RESUMEN
A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitinsâ 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Hidrocarburos Fluorados/farmacología , Proteínas Represoras/antagonistas & inhibidores , Tiazoles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Células Jurkat , Estructura Molecular , Prohibitinas , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
BACKGROUND: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. METHODS: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. RESULTS: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. CONCLUSION: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias Hipofisarias/enzimología , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Animales , Bromodesoxiuridina/farmacología , Proliferación Celular , Flavonoides/farmacología , Citometría de Flujo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Ratas , Transducción de Señal , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.
Asunto(s)
Apoptosis/efectos de los fármacos , Bromocriptina/uso terapéutico , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/patología , Prolactinoma/tratamiento farmacológico , Prolactinoma/patología , Animales , Apoptosis/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , MAP Quinasa Quinasa 2/fisiología , Masculino , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Neoplasias Hipofisarias/enzimología , Prolactinoma/enzimología , Proteína Quinasa C-delta/fisiología , Ratas , Ratas Wistar , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Lactotrofos/citología , Lactotrofos/fisiología , Proteína Quinasa C-epsilon/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Femenino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.
Asunto(s)
Estradiol/farmacología , Estrógenos/farmacología , Insulina/farmacología , Lactotrofos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Sitios de Unión , Membrana Celular/metabolismo , Proliferación Celular , Estradiol/metabolismo , Estrógenos/metabolismo , Inmunohistoquímica , Insulina/metabolismo , Lactotrofos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , Microscopía Inmunoelectrónica , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Ratas , Ratas Wistar , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Tiazoles/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Fibroblastos/metabolismo , Fibroblastos/patología , Células HT29 , Células HeLa , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Prohibitinas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
El prolactinoma es el tumor más frecuente de la hipófisis y causa una gran morbilidad debido a la hipersecreción hormonal de prolactina (PRL). Rl tratamiento de primera línea es la administración de bromocriptina (BC), un agonista de los receptores dopaminergicos que posee una efectividad de alrededor del 90%. En numerosos estudios clínicos se ha documentado que BC inhibe la transcripción, la síntesis y liberación del PRL y además, suprime la proliferacion e induce la muerte celular provocando la involucción de la masa tumoral. L apoptosis ha sido identificada como un mecanismo de muerte inducido por BC, pero su baja frecuencia no explica la reducción en el tamaño de los prolactinomas. Es por ello que el efecto citotócico de la BC puede disparar otros mecanismos no apoptóticos en las células hipofisarias que serían los principales responsables de la involución del tumor.