sequestration of endogenous TGFbeta by the expression of a soluble TGFbeta type III receptor (sRIII), can reduce malignancy in human carcinoma cells and whether the tumor-suppressive activity of sRIII is associated with the inhibition of angiogenesis. Ectopic expression of sRIII significantly inhibited the growth of tumors formed by human colon carcinoma HCT116 and breast carcinoma MDA-MB-435 cells in nude mice. It also reduced the metastatic potential of the MDA-MB-435 cells. Thus, endogenous TGFbeta appears to be necessary for the progression of these two carcinomas. Furthermore, when the tumor cells were mixed with Matrigel and embedded subcutaneously in nude mice, the blood volume in Matrigel plugs containing sRIII-expressing cells as indicated by hemoglobin levels was significantly lower than that in Matrigel plugs containing the respective control cells. Blood vessel counts in paraffin sections of the Matrigel plugs containing sRIII-expressing cells were also significantly lower than those in paraffin sections of the Matrigel plugs containing control cells. Treatment of human endothelial cells with a recombinant sRIII significantly inhibited their ability to form a capillary web structure on Matrigel. These results for the first time indicate that the sRIII-induced tumor suppression appears to be in part due to the inhibition of angiogenesis.

Asunto(s)

Inhibidores de la Angiogénesis/genética , Carcinoma/terapia , Neovascularización Patológica/terapia , Proteoglicanos/química , Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma/irrigación sanguínea , Carcinoma/patología , División Celular , Línea Celular , Células Cultivadas , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Cinética , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transfección , Células Tumorales Cultivadas

RESUMEN

PURPOSE: Whereas the early stage of prostate cancer is marked by excessive proliferation, in advanced stages of the disease, a decreased apoptotic death rate (increased cell survival) also contributes to net tumor growth. Altered regulation of the mitogen-activated protein kinase (MAPK)-regulated cell proliferation and Akt-regulated cell survival pathways are suspected causes. In this study, we wanted to determine: (a) whether the degree of Akt activation can be assessed by immunohistochemical staining of paraffin- embedded human prostate cancer biopsies with an antibody to phospho-Akt (Ser473); and (b) whether phospho-MAPK/Erk1/2 and phospho-Akt expression are altered in prostate cancer. EXPERIMENTAL DESIGN: To examine the activation status of MAPK/Erk1/2 and Akt, archival paraffin-embedded sections from 74 cases of resected prostate cancer were immunostained with antibodies to phospho-MAPK/Erk1/2 (Thr202/ Tyr204) and phospho-Akt (Ser473). RESULTS: The staining intensity for phospho-Akt was significantly greater in Gleason grades 8-10 (92% of such cases staining strongly) compared with prostatic intraepithelial neoplasia and all other grades of prostate cancer (only 10% of these cases staining strongly; P < or = 0.001). The staining intensity for phospho-MAPK/Erk, on the other hand, was significantly greater for normal, hyperplastic, and prostatic intraepithelial neoplasia lesions but declined with disease progression, reaching its lowest level of expression in high Gleason grades 8-10 (P < 0.0001). CONCLUSION: The activation state of the cell survival protein Akt can be analyzed in human prostate cancer by immunohistochemical staining of paraffin-embedded tissue with a phospho-specific Akt (Ser473) antibody. Advanced disease is accompanied by activation of Akt and inactivation of Erk.

Asunto(s)

Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Humanos , Inmunohistoquímica , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoserina/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt