RESUMO
PURPOSE: Two clinical trials were conducted to investigate the oral and perioral irritation and sensitization potential of a tooth whitening leave-on-gel alone and in combination with a whitening toothpaste, each containing 1.0% of the active ingredient potassium monopersulfate (MPS). METHODS: Both clinical trials were Institutional Review Board (IRB) approved, double-blind, randomized, and parallel group designed studies. For the MPS leave-on gel study, 200 qualifying and consented subjects were randomly assigned to two groups: (1) 0.1% hydrogen peroxide (H2O2) gel pen (34 subjects); and (2) 0.1% H2O2 + 1.0% MPS gel pen (166 subjects). Subjects used the assigned products according to instructions provided and returned on Days 22 and 36 for oral and perioral tissue examination (pre-challenge). At the Day 36 visit, the subject applied the assigned gel on site (challenge) and received oral and perioral tissue examinations 1 and 24 hours following the application to detect any post-challenge tissue reactions. For the MPS toothpaste/MPS gel pen study, 200 qualifying and consented subjects were randomly assigned to three groups: (1) Placebo toothpaste + placebo gel pen (66 subjects); (2) 1.0% MPS toothpaste + 1.0% MPS gel pen (67 subjects); and (3) 1.0% MPS toothpaste + placebo gel pen (67 subjects). The study design and procedures were the same as those for the MPS gel pen study described above. RESULTS: For the MPS gel pen study, 192 subjects completed the study. None of the eight dropouts was related to the product use. The demographic data were comparable between the two groups. No evidence of tissue irritation and sensitization was detected in any subjects at any visit, and the findings were comparable between the groups. The detected and self-reported tissue issues were minimal and minor, and they were comparable between the two groups. For the MPS toothpaste/MPS gel pen study, 200 subjects were enrolled with 12 dropped from the study, resulting in an overall dropout rate of 6%. Of the 12 that did not complete the study, none were due to product-related use. The demographic data were comparable among the three groups. The detected and self-reported tissue issues were minimal and minor, and they were comparable among the three groups. CLINICAL SIGNIFICANCE: Potassium monopersulfate (MPS) at the active concentration of 1.0% in the tooth whitening leave-on-gel and the toothpaste plus the gel did not cause oral/perioral irritation nor sensitization.
Assuntos
Clareamento Dental , Descoloração de Dente , Humanos , Cremes Dentais/uso terapêutico , Peróxido de Hidrogênio/efeitos adversos , Resultado do Tratamento , Clareamento Dental/efeitos adversos , Clareamento Dental/métodos , Método Duplo-Cego , Descoloração de Dente/tratamento farmacológicoRESUMO
Our research group demonstrated that riluzole, an inhibitor of glutamatergic signaling reduced melanoma cell proliferation in vitro and tumor progression in vivo. The underlying mechanisms of riluzole are largely unknown. Microarray analyses on two human melanoma cell lines revealed that riluzole stimulates expression of the cystine-glutamate amino acid antiporter, xCT (SLC7A11). Western immunoblot analysis from cultured human melanoma or normal melanocytic cells showed that xCT was significantly overexpressed in most melanomas, but not normal cells. Studies using human tumor biopsy samples demonstrated that overexpression of xCT was correlated with cancer stage and progression. To further investigate if xCT is involved in melanoma cell growth, we derived several stable clones through transfection of exogenous xCT to melanoma cells that originally showed very low expression of xCT. The elevated xCT expression promoted cell proliferation in vitro and inversely, these melanoma clones showed a dose-dependent decrease in cell proliferation in response to riluzole treatment. Xenograft studies showed that these clones formed very aggressive tumors at a higher rate compared to vector controls. Conversely, treatment of xenograft-bearing animals with riluzole down-regulated xCT expression suggesting that xCT is a molecular target of riluzole. Furthermore, protein lysates from tumor biopsies of patients that participated in a riluzole monotherapy phase II clinical trial showed a reduction in xCT levels in post-treatment specimens from patients with stable disease. Taken together, our results show that xCT may be utilized as a marker to monitor patients undergoing riluzole-based chemotherapies.
RESUMO
Our laboratory previously showed that ectopic expression of Grm1 is sufficient to induce spontaneous melanoma formation with 100% penetrance in transgenic mouse model, TG-3, which harbors wild-type BRaf. Studies identified Grm1 expression in human melanoma cell lines and primary to secondary metastatic melanoma biopsies having wild-type or mutated BRaf, but not in normal melanocytes or benign nevi. Grm1 expression was detected in tissues from mice genetically engineered with inducible melanocyte-specific BRafV600E. Additionally, stable clones derived from introduction of exogenous BRafV600E in mouse melanocytes also showed Grm1 expression, which was not detected in the parental or empty vector-derived cells, suggesting that expression of BRafV600E could activate Grm1 expression. Despite aberrant Grm1 expression in the inducible, melanocyte-specific BRafV600E mice, no tumors formed. However, in older mice, the melanocytes underwent senescence, as demonstrated previously by others. It was proposed that upregulated p15 and TGFß contributed to the senescence phenotype. In contrast, in older TG-3 mice the levels of p15 and TGFß remained the same or lower. Taken together, these results suggest the temporal regulation on the expression of "oncogenes" such as Grm1 or BRafV600E is critical in the future fate of the cells. If BRafV600E is turned on first, Grm1 expression can be induced, but this is not sufficient to result in development of melanoma; the cells undergo senescence. In contrast, if ectopic expression of Grm1 is turned on first, then regardless of wild-type or mutated BRaf in the melanocytes melanoma development is the consequence.
RESUMO
Metabotropic glutamate receptors (mGluRs) are widely known for their roles in synaptic signaling. However, accumulating evidence suggests roles of mGluRs in human malignancies in addition to synaptic transmission. Somatic cell homeostasis presents intriguing possibilities of mGluRs and glutamate signaling as novel targets for human cancers. More recently, aberrant glutamate signaling has been shown to participate in the transformation and maintenance of various cancer types, including glioma, melanoma skin cancer, breast cancer, and prostate cancer, indicating that genes encoding mGluRs, GRMs, can function as oncogenes. Here, we provide a review on the interactions of mGluRs and their ligand, glutamate, in processes that promote the growth of tumors of neuronal and non-neuronal origins. Further, we discuss the evolution of riluzole, a glutamate release inhibitor approved for amyotrophic lateral sclerosis (ALS), but now fashioned as an mGluR1 inhibitor for melanoma therapy and as a radio-sensitizer for tumors that have metastasized to the brain. With the success of riluzole, it is not far-fetched to believe that other drugs that may act directly or indirectly on other mGluRs can be beneficial for multiple applications. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Antineoplásicos/farmacologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Humanos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidoresRESUMO
Brain metastasis is a common endpoint in human malignant melanoma, and the prognosis for patients remains poor despite advancements in therapy. Current treatment for melanoma metastatic to the brain is grouped into those providing symptomatic relief such as corticosteroids and antiepileptic agents, to those that are disease modifying. Related to the latter group, recent studies have demonstrated that aberrant glutamate signaling plays a role in the transformation and maintenance of various cancer types, including melanoma. Glutamate secretion from these and surrounding cells have been found to stimulate regulatory pathways that control tumor growth, proliferation and survival in vitro and in vivo. The antiglutamatergic actions of an inhibitor of glutamate release, riluzole, have been detected by its ability to clear glutamate from the synapse, and it has been shown to inhibit glutamate release rather than directly inhibiting glutamate receptors. Preclinical studies have demonstrated the ability of riluzole to act as a radiosensitizing agent in melanoma. The effect of riluzole on downstream glutamatergic signaling has pointed to cross talk between the metabotropic G-protein-coupled glutamate receptors implicated in a subset of human melanomas with other signaling pathways, including apoptotic, angiogenic, ROS and cell invasion mechanisms, thus establishing its potential to be further explored in combination therapy regimens for both primary human melanoma and melanoma metastatic to the brain.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Melanoma/patologia , Fármacos Neuroprotetores/uso terapêutico , Riluzol/uso terapêutico , HumanosRESUMO
Approximately 50% of patients having metastatic melanoma develop brain metastases during the course of their illness. Evidence exists that melanoma cells have increased aptitude for the repair of sublethal DNA damage caused by ionizing radiation therapy. To address the radio-resistance of melanoma, many groups adopted radiotherapy schedules that deliver larger daily fractions of radiation, but due to the risk of neurotoxicity, these large fractions cannot be delivered to the whole brain for patients with brain metastases. Here, we used orthotopic implanted GRM1 expressing human melanoma cell xenografts in mice, to demonstrate that animals receiving concurrent glutamate signaling blockade (riluzole) and radiation led to a decrease in intracranial tumor growth compared to either modality alone. These preclinical results suggest riluzole may cause radio-sensitization that offers enhanced efficacy for a subset of human melanoma patients undergoing radiotherapy for brain metastasis.
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Neoplasias Encefálicas/secundário , Melanoma/tratamento farmacológico , Melanoma/patologia , Radiossensibilizantes/uso terapêutico , Receptores de Glutamato Metabotrópico/metabolismo , Riluzol/uso terapêutico , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Células Clonais , Modelos Animais de Doenças , Humanos , Luciferases/metabolismo , Luminescência , Camundongos , Radiossensibilizantes/farmacologia , Riluzol/farmacologia , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gain of function of the neuronal receptor, metabotropic glutamate receptor 1 (Grm1), was sufficient to induce melanocytic transformation in vitro and spontaneous melanoma development in vivo when ectopically expressed in melanocytes. The human form of this receptor, GRM1, has been shown to be ectopically expressed in a subset of human melanomas but not benign nevi or normal melanocytes, suggesting that misregulation of GRM1 is involved in the pathogenesis of certain human melanomas. Sustained stimulation of Grm1 by the ligand, glutamate, is required for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. In this study, we investigate the mechanism of an inhibitor of glutamate release, riluzole, on human melanoma cells that express metabotropic glutamate receptor 1 (GRM1). Various in vitro assays conducted show that inhibition of glutamate release in several human melanoma cell lines resulted in an increase of oxidative stress and DNA damage response markers.
Assuntos
Dano ao DNA , Melanoma/metabolismo , Melanoma/patologia , Receptores de Glutamato Metabotrópico/metabolismo , Riluzol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Biópsia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Histonas/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Melanoma/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Riluzol/uso terapêuticoRESUMO
U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.Molecular Therapy - Nucleic Acids (2013) 2, e92; doi:10.1038/mtna.2013.24; published online 14 May 2013.
RESUMO
Riluzole (1) is an approved therapeutic for the treatment of ALS and has also demonstrated anti-melanoma activity in metabotropic glutamate GRM1 positive cell lines, a mouse xenograft assay and human clinical trials. Highly variable drug exposure following oral administration among patients, likely due to variable first pass effects from heterogeneous CYP1A2 expression, hinders its clinical use. In an effort to mitigate effects of this clearance pathway and uniformly administer riluzole at efficacious exposure levels, several classes of prodrugs of riluzole were designed, synthesized, and evaluated in multiple in vitro stability assays to predict in vivo drug levels. The optimal prodrug would possess the following profile: stability while transiting the digestive system, stability towards first pass metabolism, and metabolic lability in the plasma releasing riluzole. (S)-O-Benzyl serine derivative 9 was identified as the most promising therapeutically acceptable prodrug.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Desenho de Fármacos , Melanoma/tratamento farmacológico , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Riluzol/metabolismo , Riluzol/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/metabolismo , Estabilidade de Medicamentos , Humanos , Melanoma/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Riluzol/sangue , Riluzol/síntese químicaRESUMO
We demonstrated that ectopic expression of metabotropic glutamate receptor 1 (mGluR1/Grm1) in mouse melanocytes was sufficient to induce melanoma development in vivo with 100% penetrance. We also showed that about 60% of human melanoma biopsies and cell lines, but not benign nevi or normal human melanocytes expressed mGluR1, suggesting that GRM1 may be involved in melanomagenesis. mGluR1 is expressed primarily in neurons. In various non-neuronal cells, mGluR1 expression is regulated via binding of Neuron-Restrictive-Silencer-Factor (NRSF) to a Neuron-Restrictive-Silencer-Element (NRSE). Here, we report on the possibility that aberrant mGluR1 expression in melanoma is due to alterations in NRSF and/or NRSE. We show that in human melanocytes, binding of NRSF to NRSE in the GRM1 promoter region is necessary for the suppression of mGluR1 expression. We also show that inhibiting the expression of the transcription factor Sp1 or interference with its ability to bind DNA can result in increased mGluR1 expression perhaps via its function as a negative regulator. In addition, we also provide evidence that demethylation within the promoter region of GRM1 may also be a mechanism for the derepression of mGluR1 expression in melanocytes that progress to cell transformation and tumor formation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanócitos/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Glutamato Metabotrópico/biossíntese , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Metilação de DNA/genética , Humanos , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta , Elementos Silenciadores Transcricionais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
Ectopic expression of a neuronal receptor, metabotropic glutamate receptor 1 (Grm1), in melanocytes has been implicated in melanoma development in mouse models. The human relevance of this receptor's involvement in melanoma pathogenesis was shown by detecting GRM1 expression in subsets of human melanomas, an observation lacking in benign nevi or normal melanocytes. Grm1-transformed mouse melanocytes and a conditional Grm1 transgenic mouse model confirmed a requirement for sustained expression of Grm1 for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. Here, we investigate if continued GRM1 expression is also required in human melanoma cell lines by using two inducible, silencing RNA systems: the ecdysone/Ponasterone A and tetracycline on/off approaches to regulate GRM1 expression in the presence of each inducer. Various in vitro assays were conducted to assess the consequences of a reduction in GRM1 expression on cell proliferation, apoptosis, downstream targeted signaling pathways, and in vivo tumorigenesis. We showed that suppression of GRM1 expression in several human melanoma cell lines resulted in a reduction in the number of viable cells and a decrease in stimulated mitogen-activated protein kinase (MAPK) and PI3K/AKT and suppressed tumor progression in vivo. These results reinforce earlier observations where a reduction in cell growth in vitro and tumorigenesis in vivo were correlated with decreased GRM1 activities by pharmacologic inhibitors of the receptor, supporting the notion that GRM1 plays a role in the maintenance of transformed phenotypes in human melanoma cells in vitro and in vivo and could be a potential therapeutic target for the treatment of melanoma.
Assuntos
Melanoma/genética , Receptores de Glutamato Metabotrópico/deficiência , Receptores de Glutamato Metabotrópico/genética , Neoplasias Cutâneas/genética , Animais , Apoptose/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Células HEK293 , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Distribuição Aleatória , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Melanoma is a heterogeneous disease where monotherapies are likely to fail due to variations in genomic signatures. B-RAF inhibitors have been clinically inadequate but response might be augmented with combination therapies targeting multiple signaling pathways. We investigate the preclinical efficacy of combining the multikinase inhibitor sorafenib or the mutated B-RAF inhibitor PLX4720 with riluzole, an inhibitor of glutamate release that antagonizes metabotropic glutamate receptor 1 (GRM1) signaling in melanoma cells. EXPERIMENTAL DESIGN: Melanoma cell lines that express GRM1 and either wild-type B-RAF or mutated B-RAF were treated with riluzole, sorafenib, PLX4720, or the combination of riluzole either with sorafenib or with PLX4720. Extracellular glutamate levels were determined by glutamate release assays. MTT assays and cell-cycle analysis show effects of the compounds on proliferation, viability, and cell-cycle profiles. Western immunoblotting and immunohistochemical staining showed apoptotic markers. Consequences on mitogen-activated protein kinase pathway were assessed by Western immunoblotting. Xenograft tumor models were used to determine the efficacy of the compounds in vivo. RESULTS: The combination of riluzole with sorafenib exhibited enhanced antitumor activities in GRM1-expressing melanoma cells harboring either wild-type or mutated B-RAF. The combination of riluzole with PLX4720 showed lessened efficacy compared with the combination of riluzole and sorafenib in suppressing the growth of GRM1-expressing cells harboring the B-RAF(V600E) mutation. CONCLUSIONS: The combination of riluzole with sorafenib seems potent in suppressing tumor proliferation in vitro and in vivo in GRM1-expressing melanoma cells regardless of B-RAF genotype and may be a viable therapeutic clinical combination.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácido Glutâmico/metabolismo , Melanoma/tratamento farmacológico , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Indóis/administração & dosagem , Indóis/uso terapêutico , Melanoma/metabolismo , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Riluzol/administração & dosagem , Riluzol/uso terapêutico , Sorafenibe , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We reported earlier on the oncogenic properties of Grm1 by demonstrating that stable Grm1-mouse-melanocytic clones proliferate in the absence of growth supplement and anchorage in vitro. In addition, these clones also exhibit aggressive tumorigenic phenotypes in vivo with short latency in tumor formation in both immunodeficient and syngeneic mice. We also detected strong activation of AKT in allograft tumors specifically AKT2 as the predominant isoform involved. In parallel, we assessed several human melanoma biopsy samples and found again that AKT2 was the predominantly activated AKT in these human melanoma biopsies. In cultured stable Grm1-mouse-melanocytic clones, as well as an metabotropic glutamate receptor 1 (Grm1) expressing human melanoma cell line, C8161, stimulation of Grm1 by its agonist led to the activation of AKT, while preincubation with Grm1-antagonist abolished Grm1-agonist-induced AKT activation. In addition, a reduction in tumor volume of Grm1-mouse-melanocytic-allografts was detected in the presence of small interfering AKT2 RNA (siAKT2). Taken together, these results showed that, in addition to the MAPK pathway previously reported being a downstream target of stimulated Grm1, AKT2 is another downstream target in Grm1 mediated melanocyte transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Melanoma/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/enzimologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Melanoma/genética , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/genética , Neoplasias Cutâneas/genéticaRESUMO
Previously, we reported a transgenic mouse line, TG-3, that develops spontaneous melanoma with 100% penetrance. We demonstrated that ectopic expression of Grm1 in melanocytes was sufficient to induce melanoma in vivo. In this present study, the transforming properties of Grm1 in two cultured immortalized melanocytes were investigated. We showed that, in contrast to parental melanocytes, these Grm1-clones have lost their requirement of TPA supplement for proliferation and have acquired the ability to form colonies in semi-solid medium. Xenografts of these cells formed robust tumors in both immunodeficient nude and syngeneic mice with a short latency (3-5 days). The malignancy of these cells was demonstrated by angiogenesis and invasion to the muscle and the intestine. The requirement of Grm1 expression for the maintenance of transformation was demonstrated by an inducible siRNA system. Induction of expression of siRNA for Grm1 reduced the number of proliferating/viable cells in vitro and suppressed in vivo xenografted tumor growth in comparison with control. Taken together, these results showed that expression of exogeneously introduced Grm1 is sufficient to induce full transformation of immortalized melanocytes.
Assuntos
Transformação Celular Neoplásica , Melanócitos/fisiologia , Melanoma/metabolismo , Oncogenes/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/genética , Camundongos , Camundongos Nus , Oncogenes/efeitos dos fármacos , Oncogenes/genética , RNA Interferente Pequeno/farmacologia , Receptores de Glutamato Metabotrópico/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Melanoma, the most deadly form of skin cancer, is very aggressive and resistant to present therapies. The transcription factor nuclear factor-kappa B (NF-kappaB) has been reported to be constitutively active in many types of cancer. Constitutively active NF-kappaB seen in melanoma likely plays a central role in cell survival and growth. We have established and characterized novel cell lines from our murine melanoma model. Here we report the constitutive activity of NF-kappaB in these melanoma-derived cells, as shown by electrophoretic mobility shift assay and reporter assays. We hypothesized that agents that inhibit NF-kappaB may also inhibit cell proliferation and may induce apoptosis in such melanoma cells. Curcumin has been shown to inhibit NF-kappaB activity in several cell types. In our system, curcumin selectively inhibited growth of melanoma cells, but not normal melanocytes. Curcumin induced melanoma cells to undergo apoptosis, as shown by caspase-3 activation, inversion of membrane phosphatidyl serine, and increases in cells in the sub-G1 phase. A curcumin dose-dependent inhibition of NF-kappaB-driven reporter activity correlated with decreased levels of phospho-IkappaBalpha, and decreased expression of NF-kappaB-target genes COX-2 and cyclin D1. This study demonstrates that the use of cells from our model system can facilitate studies of signaling pathways in melanoma. We furthermore conclude that curcumin, a natural and safe compound, inhibits NF-kappaB activity and the expression of its downstream target genes, and also selectively induces apoptosis of melanoma cells but not normal melanocytes. These encouraging in-vitro results support further investigation of curcumin for treatment of melanoma in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Melanoma Experimental/patologia , NF-kappa B/metabolismo , Animais , Anexina A5/metabolismo , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina D , Ciclinas/genética , Ciclinas/metabolismo , Ciclo-Oxigenase 2 , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Fase G1/efeitos dos fármacos , Luciferases/metabolismo , Melanoma Experimental/metabolismo , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Recently, several laboratories have started to investigate the involvement of glutamate signaling in cancer. In previous studies, we reported on a transgenic mouse model that develops melanoma spontaneously. Subsequent studies in these mice identified that the aberrant expression of metabotropic glutamate receptor 1 (GRM1) in melanocytes played a critical role in the onset of melanoma. Confirmation of the etiologic role of GRM1 in melanoma development was shown in a second transgenic line with GRM1 expression under the regulation of a melanocyte-specific dopachrome tautomerase promoter. Ectopic expression of GRM1 was also detected in a subset of human melanoma cell lines and biopsies, suggesting that aberrant expression of GRM1 in melanocytes may contribute to the development of human melanoma. GRM1, a seven-transmembrane domain G protein-coupled receptor, is normally expressed and functional in neuronal cells, and its ligand, glutamate, is the major excitatory neurotransmitter. Human melanoma cells are shown here to release elevated levels of glutamate, implying a possible autocrine loop. Treatment of GRM1-expressing human melanoma cells with a GRM1 antagonist (LY367385 or BAY36-7620) or a glutamate release inhibitor (riluzole) leads to a suppression of cell proliferation as well as a decrease in levels of extracellular glutamate. Treatment of human melanoma cell xenografts with riluzole for 18 days via p.o. gavage or i.v. injection leads to inhibition of tumor growth by 50% in comparison with controls. These data suggest the importance of glutamate signaling in human melanoma and imply that the suppression of glutamate signaling may be a new target for melanoma therapy.