RESUMEN
BACKGROUND/AIMS: The importance of copper in the metabolism of cancer cells has been widely studied in the last 20 years and a clear-cut association between copper levels and cancer deregulation has been established. Copper-64, emitting positrons and ß-radiations, is indicated for the labeling of a large number of molecules suitable for radionuclide imaging as well as radionuclide therapy. Glioblastoma multiforme (GBM) is the CNS tumor with the worse prognosis, characterized by high number of recurrences and strong resistance to chemo-radio therapy, strongly affecting patients survival. We have recently discovered and studied the small molecule SI113, as inhibitor of SGK1, a serine/threonine protein kinase, that affects several neoplastic phenotypes and signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation, perturbs cell cycle progression and restores chemo-radio sensibility by modulating SGK1-related substrates. In the present paper we aim to characterize the combined effects of 64CuCl2 and SI113 on human GBM cell lines with variable p53 expression. METHODS: Cell viability, cell death and stress/authopagic related pathways were then analyzed by FACS and WB-based assays, after exposure to SI113 and/or 64CuCl2. RESULTS: We demonstrate here, that i) 64CuCl2 is able to induce a time and dose dependent modulation of cell viability (with different IC50 values) in highly malignant gliomas and that the co-treatment with SI113 leads to ii) additive/synergistic effects in terms of cell death; iii) enhancement of the effects of ionizing radiations, probably by a TRC1 modulation; iv) modulation of the autophagic response. CONCLUSIONS: Evidence reported here underlines the therapeutic potential of the combined treatment with SI113 and 64CuCl2 in GBM cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Cobre/farmacología , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Sinergismo Farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The serum- and glucocorticoid-regulated kinase (SGK1) controls cell transformation and tumor progression. SGK1 affects mitotic stability by regulating the expression of RANBP1/RAN. Here, we demonstrate that SGK1 fluctuations indirectly modify the maturation of pre-miRNAs, by modulating the equilibrium of the RAN/RANBP1/RANGAP1 axis, the main regulator of nucleo-cytoplasmic transport. The levels of pre-miRNAs and mature miRNAs were assessed by qRT-PCR, in total extracts and after differential nuclear/cytoplasmic extraction. RANBP1 expression is the limiting step in the regulation of SGK1-SP1 dependent nuclear export. These results were validated in unrelated tumor models and primary human fibroblasts and corroborated in tumor-engrafted nude mice. The levels of pri-miRNAs, DROSHA, DICER and the compartmental distribution of XPO5 were documented. Experiments using RANGTP conformational antibodies confirmed that SGK1, through RANBP1, decreases the level of the GTP-bound state of RAN. This novel mechanism may play a role in the epigenomic regulation of cell physiology and fate.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , MicroARNs/metabolismo , Neoplasias Experimentales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Epigénesis Genética , Células HEK293 , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias Experimentales/genéticaRESUMEN
The serum- and glucocorticoid-regulated kinase (SGK) family consists of three members, SGK1, SGK2 and SGK3, all displaying serine/threonine kinase activity and sharing structural and functional similarities with the AKT family of kinases. SGK1 was originally described as a key enzyme in the hormonal regulation of several ion channels and pumps. Over time, growing and impressive evidence has been accumulated, linking SGK1 to the cell survival, de-differentiation, cell cycle control, regulation of caspases, response to chemical, mechanical and oxidative injury in cancer models as well as to the control of mitotic stability. Much evidence shows that SGK1 is over-expressed in a variety of epithelial tumors. More recently, many contributions to the published literature demonstrate that SGK1 can mediate chemo-and radio-resistance during the treatment of various human tumors, both in vitro and in vivo. SGK1 appears therefore as a dirty player in the stress response to chemical and radio-agents, responsible of a selective advantage that favors the uncontrolled tumor progression and the selection of the most aggressive clones. The purpose of this review is the analysis of the literature describing SGK1 as central node of the cell resistance, and a summary of the possible strategies in the pharmacological targeting of SGK1.
Asunto(s)
Antineoplásicos/uso terapéutico , Rayos gamma/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Proteínas de Neoplasias/genética , Neoplasias/terapia , Proteínas Serina-Treonina Quinasas/genética , Animales , Caspasas/genética , Caspasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Resistencia a Antineoplásicos/genética , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimioradioterapia/métodos , Glioblastoma/radioterapia , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
The SGK1 kinase is pivotal in signal transduction pathways operating in cell transformation and tumor progression. Here, we characterize in depth a novel potent and selective pyrazolo[3,4-d]pyrimidine-based SGK1 inhibitor. This compound, named SI113, active in vitro in the sub-micromolar range, inhibits SGK1-dependent signaling in cell lines in a dose- and time-dependent manner. We recently showed that SI113 slows down tumor growth and induces cell death in colon carcinoma cells, when used in monotherapy or in combination with paclitaxel. We now demonstrate for the first time that SI113 inhibits tumour growth in hepatocarcinoma models in vitro and in vivo. SI113-dependent tumor inhibition is dose- and time-dependent. In vitro and in vivo SI113-dependent SGK1 inhibition determined a dramatic increase in apoptosis/necrosis, inhibited cell proliferation and altered the cell cycle profile of treated cells. Proteome-wide biochemical studies confirmed that SI113 down-regulates the abundance of proteins downstream of SGK1 with established roles in neoplastic transformation, e.g. MDM2, NDRG1 and RAN network members. Consistent with knock-down and over-expressing cellular models for SGK1, SI113 potentiated and synergized with radiotherapy in tumor killing. No short-term toxicity was observed in treated animals during in vivo SI113 administration. These data show that direct SGK1 inhibition can be effective in hepatic cancer therapy, either alone or in combination with radiotherapy.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/radioterapia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Técnicas para Inmunoenzimas , Técnicas In Vitro , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/radioterapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/análisis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIMS: Published observations on serum and glucocorticoid regulated kinase 1 (Sgk1) knockout murine models and Sgk1-specific RNA silencing in the RKO human colon carcinoma cell line point to this kinase as a central player in colon carcinogenesis and in resistance to taxanes. METHODS: By in vitro kinase activity inhibition assays, cell cycle and viability analysis in human cancer model systems, we describe the biologic effects of a recently identified kinase inhibitor, SI113, characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold, that shows specificity for Sgk1. RESULTS: SI113 was able to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. In RKO cells, this kinase inhibitor blocked insulin-dependent phosphorylation of the Sgk1 substrate Mdm2, the main regulator of p53 protein stability, and induced necrosis and apoptosis when used as a single agent. Finally, SI113 potentiated the effects of paclitaxel on cell viability. CONCLUSION: Since SI113 appears to be effective in inducing cell death in RKO cells, potentiating paclitaxel sensitivity, we believe that this new molecule could be efficiently employed, alone or in combination with paclitaxel, in colon cancer chemotherapy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Insulina/farmacología , Células MCF-7 , Necrosis , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/química , Pirazoles/uso terapéutico , Pirimidinas/química , Pirimidinas/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The serum/glucocorticoid-inducible kinase 1 (Sgk1) has demonstrated antiapoptotic function and the capability to regulate cell survival, proliferation, and differentiation. A pivotal role of Sgk1 in carcinogenesis and in resistance to anticancer therapy has been suggested. With the aim of identifying new Sgk1 modulators, 322 pyrazolo-pyrimidine derivatives have been virtually screened with respect to a crystallographic model of Sgk1. The top five ranked compounds have been evaluated demonstrating Sgk1 inhibition in vitro and selectivity compared to RAC-alpha serine/threonine-protein kinase (Akt1).