A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Three-dimensional magnetic resonance reconstruction images before and after surgical therapy of spontaneous canine brain tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic and modifying factors that determine the risk of brain tumors

Some modifying factors may determine the risk of brain tumors. Until now, it could not be attempted to identify people at risk and also to improve significantly disease progression. Current therapy consists of surgical resection, followed by radiation therapy and chemotherapy. Despite of these treatments, the prognosis for patients is poor. In this review, we highlight general aspects concerning genetic alterations in brain tumors, namely astrocytomas, glioblastomas, oligodendrogliomas, medulloblastomas and ependymomas. The influence of these genetic alterations in patients' prognosis is discussed. Mutagen sensivity is associated with cancer risk. The convincing studies that linked DNA damages and DNA repair alterations with brain tumors are also described. Another important modifying factor is immunity. General immune response against cancer, tumor microenvironment and immune response, mechanisms of tumor escape, CNS tumor immunology, immune defects that impair anti-tumor systemic immunity in brain tumor patients and local immunosuppressive factors within CNS are also reviewed. New hope to treatment perspectives, as dendritic-cell-based vaccines is summarized too. Concluding, it seems well established that there is association between brain tumor risk and mutagen sensivity, which is highly heritable. Primary brain tumors cause depression in systemic host immunity; local immunosuppressive factors and immunological characteristics of tumor cells may explain the poor prognosis and DNA damages responses can alert immune system. However, it is necessary to clarify if individuals with both constitutional defects in immune functions and genetic instability have higher risk of developing brain tumors. Cytogenetic prospective studies and gene copy number variations analysis also must be performed in peripheral lymphocytes from brain tumor patients. © 2011 Bentham Science Publishers Ltd.

Epigenetic alterations in human brain tumors in a Brazilian population

Aberrant methylation of CpG islands located in promoter regions represents one of the major mechanisms for silencing cancer-related genes in tumor cells. We determined the frequency of aberrant CpG island methylation for several tumor-associated genes: DAPK, MGMT, p14^ARF, p16^INK4a, TP73, RB1 and TIMP-3 in 55 brain tumors, consisting of 26 neuroepithelial tumors, 6 peripheral nerve tumors, 13 meningeal tumors and 10 metastatic brain tumors. Aberrant methylation of at least one of the seven genes studied was detected in 83.6% of the cases. The frequencies of aberrant methylation were: 40% for p14^ARF, 38.2% for MGMT, 30.9% for, p16^INK4a, 14.6% for TP73 and for TIMP-3, 12.7% for DAPK and 1.8% for RB1. These data suggest that the hypermethylation observed in the genes p14^ARF, MGMT and p16^INK4a is a very important event in the formation or progression of brain tumors, since the inactivation of these genes directly interferes with the cell cycle or DNA repair. The altered methylation rate of the other genes has already been reported to be related to tumorigenesis, but the low methylation rate of RB1 found in tumors in our sample is different from that so far reported in the literature, suggesting that perhaps hypermethylation of the promoter is not the main event in the inactivation of this gene. Our results suggest that hypermethylation of the promoter region is a very common event in nervous system tumors.

Usefulness of computed tomography and magnetic resonance imaging in radiotherapy planning for the treatment of brain tumors

Advanced diagnostic techniques such as magnetic resonance imaging and computed tomography have become useful tools for confirmation of presumptive diagnosis of structural lesions in the brain such as encephalic neoplasms in small animal veterinary practice in Colombia, allowing an effective treatment planning that is more specific and less invasive for this type of pathology.

Mobile phone use and brain tumors in children and adolescents: a multicenter case-control study

It has been hypothesized that children and adolescents might be more vulnerable to possible health effects from mobile phone exposure than adults. We investigated whether mobile phone use is associated with brain tumor risk among children and adolescents.

Impact of random and systematic recall errors and selection bias in case--control studies on mobile phone use and brain tumors in adolescents (CEFALO study)

Whether the use of mobile phones is a risk factor for brain tumors in adolescents is currently being studied. Case--control studies investigating this possible relationship are prone to recall error and selection bias. We assessed the potential impact of random and systematic recall error and selection bias on odds ratios (ORs) by performing simulations based on real data from an ongoing case--control study of mobile phones and brain tumor risk in children and adolescents (CEFALO study). Simulations were conducted for two mobile phone exposure categories: regular and heavy use. Our choice of levels of recall error was guided by a validation study that compared objective network operator data with the self-reported amount of mobile phone use in CEFALO. In our validation study, cases overestimated their number of calls by 9% on average and controls by 34%. Cases also overestimated their duration of calls by 52% on average and controls by 163%. The participation rates in CEFALO were 83% for cases and 71% for controls. In a variety of scenarios, the combined impact of recall error and selection bias on the estimated ORs was complex. These simulations are useful for the interpretation of previous case-control studies on brain tumor and mobile phone use in adults as well as for the interpretation of future studies on adolescents.

The phosphoinositide 3-kinase signaling pathway as a therapeutic target in grade IV brain tumors

Brain tumors comprise a wide variety of neoplasia classified according to their cellular origin and their morphological and histological characteristics. The transformed phenotype of brain tumor cells has been extensively studied in the past years, achieving a significant progress in our understanding of the molecular pathways leading to tumorigenesis. It has been reported that the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is frequently altered in grade IV brain tumors resulting in uncontrolled cell growth, survival, proliferation, angiogenesis, and migration. This aberrant activation can be explained by oncogenic mutations in key components of the pathway or through abnormalities in its regulation. These alterations include overexpression and mutations of receptor tyrosine kinases (RTKs), mutations and deletions of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene, encoding a lipid kinase that directly antagonized PI3K activity, and alterations in Ras signaling. Due to promising results of preclinical studies investigating the PI3K/AKT pathway in grade IV brain tumors like glioblastoma and medulloblastoma, the components of this pathway have emerged as promising therapeutic targets to treat these malignant brain tumors. Although an arsenal of small molecule inhibitors that target specific components of this signaling pathway is being developed, its successful application in the clinics remains a challenge. In this article we will review the molecular basis of the PI3K/AKT signaling pathway in malignant brain tumors, mainly focusing on glioblastoma and medulloblastoma, and we will further discuss the current status and potential of molecular targeted therapies.

Chalcone JAI-51 improves efficacy of synchrotron microbeam radiation therapy of brain tumors

Microbeam radiation therapy (MRT), a preclinical form of radiosurgery, uses spatially fractionated micrometre-wide synchrotron-generated X-ray beams. As MRT alone is predominantly palliative for animal tumors, the effects of the combination of MRT and a newly synthesized chemotherapeutic agent JAI-51 on 9L gliosarcomas have been evaluated. Fourteen days (D14) after implantation (D0), intracerebral 9LGS-bearing rats received either MRT, JAI-51 or both treatments. JAI-51, alone or immediately after MRT, was administered three times per week. Animals were kept up to ∼20 weeks after irradiation or sacrificed at D16 or D28 after treatment for cell cycle analysis. MRT plus JAI-51 increased significantly the lifespan compared with MRT alone (p = 0.0367). JAI-51 treatment alone had no effect on rat survival. MRT alone or associated with JAI-51 induced a cell cycle blockade in G2/M (p < 0.01) while the combined treatment also reduced the proportion of G0/G1 cells. At D28 after irradiation, MRT and MRT/JAI-51 had a smaller cell blockade effect in the G2/M phase owing to a significant increase in tumor cell death rate (<2c) and a proportional increase of endoreplicative cells (>8c). The combination of MRT and JAI-51 increases the survival of 9LGS-bearing rats by inducing endoreduplication of DNA and tumor cell death; further, it slowed the onset of tumor growth resumption two weeks after treatment.

Brain tumors in children and adolescents and exposure to animals and farm life: a multicenter case-control study (CEFALO)

The etiology of brain tumors in children and adolescents is largely unknown, and very few environmental risk factors have been identified. The aim of this study was to examine the relationship between pre- or postnatal animal contacts or farm exposures and the risk of childhood brain tumors (CBTs), since infectious agents may pose a risk factor and a proposed mechanism is transferral of infectious agents from animals to humans.

Impact of 3,4-dihydroxy-6-18F-fluoro-L-phenylalanine PET/CT on managing patients with brain tumors: the referring physician's perspective

We investigated the impact of (18)F-DOPA brain PET/CT on the clinical management of patients with known or suspected brain tumors.

The warning-sign hierarchy between quantitative subcortical motor mapping and continuous motor evoked potential monitoring during resection of supratentorial brain tumors

Mapping and monitoring are believed to provide an early warning sign to determine when to stop tumor removal to avoid mechanical damage to the corticospinal tract (CST). The objective of this study was to systematically compare subcortical monopolar stimulation thresholds (1-20 mA) with direct cortical stimulation (DCS)-motor evoked potential (MEP) monitoring signal abnormalities and to correlate both with new postoperative motor deficits. The authors sought to define a mapping threshold and DCS-MEP monitoring signal changes indicating a minimal safe distance from the CST.

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1)

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

Determination of histopathological tumor grade in neuroepithelial brain tumors by using spectral pattern analysis of in vivo spectroscopic data

OBJECT: In this study, 1H magnetic resonance (MR) spectroscopy was prospectively tested as a reliable method for presurgical grading of neuroepithelial brain tumors. METHODS: Using a database of tumor spectra obtained in patients with histologically confirmed diagnoses, 94 consecutive untreated patients were studied using single-voxel 1H spectroscopy (point-resolved spectroscopy; TE 135 msec, TE 135 msec, TR 1500 msec). A total of 90 tumor spectra obtained in patients with diagnostic 1H MR spectroscopy examinations were analyzed using commercially available software (MRUI/VARPRO) and classified using linear discriminant analysis as World Health Organization (WHO) Grade I/II, WHO Grade III, or WHO Grade IV lesions. In all cases, the classification results were matched with histopathological diagnoses that were made according to the WHO classification criteria after serial stereotactic biopsy procedures or open surgery. Histopathological studies revealed 30 Grade I/II tumors, 29 Grade III tumors, and 31 Grade IV tumors. The reliability of the histological diagnoses was validated considering a minimum postsurgical follow-up period of 12 months (range 12-37 months). Classifications based on spectroscopic data yielded 31 tumors in Grade I/II, 32 in Grade III, and 27 in Grade IV. Incorrect classifications included two Grade II tumors, one of which was identified as Grade III and one as Grade IV; two Grade III tumors identified as Grade II; two Grade III lesions identified as Grade IV; and six Grade IV tumors identified as Grade III. Furthermore, one glioblastoma (WHO Grade IV) was classified as WHO Grade I/II. This represents an overall success rate of 86%, and a 95% success rate in differentiating low-grade from high-grade tumors. CONCLUSIONS: The authors conclude that in vivo 1H MR spectroscopy is a reliable technique for grading neuroepithelial brain tumors.