RESUMO
Cancer mediated activation of the ActRIIB-ALK4/5 heterodimer by myostatin is strongly associated with muscle wasting. We investigated in vitro and in vivo the efficacy of ALK4/5 receptor blockers SB431542 and GW788388 in preventing muscle wasting, and explored synergy with IGF-I analogue LONG R3 (LR3) IGF-I. In vitro, C2C12 skeletal muscle cells were treated with vehicle, SB431542, GW788388 and LR3 IGF-I. A C26-CD2F1 cachexia model was used to induce cachexia in vivo. Mice were allocated as non-tumour bearing (NTB) or C26 tumour-bearing (C26 TB) vehicle control, treated with SB431542, LR3 IGF-I, SB431542 and LR3 IGF-I, or GW788388 (intraperitoneally or orally). In vitro, differentiation index and mean nuclei count increased using SB431542, GW788388, LR3 IGF-I. In vivo, GW788388 was superior to SB431542 in limiting loss of bodyweight, grip-strength and gastrocnemius weight. and downregulated Atrogin-1 expression comparable to NTB mice. LR3 IGF-I treatment limited loss of muscle mass, but at the expense of accelerated tumour growth. In conclusion, treatment with GW788388 prevented cancer cachexia, and downregulated associated ubiquitin ligase Atrogin-1.
Assuntos
Benzamidas/administração & dosagem , Caquexia/prevenção & controle , Neoplasias do Colo/patologia , Dioxóis/administração & dosagem , Fator de Crescimento Insulin-Like I/análogos & derivados , Pirazóis/administração & dosagem , Receptores de Ativinas Tipo I/antagonistas & inibidores , Administração Oral , Animais , Benzamidas/farmacologia , Peso Corporal/efeitos dos fármacos , Caquexia/etiologia , Caquexia/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias do Colo/complicações , Neoplasias do Colo/metabolismo , Dioxóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Camundongos , Transplante de Neoplasias , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidoresRESUMO
INTRODUCTION: Treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. METHODS: To identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. RESULTS: We observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. CONCLUSIONS: In conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.
Assuntos
Androstenóis/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feniltioidantoína/análogos & derivados , Taxoides/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Androstenos , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Relação Dose-Resposta a Droga , Interações Medicamentosas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Microscopia Confocal , Metástase Neoplásica , Nitrilas , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Imagem com Lapso de TempoRESUMO
BACKGROUND: Gastrointestinal stromal tumours (GIST) are characterised by high expression of KIT and ETV1, which cooperate in GIST oncogenesis. Our aim was to identify microRNAs that are deregulated in GIST, have a role in GIST pathogenesis, and could potentially be used as therapeutic tool. METHODS: Differentially expressed microRNAs between primary GIST (n=50) and gastrointestinal leiomyosarcomas (GI-LMS, n=10) were determined using microarrays. Selected microRNA mimics were transfected into GIST-882 and GIST-T1 cell lines to study the effects of microRNA overexpression on GIST cells. Luciferase reporter assays were used to establish regulation of target genes by selected microRNAs. RESULTS: MiR-17-92 and miR-221/222 cluster members were significantly (P<0.01) lower expressed in GIST vs GI-LMS and normal gastrointestinal control tissues. MiR-17/20a/222 overexpression in GIST cell lines severely inhibited cell proliferation, affected cell cycle progression, induced apoptosis and strongly downregulated protein and--to a lesser extent--mRNA levels of their predicted target genes KIT and ETV1. Luciferase reporter assays confirmed direct regulation of KIT and ETV1 by miR-222 and miR-17/20a, respectively. CONCLUSION: MicroRNAs that may have an essential role in GIST pathogenesis were identified, in particular miR-17/20a/222 that target KIT and ETV1. Delivering these microRNAs therapeutically could hold great potential for GIST management, especially in imatinib-resistant disease.
Assuntos
Proteínas de Ligação a DNA/genética , Tumores do Estroma Gastrointestinal/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-kit/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Carcinogênese/genética , Processos de Crescimento Celular/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Família Multigênica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. A major impediment for the successful treatment is the development of drug resistance. The molecular processes that contribute to resistance have been extensively studied; however, there is not much known about regulation by microRNAs (miRNAs). We compared miRNA expression profiles of an isogenic cisplatin-sensitive and -resistant ovarian cancer cell line pair (A2780/A2780 DDP) and found 27 miRNAs to be differentially expressed (î¶2-fold). Five of these, including the family members miR-141/200c, showed a correlation with cisplatin sensitivity in the NCI-60 panel. Overexpression of miR-141 resulted in enhanced resistance to cisplatin in ovarian cancer cell lines. We next correlated the expression level of miR-141 in 132 primary ovarian tumors (108 serous and 24 non-serous) with response to platinum-based chemotherapy. Although no differences were observed in the serous tumors, miR-141 levels were higher in non-serous ovarian tumors that did not respond well to therapy (platinum-free interval <6 months). We demonstrate that miR-141 directly targets KEAP1, and that downregulation of KEAP1 induces cisplatin resistance. Conversely, overexpression of KEAP1 significantly enhanced cisplatin sensitivity. Expression of KEAP1 with its 3'-UTR, and a 3'-UTR in which the miR-141 target site has been mutated, revealed that miR-141 regulates KEAP1 upon exposure to cisplatin. Finally, we show that the NF-κB pathway, which can be regulated by KEAP1, is activated upon miR-141 overexpression, and that inhibition of this pathway partially reverses miR-141-mediated cisplatin resistance. These findings demonstrate that the miR-141-mediated regulation of KEAP1 has a crucial role in the cellular response to cisplatin.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Análise por Conglomerados , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Transdução de SinaisRESUMO
BACKGROUND: Solute carriers (SLCs), in particular organic cation transporters (OCTs), have been implicated in the cellular uptake of platinum-containing anticancer compounds. The activity of these carriers may determine the pharmacokinetics and the severity of side effects, including neuro- and nephrotoxicity of platinum-based chemotherapy. As decreased drug accumulation is a key mechanism of platinum resistance, SLCs may also contribute to the development of resistance. Here, we define the role of hSLC22A2 (OCT2) in the cellular uptake of platinum compounds. EXPERIMENTAL APPROACH: Human embryonic kidney (HEK) 293 cells stably expressing the hSLC22A2 gene (HEK293/hSLC22A2) were used in platinum accumulation studies. Following a 2 h exposure to various platinum compounds (100 microM), intracellular platinum levels were determined by flameless atomic absorption spectrometry. KEY RESULTS: HEK293/hSLC22A2 cells, compared with HEK293/Neo control cells, displayed significant increases in oxaliplatin (28.6-fold), Pt[DACH]Cl(2) (20.6-fold), ormaplatin (8.1-fold), tetraplatin (4.5-fold), transplatin (3.7-fold) and cisplatin (1.3-fold), but not carboplatin. SLC22A2-mediated transport could be inhibited by 1-methyl-4-phenylpyridinium. Furthermore, hSLC22A2-mediated oxaliplatin and cisplatin accumulation was time- and concentration-dependent, but non-saturable. Expression of hSLC22A2 in HEK293 cells resulted in enhanced sensitivity to oxaliplatin (12-fold) and cisplatin (1.8-fold). Although, hSLC22A2 mRNA expression was frequently found in ovarian cancer cell lines, its expression in clinical ovarian cancer specimens (n= 80) was low and did not correlate with the treatment outcome of platinum-based regimens. CONCLUSIONS AND IMPLICATIONS: The hSLC22A2 drug transporter is a critical determinant in the uptake and cytotoxicity of various platinum compounds, particularly oxaliplatin.
Assuntos
Antineoplásicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Compostos Organoplatínicos/metabolismo , Compostos de Platina/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Carboplatina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Cinética , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Oxaliplatina , Compostos de Platina/farmacologia , Compostos de Platina/uso terapêutico , RNA Mensageiro/metabolismo , Espectrofotometria Atômica , Transfecção , Resultado do TratamentoRESUMO
The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin-pathway (PI3K/AKT/mTOR-pathway) plays a role in the regulation of cell proliferation, cell survival, angiogenesis and resistance to anti-tumor treatments. In many tumor types the PI3K/AKT/mTOR-pathway is found activated through several different underlying mechanisms. Since this pathway is believed to largely drive the malignant behavior of several of these tumors, mTOR-inhibition is considered an attractive means to apply as anti-tumor treatment. Currently, four mTOR-inhibitors are explored for clinical use: rapamycin, temsirolimus (CCI-779), everolimus (RAD001) and deforolimus (AP23573). As monotherapy, mTOR-inhibitors yield interesting anti-tumor activity against various tumor types at the expense of relatively mild toxicities. This recently resulted in the registration of two mTOR-inhibitors for patients with metastatic renal cell carcinoma (RCC) while randomized studies in other tumors are currently in progress. Furthermore, mTOR-inhibitors are well-suited drugs to combine with other anti-tumor drugs as in preclinical models mTOR-inhibition overcomes chemoresistance. Consequently, mTOR-inhibitor-containing multidrug regimens are subject to clinical studies. As holds true for all anti-tumor therapies, identification of patients who are likely to respond to mTOR-inhibitor-containing therapies is of utmost importance to avoid over- or undertreatment. Preliminary results suggest that several factors reflecting activation of mTOR in tumors may be used for this purpose. This review addresses the mechanism of action and current clinical experience with mTOR-inhibitors as well as their role in overcoming resistance to conventional therapies. Additionally, potential predictors of outcome to mTOR-inhibition are discussed.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias/patologia , Proteínas Quinases/fisiologia , Divisão Celular , Sobrevivência Celular , Ensaios Clínicos como Assunto , Everolimo , Amplificação de Genes , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neovascularização Patológica/fisiopatologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/genética , Sirolimo/análogos & derivados , Sirolimo/uso terapêutico , Serina-Treonina Quinases TORRESUMO
RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD(1476) and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA.
Assuntos
Apoptose , Caspase 3/metabolismo , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Células HeLa , Humanos , Lamina Tipo A/deficiência , Lamina Tipo A/metabolismo , MicroRNAs/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteína Supressora de Tumor p14ARF/deficiência , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
The spliceosome is a ribonucleoprotein complex involved in RNA splicing, that is, the removal of non-coding introns from precursor messenger RNA. (Alternative) Splicing events may play an essential role in tumourigenesis. The recent discovery that the spliceosome is a target for novel compounds with anticancer activity opens up new therapeutic avenues.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Spliceossomos/efeitos dos fármacos , Desenho de Fármacos , Humanos , Íntrons/genética , Neoplasias/genética , Neoplasias/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Spliceossomos/metabolismoRESUMO
Although approximately half of the administered dose of irinotecan is recovered in urine, scarce data are available on the association of renal function with irinotecan pharmacokinetics and toxicity. Here, these relationships are investigated in 187 patients treated with irinotecan in a three-weekly schedule. No significant effects on irinotecan pharmacokinetics were found in these patients. However, in 131 patients treated with the registered dose, categorized renal function was related to hematological toxicity. The incidence of grade 3-4 neutropenia decreased as function of creatinine clearance, particularly in nonsmoking patients (P < 0.01). Patients with slower creatinine clearance (35-66 ml/min) had a four-times higher risk of grade 3-4 neutropenia (58% vs. 14%; P < 0.001). This study suggests that pretreatment renal function values are associated with irinotecan-induced neutropenia. A confirmatory analysis is warranted to determine whether measures of renal function should be incorporated in future attempts toward individualized treatment with irinotecan.
Assuntos
Camptotecina/análogos & derivados , Rim/metabolismo , Neutropenia/induzido quimicamente , Neutropenia/metabolismo , Adulto , Idoso , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/farmacocinética , Área Sob a Curva , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/farmacocinética , Creatinina/metabolismo , Esquema de Medicação , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Feminino , Taxa de Filtração Glomerular , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Humanos , Irinotecano , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos , Projetos de Pesquisa , Estudos RetrospectivosRESUMO
BACKGROUND: Surgical resection remains the most effective therapy for metastatic colorectal cancer confined to the liver, although the extrahepatic recurrence rate is high. AIM OF THE STUDY: To develop a mammal model in order to investigate by which mechanisms liver surgery affects distant tumour recurrence. METHODS: In this animal study the effect of partial hepatectomy (phX) on the development of tumour noduli in the lungs was evaluated. CC531 rat colon carcinoma cells were inoculated i.v. 24h before, during or 24h after surgery. Rat serum was obtained at different time-points after phX and added to in vitro CC531 cell cultures. Finally, phX was compared with an ileum resection (ilX). RESULTS: phX leads to increased tumour noduli in the lungs, compared to Sham operation (p=0.002), but only when performed directly before the injection of tumour cells and not when performed 24h before or after the inoculation. Comparable results were obtained for ilX. No growth stimulation of tumour cells after incubation with rat serum, obtained at different time-points after phX, could be detected in vitro. CONCLUSION: Not only phX, but also surgery, in general promotes distant tumour recurrence exerting the effect during the early phase of tumour cell adhesion and not during tumour outgrowth.
Assuntos
Adenocarcinoma/imunologia , Neoplasias Colorretais/cirurgia , Neoplasias Hepáticas/cirurgia , Neoplasias Pulmonares/imunologia , Adenocarcinoma/secundário , Animais , Adesão Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Hepatectomia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Neoplasias Hepáticas/secundário , Regeneração Hepática/imunologia , Neoplasias Pulmonares/secundário , Masculino , Metástase Neoplásica , Transplante de Neoplasias , Ratos , Ratos Wistar , Receptores de Fatores de Crescimento/biossínteseRESUMO
Microdialysis is a novel and minimally invasive sampling technique, based on the diffusion of analytes from the interstitial compartment through a semi-permeable membrane, and enables direct assessment of tissue disposition and penetration of drugs. Variable antitumor responses may be associated with differences in tumor vascularity, capillary permeability or tumor interstitial pressure resulting in variable delivery of anticancer agents. In preparation of pharmacokinetic studies, aimed at measuring docetaxel concentrations in healthy and malignant tissues in vivo, in pre-clinical as well as clinical studies, in vitro recovery experiments were performed. In contrast to published data, the recovery experiments suggest that docetaxel has a very low recovery as a result of non-specific binding to currently available microdialysis catheters. Here we discuss our findings with docetaxel in a historical perspective and we report on our experience using polysorbate 80 to eliminate the non-specific binding and its effects on the recovery of docetaxel.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Microdiálise/métodos , Taxoides/administração & dosagem , Taxoides/farmacocinética , Cromatografia Líquida , Protocolos Clínicos , Docetaxel , Humanos , Espectrometria de MassasRESUMO
OBJECTIVE: To assess the involvement of the multidrug resistance-associated protein 1 (MRP1) and the glutathione pathway in the multidrug resistant (MDR) phenotype of prostate cancer in vitro. MATERIALS AND METHODS: Chemoselection of human prostate cancer cell lines PC3 and DU145 with etoposide resulted in the resistant cell lines PC3-R and DU-R. Resistance against etoposide, doxorubicin and vincristine, and its reversal with leukotriene D4 antagonists MK-571 and zafirlukast, and buthionine sulfoximine (BSO), was assessed using tetrazolium-dye viability assays. Western blot analysis of MRP1 expression and glutathione content were measured, and MRP1 function assessed in fluorescence assays. RESULTS: MRP1 was increased in the MDR models; the glutathione content was significantly higher in PC3-R but there was no increase in glutathione in DU-R. Adding non-toxic doses of MK-571, zafirlukast or BSO significantly increased the sensitivity of the MDR models to cytotoxic drugs. MRP1 function was inhibited with MK-571 in the MDR models. CONCLUSION: MRP1 and glutathione mediate MDR in newly developed prostate cancer models.
Assuntos
Butionina Sulfoximina/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa/fisiologia , Antagonistas de Leucotrienos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Neoplasias da Próstata/tratamento farmacológico , Western Blotting , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Masculino , Propionatos/farmacologia , Neoplasias da Próstata/metabolismo , Quinolinas/farmacologiaRESUMO
Vaults are large ribonucleoprotein particles found in eukaryotic cells. They are composed of multiple copies of a Mr 100,000 major vault protein and two minor vault proteins of Mr 193,000 and 240,000, as well as small untranslated RNAs of 86-141 bases. The vault components are arranged into a highly characteristic hollow barrel-like structure of 35 x 65 nm in size. Vaults are predominantly localized in the cytoplasm where they may associate with cytoskeletal elements. A small fraction of vaults are found to be associated with the nucleus. As of yet, the precise cellular function of the vault complex is unknown. However, their distinct morphology and intracellular distribution suggest a role in intracellular transport processes. Here we review the current knowledge on the vault complex, its structure, components and possible functions.
Assuntos
Partículas de Ribonucleoproteínas em Forma de Abóbada , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/ultraestruturaRESUMO
Expression of the multidrug resistance proteins P-glycoprotein, encoded by the MDR1 gene, multidrug resistance-associated protein (MRP1) and the lung resistance-related protein or major vault protein (LRP/MVP) is associated with clinical resistance to chemotherapy in acute myeloid leukemia (AML). Recently, the breast cancer-resistant protein (BCRP), the equivalent of mitoxantrone-resistant protein (MXR) or placental ABC transporter (ABCP), was described in AML. We investigated MDR1, MRP1, LRP/MVP and BCRP mRNA expression simultaneously in 20 paired clinical AML samples from diagnosis and relapse or refractory disease, using quantitative Taqman analysis. In addition, standard assays for P-glycoprotein expression and function were performed. BCRP was the only resistance protein that was expressed at a significantly higher RNA level (median 1.7-fold, P = 0.04) at relapsed/refractory state as compared to diagnosis. In contrast, LRP/MVP mRNA expression decreased as disease evolved (P = 0.02), whereas MDR1 and MRP1 mRNA levels were not different at relapse as compared to diagnosis. Also, at the protein level no difference of MDR1 between diagnosis and relapse was found. A significant co-expression of BCRP and MDR1 was found at diagnosis (r = 0.47, P = 0.04). The present results suggest that BCRP, but not MDR1, MRP1 or LRP/MVP is associated with clinical resistant disease in AML.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Doença Aguda , Adolescente , Adulto , Idoso , Células da Medula Óssea/patologia , Criança , Pré-Escolar , Progressão da Doença , Humanos , Lactente , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Recidiva , Regulação para Cima , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologia , Sequência de Bases , Western Blotting , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Testes de PrecipitinaRESUMO
The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.
Assuntos
Anticorpos Monoclonais , Proteínas de Neoplasias/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Western Blotting , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Pequenas , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Fixadores , Formaldeído , Imuno-Histoquímica , Neoplasias Pulmonares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Inclusão em Parafina , Fixação de Tecidos , Células Tumorais Cultivadas , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologiaRESUMO
Human vaults are intracellular ribonucleoprotein particles believed to be involved in multidrug resistance. The complex consists of a major vault protein (MVP), two minor vault proteins (VPARP and TEP1), and several small untranslated RNA molecules. Three human vault RNA genes (HVG1-3) have been described, and a fourth was found in a homology search (HVG4). In the literature only the association of hvg1 with vaults was shown in vivo. However, in a yeast three-hybrid screen the association of hvg1, hvg2, and hvg4 with TEP1 was demonstrated. In this study we investigated the expression and vault association of different vault RNAs in a variety of cell lines, including pairs of drug-sensitive and drug-resistant cells. HVG1-3 are expressed in all cell lines examined, however, none of the cell lines expressed HVG4. This probably is a consequence of the absence of essential external polymerase III promoter elements. The bulk of the vault RNA associated with vaults was hvg1. Interestingly, an increased amount of hvg3 was bound to vaults isolated from multidrug-resistant cell lines. Our findings suggest that vaults bind the RNA molecules with different affinities in different situations. The ratio in which the vault RNAs are associated with vaults might be of functional importance.
Assuntos
Proteínas de Transporte/biossíntese , RNA/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/biossíntese , Partículas de Ribonucleoproteínas em Forma de Abóbada/fisiologia , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Citoplasma/metabolismo , Resistência a Múltiplos Medicamentos , Humanos , Dados de Sequência Molecular , RNA/análise , Proteínas de Ligação a RNA , Homologia de Sequência do Ácido Nucleico , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Glucocorticoids play an important role in the treatment of a number of hematological malignancies, such as multiple myeloma. The effects of glucocorticoids are mediated through the glucocorticoid receptor alpha, the abundance of which can be modulated by alternative splicing of the glucocorticoid receptor mRNA. Two splice variants of the glucocorticoid receptor mRNA have been described: glucocorticoid receptor beta, which reportedly has a dominant negative effect on the actions of the glucocorticoid receptor alpha, and glucocorticoid receptor P, of which the effects are unknown. In this study, we have investigated the expression levels of these two splice variants at the mRNA level in multiple myeloma cells and in a number of other hematological tumors. Although the glucocorticoid receptor beta mRNA was, if at all, expressed at very low levels, considerable amounts (up to 50% of the total glucocorticoid receptor mRNA) glucocorticoid receptor P mRNA was present in most hematological malignancies. In transient transfection studies in several cell types and in multiple myeloma cell lines, the glucocorticoid receptor P increased the activity of the glucocorticoid receptor alpha. These results suggest that the relative levels of the glucocorticoid receptor alpha and the glucocorticoid receptor P may play a role in the occurrence of glucocorticoid resistance in tumor cells during the treatment of hematological malignancies with glucocorticoids.
Assuntos
Processamento Alternativo , Neoplasias Hematológicas/metabolismo , Receptores de Glucocorticoides/biossíntese , Animais , Medula Óssea/metabolismo , Células CHO , Células COS , Cricetinae , Células HeLa , Neoplasias Hematológicas/genética , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais CultivadasRESUMO
The expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, is an independent adverse prognostic factor for response and survival in de novo acute myeloid leukemia (AML). Little is known about MDR1 expression during the development of disease. The present study investigated whether MDR1 gene- related clonal selection occurs in the development from diagnosis to relapsed AML, using a genetic polymorphism of the MDR1 gene at position 2677. Expression and function of P-gp were studied using monoclonal antibodies MRK16 and UIC2 and the Rhodamine 123 retention assay with or without PSC 833. No difference was found in the levels of P-gp function and expression between diagnosis and relapse in purified paired blast samples from 30 patients with AML. Thirteen patients were homozygous for the genetic polymorphism of MDR1 (n = 7 for guanine, n = 6 for thymidine), whereas 17 patients were heterozygous (GT). In the heterozygous patients, no selective loss of one allele was observed at relapse. Homozygosity for the MDR1 gene (GG or TT) was associated with shorter relapse-free intervals (P =.002) and poor survival rates (P =.02), compared with heterozygous patients. No difference was found in P-gp expression or function in patients with AML with either of the allelic variants of the MDR1 gene. It was concluded that P-gp function or expression is not upregulated at relapse/refractory disease and expression of one of the allelic variants is not associated with altered P-gp expression or function in AML, consistent with the fact that MDR1 gene-related clonal selection does not occur when AML evolves to recurrent disease. (Blood. 2001;97:3605-3611)