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1.
Sci Rep ; 14(1): 12363, 2024 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-38811596

RESUMEN

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.


Asunto(s)
Metilasas de Modificación del ADN , Enzimas Reparadoras del ADN , Glioblastoma , Melanoma , Tolerancia a Radiación , Proteínas Supresoras de Tumor , Humanos , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/metabolismo , Glioblastoma/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/radioterapia , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Línea Celular Tumoral , Tolerancia a Radiación/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Metilación de ADN , Reparación del ADN , Roturas del ADN de Doble Cadena/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Temozolomida/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Purinas
2.
Front Oncol ; 12: 979537, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36353533

RESUMEN

Astrocytomas are the most common subtype of brain tumors and no curative treatment exist. Longitudinal assessment of patients, usually via Magnetic Resonance Imaging (MRI), is crucial since tumor progression may occur earlier than clinical progression. MRI usually provides a means for monitoring the disease, but it only informs about the structural changes of the tumor, while molecular changes can occur as a treatment response without any MRI-visible change. Radiotherapy (RT) is routinely performed following surgery as part of the standard of care in astrocytomas, that can also include chemotherapy involving temozolomide. Monitoring the response to RT is a key factor for the management of patients. Herein, we provide plasma and tissue metabolic biomarkers of treatment response in a mouse model of astrocytoma that was subjected to radiotherapy. Plasma metabolic profiles acquired over time by Liquid Chromatography Mass Spectrometry (LC/MS) were subjected to multivariate empirical Bayes time-series analysis (MEBA) and Receiver Operating Characteristic (ROC) assessment including Random Forest as the classification strategy. These analyses revealed a variation of the plasma metabolome in those mice that underwent radiotherapy compared to controls; specifically, fumarate was the best discriminatory feature. Additionally, Nuclear Magnetic Resonance (NMR)-based 13C-tracing experiments were performed at end-point utilizing [U-13C]-Glutamine to investigate its fate in the tumor and contralateral tissues. Irradiated mice displayed lower levels of glycolytic metabolites (e.g. phosphoenolpyruvate) in tumor tissue, and a higher flux of glutamine towards succinate was observed in the radiation cohort. The plasma biomarkers provided herein could be validated in the clinic, thereby improving the assessment of brain tumor patients throughout radiotherapy. Moreover, the metabolic rewiring associated to radiotherapy in tumor tissue could lead to potential metabolic imaging approaches for monitoring treatment using blood draws.

3.
Mol Oncol ; 16(9): 1777-1794, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34856072

RESUMEN

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that make cancer cells require certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13 C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support glutathione synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine-deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, even though higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolite levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.


Asunto(s)
Cisteína , Glioma , Animales , Proliferación Celular , Cisteína/metabolismo , Cistina/metabolismo , Glutatión/metabolismo , Humanos , Ratones , Microambiente Tumoral
4.
Mol Cancer Ther ; 20(9): 1672-1679, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34158343

RESUMEN

AZD0530, a potent small-molecule inhibitor of the Src kinase family, is an anticancer drug used in the treatment of various cancers. In the case of glioblastoma (GBM), where resistance to radiotherapy frequently occurs, Src kinase is known as one of the molecules responsible for imparting radioresistance to GBM. Thus, we evaluated the effect of AZD0530 on the radiosensitivity of human GBM cells and human glioblastoma stem-like cells (GSCs). We show that Src activity of GBM and GSC is increased by radiation and inhibited by AZD0530, and using clonogenic assays, AZD0530 enhances the radiosensitivity of GBM and GSCs. Also, AZD0530 induced a prolongation of radiation-induced γH2AX without specific cell cycle and mitotic index changes, suggesting that AZD0530-induced radiosensitization in GBM cells and GSCs results from the inhibition of DNA repair. In addition, AZD0530 was shown to inhibit the radiation-induced EGFR/PI3K/AKT pathway, which is known to promote and regulate radioresistance and survival of GBM cells by radiation. Finally, mice bearing orthotopic xenografts initiated from GBM cells were then used to evaluate the in vivo response to AZD0530 and radiation. The combination of AZD0530 and radiation showed the longest median survival compared with any single modality. Thus, these results show that AZD0530 enhances the radiosensitivity of GBM cells and GSCs and suggest the possibility of AZD0530 as a clinical radiosensitizer for treatment of GBM.


Asunto(s)
Benzodioxoles/farmacología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/radioterapia , Células Madre Neoplásicas/efectos de la radiación , Quinazolinas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Ciclo Celular , Proliferación Celular , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
5.
NMR Biomed ; 34(7): e4514, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33939204

RESUMEN

Dynamic nuclear polarization (DNP) of 13 C-labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP-MRI chemical shift imaging with hyperpolarized [1-13 C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem-like cell-derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP-MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase-A expression in GBMJ1 following radiation exposure, consistent with DNP-MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP-MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.


Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Animales , Línea Celular Tumoral , Glioblastoma/diagnóstico por imagen , Humanos , Lactato Deshidrogenasa 5/metabolismo , Ácido Láctico/metabolismo , Imagen por Resonancia Magnética , Metabolómica , Ratones Desnudos , Ácido Pirúvico/metabolismo
6.
Sci Rep ; 9(1): 15458, 2019 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-31664073

RESUMEN

Cholesterol is a critical component of membranes and a precursor for hormones and other signaling molecules. Previously, we showed that unlike astrocytes, glioblastoma cells do not downregulate cholesterol synthesis when plated at high density. In this report, we show that high cell density induces ABCA1 expression in glioblastoma cells, enabling them to get rid of excess cholesterol generated by an activated cholesterol biosynthesis pathway. Because oxysterols are agonists for Liver X Receptors (LXRs), we investigated whether increased cholesterol activates LXRs to maintain cholesterol homeostasis in highly-dense glioblastoma cells. We observed that dense cells had increased oxysterols, which activated LXRß to upregulate ABCA1. Cells with CRISPR-mediated knockdown of LXRß, but not ABCA1, had decreased cell cycle progression and cell survival, and decreased feedback repression of the mevalonate pathway in densely-plated glioma cells. LXRß gene expression poorly correlates with ABCA1 in glioblastoma patients, and expression of each gene correlates with poor patient prognosis in different prognostic subtypes. Finally, gene expression and lipidomics analyses cells revealed that LXRß regulates the expression of immune response gene sets and lipids known to be involved in immune modulation. Thus, therapeutic targeting of LXRß in glioblastoma might be effective through diverse mechanisms.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/fisiología , Neoplasias Encefálicas/patología , Proliferación Celular/fisiología , Glioblastoma/patología , Metabolismo de los Lípidos , Receptores X del Hígado/fisiología , Transportador 1 de Casete de Unión a ATP/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Colesterol/metabolismo , Glioblastoma/inmunología , Glioblastoma/metabolismo , Homeostasis , Humanos , Receptores X del Hígado/metabolismo , Ácido Mevalónico/metabolismo , Transducción de Señal , Transcripción Genética
7.
Neuro Oncol ; 20(6): 799-809, 2018 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-29294092

RESUMEN

Background: Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods: Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results: PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions: Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cordoma/tratamiento farmacológico , Cordoma/patología , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Cordoma/enzimología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Cancer Lett ; 415: 217-226, 2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29199006

RESUMEN

Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Neoplasias Meníngeas/terapia , Meningioma/terapia , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quimioradioterapia/métodos , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Meningioma/metabolismo , Meningioma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Fosfatasa 2/metabolismo
9.
Radiat Res ; 187(2): 251-258, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28118115

RESUMEN

In the event of a radiological or nuclear attack, advanced clinical countermeasures are needed for screening and medical management of the exposed population. In such a scenario, minimally invasive biomarkers that can accurately quantify radiation exposure would be useful for triage management by first responders. In this murine study, we evaluated the efficacy of a novel combination of radiation responsive proteins, Flt3 ligand (FL), serum amyloid A (SAA), matrix metalloproteinase 9 (MMP9), fibrinogen beta (FGB) and pentraxin 3 (PTX3) to predict the received dose after whole- or partial-body irradiation. Ten-week-old female C57BL6 mice received a single whole-body or partial-body dose of 18 Gy from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by cardiac puncture at 24, 48, 72 h and 1 week postirradiation. Plasma protein levels were determined via commercially available ELISA assay. A multivariate discriminant analysis was utilized to generate best-fit dose prediction models for whole-body exposures using the selected biomarker panel and its potential application to partial-body exposures was examined. The combination of values from FL, SAA, MMP9, FGB and PTX3 between 24 h and 1 week postirradiation yielded novel dose-response relationships. For day 1 postirradiation, the best-fit model yielded a predictive accuracy of 81% utilizing FL alone. The use of additional proteins did not enhance the model accuracy whereas, at day 2 postirradiation, the addition of PTX3 and FGB to FL increased the accuracy to 100%. At day 3 the use of FL and PTX3 yielded a predictive accuracy of 93% and at day 7 use of FL and SAA had an accuracy of 90%. Dose prediction of partial-body exposures based on the TBI model had a higher predictive accuracy when the percentage of the body exposed to radiation increased. Our findings indicate that this novel combination of radiation responsive biomarker proteins are an efficient method for predicting radiation exposure and are more accurate when used in concert compared to using any single biomarker protein alone.


Asunto(s)
Modelos Estadísticos , Exposición a la Radiación/análisis , Irradiación Corporal Total , Animales , Biomarcadores/metabolismo , Proteína C-Reactiva/metabolismo , Femenino , Fibrinógeno/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Componente Amiloide P Sérico/metabolismo
10.
Oncotarget ; 7(47): 77365-77377, 2016 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-27764801

RESUMEN

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo. Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteína Forkhead Box M1/metabolismo , Glioblastoma/metabolismo , Tolerancia a Radiación , Factor de Transcripción STAT3/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/radioterapia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Forkhead Box M1/genética , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/radioterapia , Recombinación Homóloga , Humanos , Estimación de Kaplan-Meier , Mitosis/efectos de los fármacos , Péptidos/farmacología , Pronóstico , Unión Proteica , Transporte de Proteínas , Proteoma , Proteómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación/genética , Factor de Transcripción STAT3/genética
11.
Oncotarget ; 7(33): 52912-52927, 2016 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-25991676

RESUMEN

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-ß/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.


Asunto(s)
Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/genética , Glioblastoma/genética , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/terapia , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína smad3/metabolismo
12.
Clin Exp Metastasis ; 32(7): 717-27, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26319493

RESUMEN

Most cancer patients with brain metastases are treated with radiation therapy, yet this modality has not yet been meaningfully incorporated into preclinical experimental brain metastasis models. We applied two forms of whole brain radiation therapy (WBRT) to the brain-tropic 231-BR experimental brain metastasis model of triple-negative breast cancer. When compared to sham controls, WBRT as 3 Gy × 10 fractions (3 × 10) reduced the number of micrometastases and large metastases by 87.7 and 54.5 %, respectively (both p < 0.01); whereas a single radiation dose of 15 Gy × 1 (15 × 1) was less effective, reducing metastases by 58.4 % (p < 0.01) and 47.1 % (p = 0.41), respectively. Neuroinflammation in the adjacent brain parenchyma was due solely to a reaction from metastases, and not radiotherapy, while adult neurogenesis in brains was adversely affected following both radiation regimens. The nature of radiation resistance was investigated by ex vivo culture of tumor cells that survived initial WBRT ("Surviving" cultures). The Surviving cultures surprisingly demonstrated increased radiosensitivity ex vivo. In contrast, re-injection of Surviving cultures and re-treatment with a 3 × 10 WBRT regimen significantly reduced the number of large and micrometastases that developed in vivo, suggesting a role for the microenvironment. Micrometastases derived from tumor cells surviving initial 3 × 10 WBRT demonstrated a trend toward radioresistance upon repeat treatment (p = 0.09). The data confirm the potency of a fractionated 3 × 10 WBRT regimen and identify the brain microenvironment as a potential determinant of radiation efficacy. The data also nominate the Surviving cultures as a potential new translational model for radiotherapy.


Asunto(s)
Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundario , Irradiación Craneana/métodos , Neoplasias de la Mama Triple Negativas/radioterapia , Neoplasias de la Mama Triple Negativas/secundario , Animales , Línea Celular Tumoral , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Desnudos , Tolerancia a Radiación , Dosificación Radioterapéutica , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Radiat Res ; 184(1): 14-23, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26114330

RESUMEN

There is a need for minimally invasive biomarkers that can accurately and quickly quantify radiation exposure. Radiation-responsive proteins have applications in clinical medicine and for mass population screenings after a nuclear or radiological incident where the level of radiation exposure and exposure pattern complicate medical triage for first responders. In this study, we evaluated the efficacy of the acute phase protein serum amyloid A (SAA) as a biomarker for radiation exposure using plasma from irradiated mice. Ten-week-old female C57BL6 mice received a 1-8 Gy single whole-body or partial-body dose from a Pantak X-ray source at a dose rate of 2.28 Gy/min. Plasma was collected by mandibular or cardiac puncture at 6, 24, 48 and 72 h or 1-3 weeks postirradiation. SAA levels were determined using a commercially available ELISA assay. Data was pooled to generate SAA µg/ml threshold values correlating plasma SAA levels with radiation dose. SAA levels were statistically significant over control at all exposures between 2 and 8 Gy at 24 h postirradiation but not at 6, 48 and 72 h or 1-3 weeks postirradiation. SAA levels at 1 Gy were not significantly elevated over control at all time points. Total-body-irradiated (TBI) SAA levels at 24 h were used to generate a dose prediction model that successfully differentiated TBI mice into dose received cohorts of control/1 Gy and ≥ 2 Gy groups with a high degree of accuracy in a blind study. Dose prediction of partial-body exposures based on the TBI model correlated increasing predictive accuracy with percentage of body exposure to radiation. Our findings indicate that plasma SAA levels might be a useful biomarker for radiation exposure in a variety of total- and partial-body irradiation settings.


Asunto(s)
Monitoreo de Radiación/métodos , Proteína Amiloide A Sérica/análisis , Animales , Biomarcadores , Línea Celular , Femenino , Histonas/análisis , Humanos , Inflamación/sangre , Inflamación/etiología , Ratones , Ratones Endogámicos C57BL , Irradiación Corporal Total
14.
Mol Cancer Res ; 13(5): 852-62, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25722303

RESUMEN

UNLABELLED: To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. IMPLICATIONS: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies.


Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Reparación del ADN/efectos de la radiación , Glioblastoma/genética , Glioblastoma/radioterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/enzimología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Femenino , Glioblastoma/enzimología , Humanos , Ratones Desnudos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Tolerancia a Radiación , Distribución Aleatoria , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Clin Cancer Res ; 20(1): 110-9, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24198241

RESUMEN

PURPOSE: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines. EXPERIMENTAL DESIGN: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay. RESULTS: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation. CONCLUSION: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair.


Asunto(s)
Benzoxazoles/farmacología , Neoplasias Pancreáticas/radioterapia , Pirimidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Femenino , Humanos , Ratones , Ratones Desnudos , Biosíntesis de Proteínas/efectos de la radiación , Tolerancia a Radiación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias Pancreáticas
16.
Neuro Oncol ; 16(1): 29-37, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24311635

RESUMEN

BACKGROUND: The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs). METHODS: mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation. RESULTS: Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments. CONCLUSIONS: These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.


Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Morfolinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Células Madre Neoplásicas/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzamidas , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/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 , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Femenino , Técnica del Anticuerpo Fluorescente , Glioblastoma/patología , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Células Tumorales Cultivadas , Terapia por Rayos X , Ensayos Antitumor por Modelo de Xenoinjerto
17.
Transl Oncol ; 6(3): 355-62, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23730416

RESUMEN

The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according γH2AX foci. PP242 exposure did not influence the initial level of γH2AX foci after irradiation but did significantly delay the dispersal of radiation-induced γH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.

18.
Eur J Cancer ; 49(14): 3020-8, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23790466

RESUMEN

Glioblastoma multiforme (GBM) is the most common primary brain tumour in the United States of America (USA) with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signalling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumour growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.


Asunto(s)
Proteínas de Ciclo Celular/genética , Glioblastoma/genética , Mitosis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Femenino , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Ratones , Ratones Desnudos , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Tiofenos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1
19.
PLoS One ; 8(3): e58558, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23505536

RESUMEN

PURPOSE: Ablative bone marrow irradiation is an integral part of hematopoietic stem cell transplantation. These treatment regimens are based on classically held models of radiation dose and the bone marrow response. Flt-3 ligand (FL) has been suggested as a marker of hematopoiesis and bone marrow status but the kinetics of its response to bone marrow irradiation has yet to be fully characterized. In the current study, we examine plasma FL response to total body and partial body irradiation in mice and its relationship with irradiation dose, time of collection and pattern of bone marrow exposure. MATERIALS/METHODS: C57BL6 mice received a single whole body or partial body irradiation dose of 1-8 Gy. Plasma was collected by mandibular or cardiac puncture at 24, 48 and 72 hr post-irradiation as well as 1-3 weeks post-irradiation. FL levels were determined via ELISA assay and used to generate two models: a linear regression model and a gated values model correlating plasma FL levels with radiation dose. RESULTS: At all doses between 1-8 Gy, plasma FL levels were greater than control and the level of FL increased proportionally to the total body irradiation dose. Differences in FL levels were statistically significant at each dose and at all time points. Partial body irradiation of the trunk areas, encompassing the bulk of the hematopoietically active bone marrow, resulted in significantly increased FL levels over control but irradiation of only the head or extremities did not. FL levels were used to generate a dose prediction model for total body irradiation. In a blinded study, the model differentiated mice into dose received cohorts of 1, 4 or 8 Gy based on plasma FL levels at 24 or 72 hrs post-irradiation. CONCLUSION: Our findings indicate that plasma FL levels might be used as a marker of hematopoietically active bone marrow and radiation exposure in mice.


Asunto(s)
Médula Ósea/efectos de la radiación , Proteínas de la Membrana/sangre , Dosis de Radiación , Animales , Relación Dosis-Respuesta en la Radiación , Femenino , Ratones , Factores de Tiempo , Irradiación Corporal Total
20.
J Vis Exp ; (61): e3397, 2012 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-22415465

RESUMEN

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 µl every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.


Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Glioblastoma/patología , Glioblastoma/terapia , Trasplante de Neoplasias/métodos , Técnicas Estereotáxicas , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Craneotomía , Modelos Animales de Enfermedad , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto
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