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Background: Cannabidiol (CBD), a nonpsychoactive cannabinoid with a low toxicity profile, has been shown to produce antitumor activity across cancers in part through selective production of reactive oxygen species (ROS) in tumor cells. The alkylating agent, temozolomide (TMZ), is standard of care for treatment of glioblastoma (GBM). It can trigger increased ROS to induce DNA damage. It has also been reported that downregulating the expression of RAD51, an important DNA damage repair protein, leads to sensitization of GBM to TMZ. Methods: We determined the extent to which CBD enhanced the antitumor activity of TMZ in multiple orthotopic models of GBM. In addition, we investigated the potential for CBD to enhance the antitumor activity of TMZ through production of ROS and modulation of DNA repair pathways. Results: CBD enhanced the activity of TMZ in U87 MG and U251 GBM cell lines and in patient-derived primary GBM163 cells leading to stimulation of ROS, activation of the ROS sensor AMP-activated protein kinase (AMPK), and upregulation of the autophagy marker LC3A. CBD produced a sensitization of U87 and GBM163-derived intracranial (i.c.) tumors to TMZ and significantly increased survival of tumor-bearing mice. However, these effects were not observed in orthotopic models derived from GBM with intact methylguanine methyltransferase (MGMT) expression. We further demonstrate that CBD inhibited RAD51 expression in MGMT-methylated models of GBM, providing a potential mechanism for tumor sensitization to TMZ by CBD. Conclusion: These data support the potential therapeutic benefits of using CBD to enhance the antitumor activity of TMZ in GBM patients.
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Background: We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, downregulated the expression of the prometastatic gene inhibitor of DNA binding 1 (ID1) in cancer cells, leading to inhibition of tumor progression in vivo. While CBD is broadly used, including in the self-medication of cancer patients, and CBD-based therapies are undergoing clinical evaluation for cancer treatment, its mechanisms of action are still poorly understood. Methods: In this study, using microarray analysis and Western blot analysis for validation, we attempted to identify the full spectrum of genes regulated by CBD across various aggressive cancer cell lines, including the breast, brain, head and neck, and prostate. Results: We confirmed that ID1 was a major target downregulated by CBD and also discovered that CBD inhibited FOXM1 (Forkhead box M1), a transcriptional activator involved in cell proliferation, while simultaneously upregulating GDF15 (growth differentiation factor 15), a cytokine associated with tissue differentiation. Conclusion: Our results suggest that, by modulating expression of shared key cancer-driving genes, CBD could represent a promising nontoxic therapeutic for treating tumors of various origins.
Assuntos
Canabidiol , Regulação Neoplásica da Expressão Gênica , Neoplasias , Canabidiol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Humanos , Masculino , Neoplasias/tratamento farmacológico , OncogenesRESUMO
Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers. SIGNIFICANCE: These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3606/F1.large.jpg.
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Carcinogênese/metabolismo , Carcinoma de Células Escamosas/patologia , Senescência Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias Cutâneas/patologia , Animais , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/metabolismoRESUMO
Neurodegeneration is a major age-related pathology. Cognitive decline is characteristic of patients with Alzheimer's and related dementias and cancer patients after chemo- or radio-therapies. A recently emerged driver of these and other age-related pathologies is cellular senescence, a cell fate that entails a permanent cell cycle arrest and pro-inflammatory senescence-associated secretory phenotype (SASP). Although there is a link between inflammation and neurodegenerative diseases, there are many open questions regarding how cellular senescence affects neurodegenerative pathologies. Among the various cell types in the brain, astrocytes are the most abundant. Astrocytes have proliferative capacity and are essential for neuron survival. Here, we investigated the phenotype of primary human astrocytes made senescent by X-irradiation, and identified genes encoding glutamate and potassium transporters as specifically downregulated upon senescence. This down regulation led to neuronal cell death in co-culture assays. Unbiased RNA sequencing of transcripts expressed by non-senescent and senescent astrocytes confirmed that glutamate homeostasis pathway declines upon senescence. Our results suggest a key role for cellular senescence, particularly in astrocytes, in excitotoxicity, which may lead to neurodegeneration including Alzheimer's disease and related dementias.
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Doença de Alzheimer/genética , Astrócitos/metabolismo , Senescência Celular/genética , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sistema X-AG de Transporte de Aminoácidos/genética , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/patologia , Cultura Primária de Células , Raios XRESUMO
Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.
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Senescência Celular/fisiologia , Neuropatologia/métodos , Paraquat/efeitos adversos , Doença de Parkinson/etiologia , Animais , Humanos , Camundongos , Doença de Parkinson/patologia , Fatores de RiscoRESUMO
The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.
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Interleucina-1alfa/metabolismo , Neoplasias da Próstata/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/genética , Interleucina-6/metabolismo , Masculino , Camundongos SCID , Mitoxantrona/farmacologia , NF-kappa B/metabolismo , Fenótipo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína 1 Inibidora de Diferenciação/fisiologia , Invasividade Neoplásica/genética , Animais , Neoplasias Encefálicas/patologia , Canabidiol/farmacologia , Linhagem Celular Tumoral , Feminino , Glioblastoma/patologia , Humanos , Proteína 1 Inibidora de Diferenciação/antagonistas & inibidores , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Camundongos Nus , Neurospora , Interferência de RNA , Transplante Heterólogo , Regulação para CimaRESUMO
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.
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Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canabidiol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Proteína 1 Inibidora de Diferenciação/agonistas , Proteína 1 Inibidora de Diferenciação/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Transplante Isogênico , alfa-Tocoferol/farmacologiaRESUMO
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.