RESUMEN
Glioblastoma is the most common form of primary adult brain tumors. A majority of glioblastomas grow invasively into distant brain tissue, leading to tumor recurrence, which is ultimately incurable. It is, therefore, essential to discover master regulators that control glioblastoma invasiveness and target them therapeutically. We show here that the transcriptional regulator Id-1 plays a critical role in modulating the invasiveness of glioblastoma cell lines and primary glioblastoma cells. Id-1 expression levels positively correlate with glioma cell invasiveness in culture and with histopathologic grades in patient biopsies. Id-1 knockdown dramatically reduces glioblastoma cell invasion that is accompanied by profound morphologic changes and robust reduction in expression levels of "mesenchymal" markers, as well as inhibition of self-renewal potential and downregulation of glioma stem cell markers. Importantly, genetic knockdown of Id-1 leads to a significant increase in survival in an orthotopic model of human glioblastoma. Furthermore, we show that a nontoxic compound, cannabidiol, significantly downregulates Id-1 gene expression and associated glioma cell invasiveness and self-renewal. In addition, cannabidiol significantly inhibits the invasion of glioblastoma cells through an organotypic brain slice and glioma progression in vivo. Our results suggest that Id-1 regulates multiple tumor-promoting pathways in glioblastoma and that drugs targeting Id-1 represent a novel and promising strategy for improving the therapy and outcome of patients with glioblastoma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína 1 Inhibidora de la Diferenciación/fisiología , Invasividad Neoplásica/genética , Animales , Neoplasias Encefálicas/patología , Cannabidiol/farmacología , Línea Celular Tumoral , Femenino , Glioblastoma/patología , Humanos , Proteína 1 Inhibidora de la Diferenciación/antagonistas & inhibidores , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Ratones , Ratones Desnudos , Neurospora , Interferencia de ARN , Trasplante Heterólogo , Regulación hacia ArribaRESUMEN
Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cannabidiol/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Proteína 1 Inhibidora de la Diferenciación/agonistas , Proteína 1 Inhibidora de la Diferenciación/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación/efectos de los fármacos , Trasplante Isogénico , alfa-Tocoferol/farmacologíaRESUMEN
The cannabinoid 1 (CB(1)) and cannabinoid 2 (CB(2)) receptor agonist Delta(9)-tetrahydrocannabinol (THC) has been shown to be a broad-range inhibitor of cancer in culture and in vivo, and is currently being used in a clinical trial for the treatment of glioblastoma. It has been suggested that other plant-derived cannabinoids, which do not interact efficiently with CB(1) and CB(2) receptors, can modulate the actions of Delta(9)-THC. There are conflicting reports, however, as to what extent other cannabinoids can modulate Delta(9)-THC activity, and most importantly, it is not clear whether other cannabinoid compounds can either potentiate or inhibit the actions of Delta(9)-THC. We therefore tested cannabidiol, the second most abundant plant-derived cannabinoid, in combination with Delta(9)-THC. In the U251 and SF126 glioblastoma cell lines, Delta(9)-THC and cannabidiol acted synergistically to inhibit cell proliferation. The treatment of glioblastoma cells with both compounds led to significant modulations of the cell cycle and induction of reactive oxygen species and apoptosis as well as specific modulations of extracellular signal-regulated kinase and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. Our results suggest that the addition of cannabidiol to Delta(9)-THC may improve the overall effectiveness of Delta(9)-THC in the treatment of glioblastoma in cancer patients.
Asunto(s)
Cannabidiol/farmacología , Dronabinol/farmacología , Glioblastoma/patología , Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Glioblastoma/enzimología , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
The inhibitor of basic helix-loop-helix transcription factors, Id-1, is an important gene whose expression increases during prostate cancer progression and that upregulates proliferation, migration and invasion. We used microarray analysis to identify the downstream genes whose transcriptional expression is modulated by Id-1 protein. We compared gene expression in control LNCaP cells and Id-1-transduced LNCaP cells, which become significantly more aggressive after Id-1 overexpression, thus mimicking the high levels of Id-1 detected in metastatic cell lines. We used the Affy HTA U133A Expression Arrays with 45,000 probe sets representing more than 39,000 transcripts. We found that one of the most significantly downregulated genes on Id-1 expression was kallikrein 3 [also called prostate specific antigen (PSA)], the most commonly used biomarker of prostate cancer. Here, we show that the reduction in PSA mRNA and protein expression associated with high-grade prostate cancers, which generally express high levels of Id-1, could be the consequence of Id-1 overexpression.