RESUMEN
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions, including cell cycle progression; cell migration, invasion, and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene, which is located downstream of PI3K, have been shown in a variety of cancers, including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways, including inhibitors of the PI3K pathway, have shown antitumor activity, but inhibitors of Akt have not been examined. In this study, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors, KP-372-1 and KP-372-2 (herein called KP-1 and KP-2), effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L, which, in turn, reduced the activation of intracellular downstream targets of Akt, including GSK-3beta and p70s6k. Furthermore, the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively, these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas.
Asunto(s)
Neoplasias del Sistema Nervioso Central/enzimología , Glioblastoma/enzimología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Tetrazoles/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fosforilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina/metabolismo , Transducción de SeñalRESUMEN
The phosphatidylinositol 3-kinase pathway is an important regulator of a wide spectrum of tumor-related biological processes, including cell proliferation, survival, and motility, as well as neovascularization. Protein kinase B/Akt is activated in a complex manner through the phosphorylation of protein kinase B/Akt on Thr308 and Ser473. Although protein-dependent kinase-1 has been shown to phosphorylate Akt at Thr308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Akt at Ser473. A possible candidate is integrin-linked kinase (ILK), which has been shown to phosphorylate Akt at Ser473 in vitro. ILK is a multidomain focal adhesion protein that is believed to be involved in signal transmission from integrin and growth factor receptors. Further, ILK is implicated in the regulation of anchorage-dependent cell growth/survival, cell cycle progression, invasion and migration, and tumor angiogenesis. In this study, we tested the hypothesis that ILK inhibition would inhibit these processes in gliomas in which it is constitutively expressed. We found that a newly developed small-molecule compound (QLT0267) effectively inhibited signaling through the ILK/Akt cascade in glioma cells by blocking the phosphorylation of Akt and downstream targets, including mammalian target of rapamycin and glycogen synthase kinase-3beta. Treatment of glioma cells with 12.5 micromol/L QLT0267 inhibited cell growth by 50% at 48 hours. An anchorage-dependent cell growth assay confirmed the cell growth-inhibitory effect of QLT0267. Further, the decrease in cell growth was associated with a dramatic accumulation of cells in the G2-M phase of the cell cycle. Although the cell growth-inhibitory effects of the ILK inhibitor were achieved only at a high concentration, the QLT0267 was able to reduce cellular invasion and angiogenesis at much lower concentrations as shown by in vitro invasion assays and vascular endothelial growth factor secretion. Thus, blocking the ILK/Akt pathway is a potential strategy for molecular targeted therapy for gliomas.
Asunto(s)
Glioblastoma/enzimología , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Western Blotting , Ciclo Celular , División Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Fase G2 , Glioma/terapia , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Serina/química , Treonina/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vitronectina/química , Vitronectina/metabolismoRESUMEN
Point mutations and deletions in mitochondrial DNA (mtDNA) accumulate as a result of oxidative stress, including ionizing radiation. As a result, dysfunctional mitochondria suffer from a decline in oxidative phosphorylation and increased release of superoxides and other reactive oxygen species (ROS). Through this mechanism, mitochondria have been implicated in a host of degenerative diseases. Associated with this type of damage, and serving as a marker of total mtDNA mutations and deletions, the accumulation of a specific 4977-bp deletion, known as the common deletion (Delta-mtDNA(4977)), takes place. The Delta-mtDNA(4977) has been reported to increase with age and during the progression of mitochondrial degeneration. The purpose of this study was to investigate whether ionizing radiation induces the formation of the common deletion in a variety of human cell lines and to determine if it is associated with cellular radiosensitivity. Cell lines used included eight normal human skin fibroblast lines, a radiosensitive non-transformed and an SV40 transformed ataxia telangiectasia (AT) homozygous fibroblast line, a Kearns Sayre Syndrome (KSS) line known to contain mitochondrial deletions, and five human tumor lines. The Delta-mtDNA(4977) was assessed by polymerase chain reaction (PCR). Significant levels of Delta-mtDNA(4977) accumulated 72 h after irradiation doses of 2, 5, 10 or 20 Gy in all of the normal lines with lower response in tumor cell lines, but the absolute amounts of the induced deletion were variable. There was no consistent dose-response relationship. SV40 transformed and non-transformed AT cell lines both showed significant induction of the deletion. However, the five tumor cell lines showed only a modest induction of the deletion, including the one line that was deficient in DNA damage repair. No relationship was found between sensitivity to radiation-induced deletions and sensitivity to cell killing by radiation.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/efectos de la radiación , Fibroblastos/efectos de la radiación , Eliminación de Secuencia , Adolescente , Adulto , Secuencia de Bases , Línea Celular , Niño , Preescolar , Cartilla de ADN , Femenino , Fibroblastos/fisiología , Humanos , Rayos Infrarrojos , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Radiación Ionizante , Piel/citologíaRESUMEN
Inhibition of poly(ADP-ribose) polymerase (PARP) by a novel, potent inhibitor, INO-1001, was examined in two rodent and one human fibroblast cell lines, after single and fractionated radiation treatments. Since PARP plays a role in the early events following DNA damage and influences the effectiveness of DNA repair, its inhibition has been proposed to constitute a drug target for the development of novel radiosensitizers. We found that INO-1001 effectively inhibited PARP activity at non-cytotoxic concentrations. Combination treatment of 10 microM INO-1001 and a single dose of radiation resulted in significant radiosensitization of all three cells lines (enhancement ratios 1.4-1.6). This radioenhancement was even greater when the drug and radiation were given as fractionated treatments (enhancement ratio 8.0). Apoptosis (as evaluated by TUNEL staining) was not enhanced by the treatments, suggesting that inhibiting PARP enzyme activity by INO-1001 enhanced radiation-induced cell killing by interfering with DNA repair mechanisms, resulting in necrotic cell death. INO-1001 therefore, appears to have potential as a potent enhancer of radiation sensitivity, without any intrinsic cytotoxicity from the drug alone.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Células CHO , Cricetinae , Reparación del ADN , Humanos , Poli(ADP-Ribosa) Polimerasas/fisiologíaRESUMEN
The BRCA1 and BRCA2 gene products are thought to play important roles in the processing of DNA damage. To assess whether heterozygous mutations in these genes are associated with cellular radiosensitivity, we performed an in vitro radiation clonogenic survival assay on dermal fibroblasts obtained from 8 sequence-proven BRCA heterozygotes (6 BRCA1, 2 BRCA2). These data were compared to results obtained from a previous set of 17 prospectively studied cancer patients who had a negligible risk for a BRCA mutation. In addition, results from radiation-induced chromatid break assay performed on lymphocytes obtained from 9 BRCA heterozygotes (8 BRCA1, 1 BRCA2) were compared to results from a control group of 18 women with no cancer history. Results from both assays suggested that cells containing a heterozygous mutation in BRCA1 or BRCA2 were more radiosensitive than controls. For the fibroblast studies, the mean surviving fraction at 2 Gy (SF2) for carriers was 0.279 vs. 0.348 for the control set (p = 0.007). For the lymphocyte studies, the mean number of chromatid breaks after 125 cGy of radiation was 0.79 breaks per cell for the carriers vs. 0.45 for the controls (p = 0.0005). There was no apparent difference in the radiosensitivity between cells with BRCA1 vs. BRCA2 mutations (p = 0.769), although the small sample size minimizes the certainty of this observation. These preliminary results are consistent with a relationship between a germline mutation in BRCA1 or BRCA2 and a hypersensitivity to radiation. This phenotype could possibly predispose to an increased risk of radiation-induced mutagenesis and carcinogenesis.