RESUMO
BACKGROUND: Glucose regulated protein 78 (GRP78) functions as a sensor of endoplasmic reticulum (ER) stress. The aim of this study was to test the hypothesis that molecules that bind to GRP78 induce the unfolded protein response (UPR) and enhance cell death in combination with ER stress inducers. METHODS: Differential scanning calorimetry (DSC), measurement of cell death by flow cytometry and the induction of ER stress markers using western blotting. RESULTS: Epigallocatechin gallate (EGCG), a flavonoid component of Green Tea Camellia sinensis, and honokiol (HNK), a Magnolia grandiflora derivative, bind to unfolded conformations of the GRP78 ATPase domain. Epigallocatechin gallate and HNK induced death in six neuroectodermal tumour cell lines tested. Levels of death to HNK were twice that for EGCG; half-maximal effective doses were similar but EGCG sensitivity varied more widely between cell types. Honokiol induced ER stress and UPR as predicted from its ability to interact with GRP78, but EGCG was less effective. With respect to cell death, HNK had synergistic effects on melanoma and glioblastoma cells with the ER stress inducers fenretinide or bortezomib, but only additive (fenretinide) or inhibitory (bortezomib) effects on neuroblastoma cells. CONCLUSION: Honokiol induces apoptosis due to ER stress from an interaction with GRP78. The data are consistent with DSC results that suggest that HNK binds to GRP78 more effectively than EGCG. Therefore, HNK may warrant development as an antitumour drug.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/uso terapêutico , Catequina/análogos & derivados , Proteínas de Choque Térmico/metabolismo , Lignanas/uso terapêutico , Neoplasias/tratamento farmacológico , Antineoplásicos Fitogênicos/metabolismo , Compostos de Bifenilo/metabolismo , Catequina/metabolismo , Catequina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/antagonistas & inibidores , Humanos , Lignanas/metabolismo , Terapia de Alvo Molecular , Peso Molecular , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: MYCN is the most commonly amplified gene in human neuroblastomas. This proto-oncogene has been overexpressed in a mouse model of the disease in order to explore the role of MYCN in this tumour. AIMS: To report the histopathological features of neuroblastomas from MYCN transgenic mice. METHODS: 27 neuroblastomas from hemizygous transgenic mice and four tumours from homozygous mice were examined histologically; Ki67 and MYCN immunocytochemistry was performed in 24 tumours. RESULTS: Tumours obtained from MYCN transgenic mice resembled human neuroblastomas, displaying many of the features associated with stroma-poor neuroblastoma, including heterogeneity of differentiation (but no overt ganglionic differentiation was seen), low levels of Schwannian stroma and a high mitosis karyorrhexis index. The tumours had a median Ki67 labelling index of 70%; all tumours expressed MYCN with a median labelling index of 68%. The most striking difference between the murine and human neuroblastomas was the presence of tingible body macrophages in the transgenic mouse tumours reflecting high levels of apoptosis. This has not previously been described in human or other murine neuroblastoma models. CONCLUSIONS: These studies highlight the histological similarities between tumours from MYCN transgenic mice and human neuroblastomas, and reaffirm their role as a valuable model to study the biology of aggressive human neuroblastoma.
Assuntos
Neoplasias Abdominais/patologia , Neuroblastoma/patologia , Proteínas Nucleares , Proteínas Oncogênicas , Neoplasias Abdominais/genética , Animais , Biomarcadores/análise , Western Blotting , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Masculino , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Oncogênicas/análise , Proteínas Oncogênicas/genética , Proto-Oncogene Mas , Ubiquitina Tiolesterase/análiseRESUMO
The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2'-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5'-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.
Assuntos
Metilação de DNA/efeitos dos fármacos , Mercaptopurina/farmacologia , Metiltransferases/metabolismo , Tioguanina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , DNA/genética , DNA/isolamento & purificação , Primers do DNA , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Rim/enzimologia , Cinética , Metiltransferases/efeitos dos fármacos , Metiltransferases/genética , S-Adenosil-Homocisteína/metabolismoRESUMO
Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg(-1)) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA.
Assuntos
Benzotiazóis/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Imidazóis/farmacologia , Neuroblastoma/metabolismo , Tretinoína/metabolismo , Animais , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450 , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Neuroblastoma/patologia , RNA Interferente Pequeno/farmacologia , Ácido Retinoico 4 Hidroxilase , Transplante Heterólogo , Tretinoína/farmacocinéticaRESUMO
Endoplasmic reticulum (ER) malfunction, leading to ER stress, can be a consequence of genome instability and hypoxic tissue environments. Cancer cells survive by acquiring or enhancing survival mechanisms to counter the effects of ER stress and these homeostatic responses may be new therapeutic targets. Understanding the links between ER stress and apoptosis may be approached using drugs specifically to target ER stress responses in cancer cells. The retinoid analogue fenretinide [N-(4-hydroxyphenyl) retinamide] is a new cancer preventive and chemotherapeutic drug, that induces apoptosis of some cancer cell types via oxidative stress, accompanied by induction of an ER stress-related transcription factor, GADD153. The aim of this study was to test the hypothesis that fenretinide induces ER stress in neuroectodermal tumour cells, and to elucidate the role of ER stress responses in fenretinide-induced apoptosis. The ER stress genes ERdj5, ERp57, GRP78, calreticulin and calnexin were induced in neuroectodermal tumour cells by fenretinide. In contrast to the apoptosis-inducing chemotherapeutic drugs vincristine and temozolomide, fenretinide induced the phosphorylation of eIF2alpha, expression of ATF4 and splicing of XBP-1 mRNA, events that define ER stress. In these respects, fenretinide displayed properties similar to the ER stress inducer thapsigargin. ER stress responses were inhibited by antioxidant treatment. Knockdown of ERp57 or ERdj5 by RNA interference in these cells increased the apoptotic response to fenretinide. These data suggest that downregulating homeostatic ER stress responses may enhance apoptosis induced by oxidative stress-inducing drugs acting through the ER stress pathway. Therefore, ER-resident proteins such as ERdj5 and ERp57 may represent novel chemotherapeutic targets.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fenretinida/farmacologia , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Processamento Alternativo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP40 , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tumores Neuroectodérmicos/tratamento farmacológico , Tumores Neuroectodérmicos/metabolismo , Tumores Neuroectodérmicos/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína 1 de Ligação a X-BoxAssuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neuroblastoma/metabolismo , Receptores do Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fenretinida/farmacologia , Humanos , Neuroblastoma/patologia , Receptores do Ácido Retinoico/genética , Transfecção , Receptor gama de Ácido RetinoicoRESUMO
Fenretinide induces apoptosis in SH-SY5Y neuroblastoma cells via a signaling pathway involving the production of reactive oxygen species (ROS), 12-lipoxygenase activity and the induction of the GADD153 transcription factor. NF-kappa B is a key element of many cell signaling pathways and adopts a pro- or anti-apoptotic role in different cell types. Studies have suggested that NF-kappa B may play a pro-apoptotic role in SH-SY5Y cells, and in other cell types NF-kappa B activation may be linked to lipoxygenase activity. The aim of this study was to test the hypothesis that NF-kappa B activity mediates fenretinide-induced apoptosis in SH-SY5Y neuroblastoma cells. Using a dominant-negative construct for Ikappa Balpha stably transfected into SH-SY5Y cells, we show that apoptosis, but not the induction of ROS, in response to fenretinide was blocked by abrogation of NF-kappa B activity. In parental SH-SY5Y cells, fenretinide induced NF-kappa B activity and Ikappa Balpha phosphorylation. These results suggest that NF-kappa B activity links fenretinide-induced ROS to the induction of apoptosis in SH-SH5Y cells, and may be a target for the future development of drugs for neuroblastoma therapy.
Assuntos
Apoptose/efeitos dos fármacos , Fenretinida/farmacologia , NF-kappa B/fisiologia , Citometria de Fluxo , Humanos , Proteínas I-kappa B/biossíntese , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Neuroblastoma , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.
Assuntos
Antineoplásicos/metabolismo , Isotretinoína/metabolismo , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Tretinoína/metabolismo , Acitretina/farmacologia , Antineoplásicos/farmacologia , Benzotiazóis , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Imidazóis/farmacologia , Isotretinoína/farmacologia , Ácido Retinoico 4 Hidroxilase , Proteínas de Ligação ao Retinol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologia , Tretinoína/farmacologiaRESUMO
BACKGROUND: The anabolic effects of insulin are well recognized but the mechanism by which insulin decreases muscle protein degradation in human is unclear. However, in a variety of catabolic conditions it is believed to be changes in the activity of the ATP-dependent ubiquitin proteolytic pathway that are responsible for changes in protein degradation in skeletal muscle. The aim of this study was to test the hypothesis that insulin regulates the ATP-dependent ubiquitin proteolytic pathway in human muscle. MATERIAL AND METHODS: The effects of insulin and acidosis on protein degradation were measured in human myocytes using L-[14C]phenylalanine. The effect of insulin on the activity of the ATP-dependent ubiquitin pathway was assessed from the mRNA expression of ubiquitin and the ubiquitin-conjugating enzyme E214k in human myocytes. RESULTS AND CONCLUSIONS: Coincubation of human myocytes with 100 nM of insulin was associated with a significant reduction in protein degradation. Metabolic acidosis is known to increase skeletal muscle protein degradation rates, and in our experiments protein degradation at a pH of 7.0 was significantly higher than pH 7.35. Eight-hour exposure to 100 nM of insulin resulted in a significant reduction in the expression of E214k but no change in the expression of ubiquitin. CONCLUSIONS: In human muscle we have demonstrated regulation by insulin of the ATP-dependent ubiquitin pathway at the level of ubiquitin conjugation.
Assuntos
Insulina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
BACKGROUND: In chronic renal failure, metabolic acidosis is associated with increased whole body protein degradation. In rats this effect of acidosis occurs in skeletal muscle and is associated with increased ubiquitin mRNA expression. This has not been demonstrated in humans. MATERIALS AND METHODS: Six patients with chronic renal failure and acidosis underwent muscle biopsy before and after 1 month's treatment with sodium bicarbonate. RNA was extracted from the biopsy, and the expression of the genes for ubiquitin and the proteasome component, C2, were measured by Northern blotting. RESULTS AND CONCLUSIONS: There was no significant difference in the expression of ubiquitin or C2 after bicarbonate treatment. This is contrast with results from animal models of acidosis and some other catabolic conditions in humans. This may reflect the complexity of the ubiquitin-dependent pathway, and it may be that changes in ubiquitin expression are only seen with more severe and/or acute changes in pH.
Assuntos
Falência Renal Crônica/tratamento farmacológico , Músculo Esquelético/metabolismo , RNA/análise , Bicarbonato de Sódio/uso terapêutico , Ubiquitina/genética , Acidose/tratamento farmacológico , Adulto , Biópsia , Northern Blotting/métodos , Complemento C2/genética , Feminino , Expressão Gênica , Humanos , Falência Renal Crônica/metabolismo , Masculino , Pessoa de Meia-Idade , Bicarbonato de Sódio/metabolismo , Falha de TratamentoRESUMO
RXR beta is predominantly involved in retinoid responses in neuroblastoma cells, in particular the N-type SH SY 5Y cells and the S-type SH S EP cells, both derivatives of a mixed phenotype neuroblastoma cell line. The aim of this study was to identify RXR beta isoforms expressed in neuroblastoma cells and to characterise a putative novel RXR beta transcript. RXR beta 1 and RXR beta 2 were expressed in these neuroblastoma cells. An isoform with an insertion into the ligand binding domain, RXR beta(SLSR) (referred to in previous studies as RXR beta 3), was expressed at a similar level to RXR beta. A novel RXR beta transcript was identified by RNase protection assays and was at least as abundant as the expected RXR beta transcript and expressed in other cell types. Evidence suggests that this novel transcript was transcribed from an internal promoter between exons 5 and 6, contained a retained intron (intron 6) and was alternatively spliced with and without the SLSR insertion. These data show that the pattern of RXR beta expression is complex. The relative abundance of the novel RXR beta transcript suggests that it may be an important aspect of RXR beta function or regulation in a range of cell types.
Assuntos
Neuroblastoma/patologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Western Blotting , Primers do DNA , Humanos , Isoformas de Proteínas/genética , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
BACKGROUND AND PROCEDURE: The CD95/CD95 ligand (CD95L) system is a key regulator of apoptosis. To evaluate a possible role of the CD95/CD95L system in human neuroblastoma (NB) cells, we investigated the constitutive and interferongamma (INFgamma)-induced expression of CD95 and CD95L, and CD95-mediated cell death in the SK-N-BE(2) cell line. RESULTS: Modulation of CD95/CD95L expression and triggering of an autocrine apoptotic mechanism by IFNgamma suggest a potential role for INFgamma as a therapeutic agent for NB. CONCLUSIONS: The evidence that retinoids induce apoptosis via tissue transglutaminase (tTG) and that N-methyl-D-aspartate (NMDA) and gp120 act through the nitric oxide synthase (NOS) activation pathway, indicates the existence of different molecular mechanisms of action, whose pharmacological exploitation might be used in an additive fashion.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Neuroblastoma/patologia , Receptor fas/fisiologia , Cálcio/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinergismo Farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteína Ligante Fas , Proteínas de Ligação ao GTP/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , Humanos , Transporte de Íons , Glicoproteínas de Membrana/genética , N-Metilaspartato/farmacologia , Proteínas de Neoplasias/genética , Neuroblastoma/metabolismo , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/fisiologia , Proteína 2 Glutamina gama-Glutamiltransferase , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Transglutaminases/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Receptor fas/genéticaRESUMO
BACKGROUND: Neuroblastoma, a childhood tumour of the sympathetic nervous system, may undergo spontaneous differentiation or regression due to apoptosis after no or minimal therapy. However, the majority of neuroblastomas are diagnosed as metastatic tumours with a poor prognosis in spite of intensive multimodal therapy. Vitamin A and its analogues (retinoic acid, RA) play an important role in normal cel lular differentiation and programmed cell death. RA regulates neuroblastoma growth and differentiation in vitro, and has shown activity against human neuroblastoma in vivo. PROCEDURE: Recently, 9-cis RA was shown to induce apoptosis in vitro in neuroblastoma using a 5 days short-term treatment and subsequent washout. In the present study, nude rats with human neuroblastoma SH-SY5Y xenografts were treated with 13-cis RA (4 mg po daily), 9-cis RA (5 mg po daily) or the novel analogue Ro 13-6307 (0.3 mg po daily) using either a continuous or short-term schedule. RESULTS: ALL three different retinoids decreased neuroblastoma growth significantly in terms of tumour weight after 8-12 days when compared to untreated controls (P < 0.05). Minor signs of toxicity in 13-cis RA treated rats were observed. However, severe toxicity with significant weight loss was seen in all rats treated with 9-cis RA and Ro 13-6307. Toxicity was more pronounced with the continuous regimen. CONCLUSIONS: We conclude that different retinoids reduce neuroblastoma tumour growth in vivo. Drug scheduling and dosage may affect both therapeutic efficacy and toxic side effects. Further in vivo studies are warranted, including pharmacokinetic and molecular analyses, before clinical trials with promising retinoids like 9-cis RA and Ro 13-6307 can be started in children with neuroblastoma.
Assuntos
Antineoplásicos/uso terapêutico , Ácidos Graxos Insaturados/uso terapêutico , Isotretinoína/uso terapêutico , Neuroblastoma/tratamento farmacológico , Tretinoína/uso terapêutico , Alitretinoína , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Peso Corporal/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Diarreia/induzido quimicamente , Esquema de Medicação , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/farmacologia , Ácidos Graxos Insaturados/toxicidade , Feminino , Humanos , Isotretinoína/administração & dosagem , Isotretinoína/farmacologia , Isotretinoína/toxicidade , Masculino , Camundongos , Transplante de Neoplasias , Neuroblastoma/patologia , Ratos , Ratos Nus , Tretinoína/administração & dosagem , Tretinoína/farmacologia , Tretinoína/toxicidade , Células Tumorais Cultivadas/transplante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: 9-cis retinoic acid (RA) is more effective than all-trans RA at inducing neuroblastoma differentiation in vitro, and has distinct biological properties with respect to its ability to promote apoptosis in N-type neuroblastoma cells. The cellular effects of 9-cis RA may, in part, result from activation of retinoid X receptor (RXR) homodimers. If this hypothesis is correct, 9-cis RA may control the expression of a different subset of retinoid-regulated genes compared to all-trans RA. PROCEDURE: We have therefore used differential mRNA display to identify genes differentially expressed in neuroblastoma cells in response to all-trans and 9-cis RA. RESULTS: The majority of cDNAs differentially expressed in response to all-trans or 9-cis RA matched to nonredundant Genbank sequences or EST database sequences. Differential-display profiles were similar in SH SY 5Y and SH S EP cells, clonal derivatives of the mixed neuroblastoma cell line SK N SH, although there were apparent differences between these cell lines with respect to the retinoid-regulation of specific RT-PCR cDNA fragments. CONCLUSIONS: These data support the view that 9-cis and all-trans RA act via different receptor mechanisms.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Tretinoína/farmacologia , Alitretinoína , Apoptose/efeitos dos fármacos , DNA Complementar/genética , Etiquetas de Sequências Expressas , Humanos , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Considerable evidence suggests that diabetes mellitus and hypertension are influenced by genetic factors. Studies in humans have associated glucocorticoid receptor (GR) polymorphisms with high blood pressure, insulin sensitivity, body mass index, increased visceral fat, and variations in tissue-specific steroid sensitivity. The N363S polymorphism of the GR results in an asparagine to serine amino acid substitution in a modulatory region of the receptor. Phosphorylation of serine residues in this region has been shown to enhance transactivation of GR responsive genes. The aim of this study was to investigate the association between the 363S allele and risk factors for coronary heart disease and diabetes mellitus in a population of European origin living in the northeast of the United KINGDOM: Blood samples from 135 males and 240 females were characterized for 363 allele status. The overall frequency of the 363S allele was 3.0%, 23 heterozygotes (7 males and 16 females) but no 363S homozygotes were identified. The data show a significant association of the 363S allele with increased waist to hip ratio in males but not females. This allele was not associated with blood pressure, body mass index, serum cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein cholesterol levels, and glucose tolerance status. The results of this study suggest that this GR polymorphism may contribute to central obesity in men. Further studies are required to elucidate the properties of GR(363S) at a molecular level.
Assuntos
Doença das Coronárias/etiologia , Diabetes Mellitus/etiologia , Obesidade/genética , Polimorfismo Genético , Receptores de Glucocorticoides/genética , Adulto , Idoso , Alelos , Pressão Sanguínea , Índice de Massa Corporal , Feminino , Humanos , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Glucocorticoids are used in the treatment of acute lymphoblastic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL) but many patients develop glucocorticoid resistance on relapse. The ligand-activated glucocorticoid receptor inhibits activity of the AP-1 transcription factor and the purpose of this study was to test the hypothesis that up-regulation or overexpression of AP1-binding activity may be an important mechanism of glucocorticoid resistance in ALL and CLL. In vitro sensitivity of patient blasts to prednisolone was measured using th
Assuntos
Resistencia a Medicamentos Antineoplásicos , Glucocorticoides/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fator de Transcrição AP-1/fisiologia , Adulto , Sequência de Bases , Northern Blotting , Western Blotting , Pré-Escolar , Primers do DNA , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
The RARbeta/gamma-selective retinoids fenretinide and CD437 induce caspase-dependent apoptosis but generate free radicals independently of caspases. Apoptosis, but not free radical generation, induced by these retinoids is inhibited by RARbeta/gamma-specific antagonists. Both fenretinide and CD437 induce apoptosis synergistically with cisplatin, carboplatin, or etoposide. However, antioxidants inhibit this synergy to the level obtained with chemotherapeutic drugs alone, and this implies that free radical generation is important in the synergistic response. Since apoptosis induced by fenretinide or CD437 is mediated by apoptotic pathways involving RARs and/or mitochondria and differs from mechanisms of chemotherapy-induced apoptosis this may explain the strong synergistic response seen between these synthetic retinoids and chemotherapeutic drugs. These results suggest that fenretinide or CD437 may be useful adjuncts to neuroblastoma therapy.
Assuntos
Antineoplásicos/uso terapêutico , Fenretinida/uso terapêutico , Neuroblastoma/tratamento farmacológico , Retinoides/uso terapêutico , Apoptose/efeitos dos fármacos , Criança , Sinergismo Farmacológico , Radicais Livres/metabolismo , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais CultivadasRESUMO
Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Carboplatina/uso terapêutico , Sobrevivência Celular , Cisplatino/uso terapêutico , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Etoposídeo/uso terapêutico , Fenretinida/administração & dosagem , Citometria de Fluxo , Radicais Livres/análise , Humanos , Neuroblastoma/fisiopatologia , Retinoides/administração & dosagem , Fatores de Tempo , Tretinoína/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fenretinida/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/fisiologia , Inibidores de Caspase , Caspases/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , Radicais Livres/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
The aim of this study was to clarify retinoid receptor mechanisms mediating the effects of 9-cis retinoic acid (RA) and investigate the ability of RAR- and RXR-specific analogues to induce differentiation and inhibit proliferation in neuroblastoma cells. Differentiation and the inhibition of proliferation by 9-cis RA, but not all-trans RA, were inhibited by the RXR-homodimer antagonist LG745. The RXR-specific agonist LGD1069 was ineffective at inducing differentiation or inhibiting proliferation, but showed marked synergism with RAR-specific agonists with respect to inhibiting proliferation. These data suggest that the effects of 9-cis RA are mediated via both RXR-homodimers and heterodimers. However, combinations of RAR- and RXR-selective analogues were not as effective at promoting differentiation. This study indicates that different receptor mechanisms are involved in retinoid-induced differentiation and inhibition of proliferation in neuroblastoma cells.