RESUMEN
The 90-kDa heat-shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective antineoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, that exhibits potent antiproliferative and cytotoxic activity along with client protein degradation without induction of the heat-shock response in both androgen-dependent and -independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was used to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90ß, and Hsc70 proteins. The binding affinity (Kd) for Hsp90α was determined to be 191 µM, whereas the Kd for Hsp90ß was 726 µM, demonstrating a preference for Hsp90α. Western blot experiments with four different prostate cancer cell lines treated with KU675 supported this selectivity by inducing the degradation of Hsp90α -: dependent client proteins. KU675 also displayed binding to Hsc70 with a Kd value at 76.3 µM, which was supported in cellular by lower levels of Hsc70-specific client proteins on Western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C-terminal inhibitor, as well as a dual inhibitor of Hsc70, and may have potential use for the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Cumarinas/farmacología , Proteínas del Choque Térmico HSC70/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Novobiocina/análogos & derivados , Compuestos de Fenilurea/farmacología , Neoplasias de la Próstata/metabolismo , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cumarinas/síntesis química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Compuestos de Fenilurea/síntesis química , Unión ProteicaRESUMEN
Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Novobiocina/farmacología , Línea Celular , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Unión Proteica , Pliegue de Proteína , Isoformas de ProteínasRESUMEN
Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.
Asunto(s)
Carcinogénesis/genética , Fosfolipasas A2 Grupo II/genética , Polimorfismo Genético , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Alelos , Animales , Clonación Molecular , Genotipo , Fosfolipasas A2 Grupo II/metabolismo , Células HCT116 , Humanos , Intestinos/patología , Ratones , Ratones Desnudos , Degradación de ARNm Mediada por Codón sin Sentido , Plásmidos/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.
Asunto(s)
Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Luciferasas/química , Replegamiento Proteico/efectos de los fármacos , Animales , Bioensayo , Western Blotting , Línea Celular Tumoral , Colorantes , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Luciferasas/antagonistas & inhibidores , Novobiocina/análogos & derivados , Novobiocina/farmacología , Desnaturalización Proteica , Conejos , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Rodaminas , Bibliotecas de Moléculas PequeñasRESUMEN
In treating cancer with clinically approved chemotherapies, the high systemic toxicity and lack of selectivity for malignant cells often result in an overall poor response rate. One pharmacological approach to improve patient response is to design targeted therapies that exploit the cancer milieu by reductively activating prodrugs, which results in the selective release of the free drug in the tumor tissue. Previously, we characterized prodrugs of seco-CBI-indole 2 (CBI-indole 2) designed to be activated in hypoxic tumor microenvironments, wherein the tumor maintains higher concentrations of "reducing" nucleophiles capable of preferentially releasing the free drug by nucleophilic attack on a weak N-O bond. Of these prodrugs, BocNHO-CBI-indole 2 (BocNHO) surpassed the efficacy of the free drug, CBI-indole 2, when examined in vivo in the murine L1210 leukemia model and demonstrated reduced toxicity suggesting a targeted or sustained release in vivo. Herein, we further examine the biological activity of the BocNHO prodrug in murine breast cancer, as well as human prostate and lung cancer cell lines, in vitro. Notably, BocNHO manifests potent antiproliferative and cytotoxic activity in all three tumor cell lines. However, in comparison to the activity observed in the murine cancer cell line, the human cancer cell lines were less sensitive, especially at early timepoints for cytotoxicity. Based on these findings, BocNHO was tested in a more clinically relevant orthotopic lung tumor model, revealing significant efficacy and reduced toxicity compared with the free drug. The data suggests that this pharmacological approach to designing targeted therapies is amenable to human solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Carbamatos/farmacología , Indoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Profármacos/farmacología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carbamatos/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Indoles/uso terapéutico , Ratones , Ratones Desnudos , Oxidación-Reducción , Profármacos/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Two novel cyclic N-acyl O-amino phenol prodrugs are reported as new members of a unique class of reductively cleaved prodrugs of the duocarmycin family of natural products. These prodrugs were explored with the expectation that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles and were designed to liberate the free drug without the release of an extraneous group. In vivo evaluation of the prodrug 6 showed that it exhibits extraordinary efficacy (T/C > 1500, L1210; 6/10 one year survivors), substantially exceeding that of the free drug, that its therapeutic window of activity is much larger, permitting a dosing ≥ 40-fold higher than the free drug, and yet that it displays a potency in vivo that approaches the free drug (within 3-fold). Clearly, the prodrug 6 benefits from either its controlled slow release of the free drug or its preferential intracellular reductive cleavage.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Indoles/administración & dosificación , Profármacos , Alquilación , Animales , Antibióticos Antineoplásicos/química , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Cromatografía Líquida de Alta Presión , ADN/química , Preparaciones de Acción Retardada , Duocarmicinas , Indicadores y Reactivos , Indoles/química , Leucemia L1210/tratamiento farmacológico , Ratones , Pirrolidinonas/administración & dosificación , Pirrolidinonas/química , Espectrofotometría Ultravioleta , Estereoisomerismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A unique heterocyclic carbamate prodrug of seco-CBI-indole(2) that releases no residual byproduct is reported as a new member of a class of hydrolyzable prodrugs of the duocarmycin and CC-1065 family of natural products. The prodrug was designed to be activated by hydrolysis of a carbamate releasing the free drug without the cleavage release of a traceable extraneous group. Unlike prior carbamate prodrugs examined that are rapidly cleaved in vivo, the cyclic carbamate was found to be exceptionally stable to hydrolysis under both chemical and biological conditions providing a slow, sustained release of the exceptionally potent free drug. An in vivo evaluation of the prodrug found that its efficacy exceeded that of the parent drug, that its therapeutic window of efficacy versus toxicity is much larger than the parent drug, and that its slow free drug release permitted the safe and efficacious use of doses 150-fold higher than the parent compound.
Asunto(s)
Antineoplásicos/metabolismo , Carbamatos/química , Diseño de Fármacos , Indoles/metabolismo , Profármacos/metabolismo , Profármacos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Ciclopropanos/química , Duocarmicinas , Humanos , Hidrólisis , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Ratones , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heat shock protein 90 (Hsp90) has emerged as a promising therapeutic target for the treatment of cancer. Several Hsp90 inhibitors have entered clinical trials. However, some toxicological detriments have arisen, such as cardiotoxicity resulting from hERG inhibition following the administration of Hsp90 inhibitors. We sought to investigate this toxicity as hERG has been previously reported as a client protein that depends upon Hsp90 for its maturation and functional trafficking. In this study we show that hERG depends upon a single Hsp90 isoform. hERG preferentially co-immunoprecipitated with Hsp90α, and genetic knockdown of Hsp90α, but not Hsp90ß, resulted in a trafficking-defective hERG channel. This study demonstrates the importance of delineating the isoform dependence of Hsp90 client proteins and provides rationale for the design of isoform-selective Hsp90 inhibitors that avoid detrimental effects.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Isoformas de Proteínas/metabolismo , Western Blotting , Línea Celular , Densitometría , Canales de Potasio Éter-A-Go-Go/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Inmunoprecipitación , Espectrometría de Masas , Modelos Biológicos , Unión Proteica , Isoformas de Proteínas/genética , Transporte de Proteínas/fisiología , ARN Interferente PequeñoRESUMEN
BACKGROUND: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. METHODS: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. RESULTS: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. CONCLUSIONS: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Novobiocina/farmacología , Unión Proteica/efectos de los fármacos , RatasRESUMEN
A series of N-acyl O-amino derivatives of seco-CBI-indole(2) are reported and examined as prototypical members of a unique class of reductively activated (cleaved) prodrugs of the duocarmycin and CC-1065 family of antitumor agents. These prodrugs were designed to be potentially preferentially activated in hypoxic tumor environments which carry an intrinsically higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives by nucleophilic cleavage of a weak N-O bond. A remarkable range of stabilities and a resulting direct correlation with in vitro/in vivo biological potencies was observed for these prodrugs, even enlisting subtle variations in the electronic and steric environment around the weak N-O bond. An in vivo evaluation of several of the prodrugs demonstrates that some approach the potency and exceed the efficacy of the free drug itself (CBI-indole(2)), suggesting the prodrugs may offer an additional advantage related to a controlled or targeted release.
Asunto(s)
Antineoplásicos Alquilantes/síntesis química , Indoles/síntesis química , Fenoles/síntesis química , Profármacos/síntesis química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Duocarmicinas , Indoles/química , Indoles/farmacología , Leucemia L1210 , Ratones , Ratones Endogámicos DBA , Trasplante de Neoplasias , Fenoles/química , Fenoles/farmacología , Profármacos/química , Profármacos/farmacología , Pirroles/síntesis química , Pirroles/química , Pirroles/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
PURPOSE: Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG. MATERIALS AND METHODS: LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface plasmon resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90. RESULTS: F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (K(d)) of 100 microM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis. CONCLUSIONS: These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores de Crecimiento/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Novobiocina/análogos & derivados , Novobiocina/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/patología , SpodopteraRESUMEN
The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G(2)/M arrest, whereas cells treated with 17-AAG accumulated in G(1). Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x(L) overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Novobiocina/análogos & derivados , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Cromatografía de Afinidad , Citometría de Flujo , Humanos , Células Jurkat , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Novobiocina/metabolismo , Novobiocina/farmacología , Resonancia por Plasmón de SuperficieRESUMEN
N-Acyl O-amino phenol derivatives of CBI-TMI and CBI-indole2 are reported as prototypical members of a new class of reductively activated prodrugs of the duocarmycin and CC-1065 class of antitumor agents. The expectation being that hypoxic tumor environments, with their higher reducing capacity, carry an intrinsic higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives (tunable N-O bond cleavage) and increasing their sensitivity to the prodrug treatment. Preliminary studies indicate the prodrugs effectively release the free drug in functional cellular assays for cytotoxic activity approaching or matching the activity of the free drug, yet remain essentially stable and unreactive to in vitro DNA alkylation conditions (<0.1-0.01% free drug release) and pH 7.0 phosphate buffer, and exhibit a robust half-life in human plasma (t1/2 = 3 h). Characterization of a representative O-(acylamino) prodrug in vivo indicates that they approach the potency and exceed the efficacy of the free drug itself (CBI-indole2), indicating that not only is the free drug effectively released from the inactive prodrug but also that they offer additional advantages related to a controlled or targeted release in vivo.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Indoles/farmacología , Profármacos/farmacología , Alquilación/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/química , Línea Celular Tumoral , ADN/efectos de los fármacos , ADN/metabolismo , Duocarmicinas , Humanos , Indoles/química , Concentración 50 Inhibidora , Leucemia L1210/tratamiento farmacológico , Ratones , Ratones Endogámicos DBA , Fenoles/química , Fenoles/farmacologíaRESUMEN
FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2alpha (PGF2alpha). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2alpha. Following the removal of PGF2alpha, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2alpha-could activate T-cell factor (Tcf)/beta-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF2alpha in cells expressing either the FPA or FPB isoforms. However, PGE2 has much lower efficacy as compared with PGF2alpha for the activation of Tcf/beta-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2alpha-stimulated Tcf/beta-catenin signaling in a dose-dependent manner while having no effect on PGF2alpha-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2alpha has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Dinoprost/farmacología , Dinoprostona/farmacología , Receptores de Prostaglandina/fisiología , Transducción de Señal/efectos de los fármacos , Transactivadores/fisiología , Línea Celular , Proteínas del Citoesqueleto/antagonistas & inhibidores , Genes Reporteros , Humanos , Riñón , Subunidades de Proteína/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Transactivadores/antagonistas & inhibidores , Transfección , beta CateninaRESUMEN
FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.
Asunto(s)
Empalme Alternativo , ARN Mensajero , Receptores de Prostaglandina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biología Computacional , Endotelio/metabolismo , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Placenta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Venas Umbilicales/metabolismoRESUMEN
Prostaglandin F(2alpha) (PGF(2alpha)) modulates clonal selection processes in the mouse skin model of carcinogenesis. In this study we investigated whether JB6 mouse epidermal cells expressed a functional PGF(2alpha) receptor (FP) coupled with a cell-transformation response. Treatment of JB6 cells with an FP agonist (fluprostenol) potently (pM-nM) increased anchorage-dependent and anchorage-independent growth. Inositol phospholipid accumulation and extracellular signal-regulated kinase (Erk) activity were increased in cells treated with FP agonists, consistent with established FP-related signal transduction. FP mRNA was detected by reverse transcription-polymerase chain reaction, and the average specific [(3)H]PGF(2alpha) binding was 8.25 +/- 0.95 fmol/mg protein. Erk activity and colony size were increased by cotreatment of JB6 cells with epidermal growth factor (EGF) and fluprostenol to a greater extent than with either treatment alone, whereas the cotreatment effect on colony number appeared to be simply additive. Collectively, our data indicated that JB6 cells expressed a functional FP coupled with transformation-related signal transduction and the regulation of clonal selection processes. Erk activity appears to be a convergence point in the EGF and FP pathways. The data raise the possibility that the FP contributes to clonal selection processes but probably plays a more important role as a response modifier.