RESUMO
Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.
Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/farmacologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neovascularização Patológica/patologia , Angiopoietina-1/análise , Angiopoietina-2/metabolismo , Animais , Linhagem Celular Tumoral , Células Endoteliais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Pericitos/patologia , Fenótipo , Receptor TIE-2/metabolismo , Transplante HeterólogoRESUMO
PURPOSE: This study aimed to identify and evaluate molecular targets for the development of a novel combination chemotherapy to treat refractory and recurrent diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGN: Lymphoma samples from 38 cases of primary and recurrent DLBCL were analyzed using real-time quantitative PCR of the RPS6KB1 and CDC2 genes, and immunohistochemistry for their gene products p70S6K/p85S6K and cdc2/cdk1. The Farage, Karpas422, Pfeiffer, and Toledo DLBCL cell lines were subsequently treated with rapamycin and UCN-01 alone or in combination. Cell proliferation, apoptosis, and cell cycle progression were analyzed after the drug treatment. In addition, the levels of several key protein kinases involved in the phosphoinositide 3'-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, apoptosis, and cell cycle progression were analyzed in the presence and absence of the drugs. RESULTS: Amplification of the RPS6KB1 and CDC2 genes was found in both primary and recurrent DLBCL. Moreover, the vast majority of these lymphomas (approximately 94%) were strongly positive for phospho-p70S6K and cdc2/cdk1 proteins. The combination of rapamycin and UCN-01 synergistically inhibited the DLBCL cell proliferation by inducing G1 arrest as well as apoptosis by suppressing the phosphorylation of p70S6K/p85S6K and CDC2 expression. CONCLUSION: RPS6KB1 and CDC2 overexpression is common in DLBCL. Simultaneously targeting the RPS6KB1 and CDC2 products phospho-p70S6K/p85S6K and cdc2/cdk1 is very effective in inhibiting DLBCL proliferation and overcoming drug resistance. This work suggests that multilevel inhibition of the PI3K/Akt/mTOR pathway and double-block of cell cycle progression are effective strategies for DLBCL therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Recidiva Local de Neoplasia/patologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Western Blotting , Proteína Quinase CDC2/genética , Proliferação de Células/efeitos dos fármacos , Ciclina B/genética , Quinases Ciclina-Dependentes , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Sirolimo/administração & dosagem , Estaurosporina/administração & dosagem , Estaurosporina/análogos & derivados , Serina-Treonina Quinases TOR , Células Tumorais CultivadasRESUMO
Human E1 is a key player in protein ubiquitination, however the E1 structure is not available. In this paper, we describe the derivation of a human E1 structure using molecular modelling based on the crystal structure of S. cerevisiae E1 and M. Musculus E1. Key interactions between our E1 model and ubiquitin are also discussed.
RESUMO
PURPOSE: This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. MATERIALS AND METHODS: HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to alpha(v)beta(3) integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. RESULTS: HPMA copolymers containing AH-GDM and RGDfK showed active binding to the alpha(v)beta(3) integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer-AH-GDM-RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. CONCLUSION: Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer-AH-GDM-RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.
Assuntos
Acrilamidas/química , Benzoquinonas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lactamas Macrocíclicas/administração & dosagem , Peptídeos Cíclicos/química , Neoplasias da Próstata/tratamento farmacológico , Acrilamidas/metabolismo , Acrilamidas/toxicidade , Animais , Benzoquinonas/metabolismo , Benzoquinonas/toxicidade , Catepsina B/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proposta de Concorrência , Feminino , Humanos , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Lactamas Macrocíclicas/metabolismo , Lactamas Macrocíclicas/toxicidade , Masculino , Camundongos , Camundongos Nus , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/toxicidade , Testes de ToxicidadeRESUMO
PURPOSE: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001 and investigated its effects on telomerase and telomeres. EXPERIMENTAL DESIGN: We compared telomere length to KML001 cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species-related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: We established a significant inverse correlation (r(2) = 0.9) between telomere length and cytotoxicity. KML001 exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by gamma-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. CONCLUSIONS: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.
Assuntos
Antineoplásicos/farmacologia , Arsenitos/farmacologia , Dano ao DNA/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Compostos de Sódio/farmacologia , Telômero/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Hibridização In Situ , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Telomerase/efeitos dos fármacosRESUMO
Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.
Assuntos
Androstadienos/farmacologia , Benzimidazóis/farmacologia , Retículo Endoplasmático/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Androgênios/metabolismo , Androstadienos/química , Animais , Benzimidazóis/química , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Tapsigargina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Androgênios/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neuregulina-1/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The process of tumour invasion and subsequent metastasis represents the most lethal aspect of cancer. In this study the invasive and migratory activity of four human breast cancer cell lines; MCF-7, MDA-MB-231, BT474 and Hs578T was investigated. Isogenic subclones were isolated from the Hs578T cell line using sequential passages through the BD Matrigel Invasion Chamber assay system. A new invasive subclone designated, Hs578Ts(i)8 was isolated and shown to be 3-fold more invasive and 2.5-fold more migratory than the parental cell line. The variant cells formed up to 25 times more colonies in soft agar and also produced tumours in vivo in nude mice. Flow cytometry analysis showed that the Hs578Ts(i)8 cells had 30% more CD44+/CD24-/low cells than the parental Hs578T cell line. The presence of a breast cancer stem cell population within the variant cell line may provide an explanation for the increased anchorage independent growth and tumourigenicity.
Assuntos
Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Células-Tronco/patologia , Animais , Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Células-Tronco/metabolismoRESUMO
Four nucleoside analogues ( 1- 4) containing a common heterocyclic base, 4(7)-amino-6(5) H-imidazo[4,5- d]pyridazin-7(4)one, were screened against calf-intestine adenosine deaminase. Compounds 1 and 3 with K(i) values of 10-12 microM are more than four times as potent inhibitors of ADA compared with 2 and 4, with K(i) values of 51-52 microM. Also, 3 is not a substrate of ADA. Nucleosides 3 and 4 also exhibit moderate in vitro activity against breast cancer cell lines, while all four are only minimally or nontoxic to the normal cells.
Assuntos
Inibidores de Adenosina Desaminase , Adenosina/análogos & derivados , Adenosina/síntese química , Imidazóis/química , Inosina/análogos & derivados , Inosina/síntese química , Piridazinas/química , Adenosina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Inosina/farmacologia , Relação Estrutura-AtividadeRESUMO
An increased understanding of stem-cell biology at the molecular level, as well as the isolation and characterization of a rare subset of cells with tumor-initiating properties from several human tumor types, have renewed interest in the exploitation of cancer stem cells (CSCs) as therapeutic targets. Although CSCs share various characteristics with normal stem cells, including self-renewal, asymmetric cell division, indefinite proliferative capacity, and self-protection mechanisms, they also contain unique and disease-specific features suitable for exploitation as therapeutic targets. Several existing anticancer agents and experimental therapeutics can inhibit pathways critical to CSC maintenance, and could, therefore, be utilized to eradicate CSCs. In this review, general and tumor-type specific cancer-stem-cell targets are highlighted. In addition, known inhibitors of these targets that could be utilized in the design of clinical protocols together with conventional cytotoxics as debulking agents are described.
Assuntos
Antineoplásicos/farmacologia , Neoplasias , Células-Tronco Neoplásicas , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Neoplasias/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/fisiologiaRESUMO
An emerging concept in cancer biology is that a rare population of cancer stem cells exists among the heterogeneous cell mass that constitutes a tumor. This concept is best understood in human myeloid leukemia. Normal and malignant hematopoietic stem cell functions are defined by a common set of critical stemness genes that regulate self-renewal and developmental pathways. Several stemness factors, such as Notch or telomerase, show differential activation in normal hematopoietic versus leukemia stem cells. These differences could be exploited therapeutically even with drugs that are already in clinical use for the treatment of leukemia. The translation of novel and existing leukemic stem cell-directed therapies into clinical practice, however, will require changes in clinical trial design and the inclusion of stem cell biomarkers as correlative end points.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Animais , Humanos , Camundongos , Transdução de SinaisRESUMO
As new target-directed anticancer agents emerge, preclinical efficacy studies need to integrate target-driven model systems. This approach to drug development requires rapid and reliable characterization of the new targets in established tumor models, such as xenografts and cell lines. Here, we have applied tissue microarray technology to patient-derived, re-growable human tumor xenografts. We have profiled the expression of five proteins involved in cell migration and/or angiogenesis: vascular endothelial growth factor (VEGF), matrix metalloproteinase 1 (MMP1), protease activated receptor (PAR1), cathepsin B, and beta1 integrin in a panel of over 150 tumors and compared their expression levels to available patient outcome data. For each protein, several target overexpressing xenografts were identified. They represent a subset of tumor models prone to respond to specific inhibitors and are available for future preclinical efficacy trials. In a "proof of concept" experiment, we have employed tissue microarrays to select in vivo models for therapy and for the analysis of molecular changes occurring after treatment with the anti-VEGF antibody HuMV833 and gemcitabine. Whereas the less angiogenic pancreatic cancer PAXF736 model proved to be resistant, the highly vascularized PAXF546 xenograft responded to therapy. Parallel analysis of arrayed biopsies from the different treatment groups revealed a down-regulation of Ki-67 and VEGF, an altered tissue morphology, and a decreased vessel density. Our results demonstrate the multiple advantages of xenograft tissue microarrays for preclinical drug development.
Assuntos
Movimento Celular/genética , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neoplasias Pancreáticas/genética , Análise Serial de Tecidos/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antimetabólitos Antineoplásicos/uso terapêutico , Catepsina B/genética , Catepsina B/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Técnicas Imunoenzimáticas , Integrina beta1/genética , Integrina beta1/metabolismo , Metaloproteinase 1 da Matriz/genética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/tratamento farmacológico , Receptor PAR-1/genética , Ribonucleotídeo Redutases/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Mouse models of cancer have consistently been used to qualify new anticancer drugs for study in human clinical trials. The most used models include transplantable murine tumors grown in syngeneic hosts and xenografts of human tumors grown in immunodeficient mice. For the latter systems, retrospective preclinical-clinical correlation studies are available, which suggest that improvements must be made to increase their value. Transgenic, knock-out, and knock-in mouse models and their intercrosses are more recent developments that mirror defined steps of human carcinogenesis. However, their value in predicting clinical results remains to date poorly defined. We take the position that properly used and interpreted human tumor xenografts grown in immunodeficient mice can be useful, although not absolutely predictive of behavior in the clinic, and continue to make contributions to critical clinical development choices.
Assuntos
Antineoplásicos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , Camundongos , Camundongos SCID , Camundongos TransgênicosRESUMO
The past two decades have seen a dramatic change in cancer treatment paradigms. Anticancer agents are no longer being developed based on empiricism and serendipity, but are now being aimed to inhibit a validated target that is relatively specific for tumours rather than normal cells. The vast majority of cancers arise from multiple genetic lesions; thus, sophisticated drug cocktails, or single drugs acting on multiple downstream targets will be needed for successful cancer therapy. Three emerging concepts that are addressing these therapeutic needs and that are key to blocking steps in tumourigenesis will be highlighted in this review: (a) attacking cancer cell immortality by targeting the telomere/telomerase complex; (b) targeting oncogene activation by inhibiting the molecular chaperone Hsp90; and (c) stabilizing tumour suppressor proteins by modulating the ubiquitin-proteasome system.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Ubiquitina/metabolismoRESUMO
Approximately 50% of all cancer patients develop cachexia, a paraneoplastic syndrome that is characterized by wasting of adipose tissue and skeletal muscle mass. Cytokines, including TNF-alpha, interleukins-1, -6, and interferon-A are known mediators of the cachectic process. The latter however represent only one of many imbalanced systems in cancer cachexia. The aim of this study was to further delineate the pathogenesis of cachexia by molecular profiling. Human renal cancer xenografts that do and do not induce cachexia in mice were used as disease models. Cachexia-associated gene expression was studied on Human Genome U95 Affymetrix arrays and revealed several new genes such as TNF-alpha ligand superfamily protein, interferon-A treatment inducible protein, and DKFZ5641I1922. The expression of the IL-8 gene was also elevated in cachexia inducing xenografts (CIX). At the protein level, TNF-alpha was found expressed only in CIX, whereas IL-1 and IL-6 were not cachexia specific. Levels of parathyroid hormone-related protein were elevated in CIX and accompanied by hypercalcemia. COX-2 and prostaglandin E2 were also found to be over expressed. By using the COX-2 inhibitors rofecoxib and nimesulide, we were able to delay tumor-mediated wasting in vivo. Overall, our results suggest that cachexia is a multigenetic disease that will require complex combinations of drugs for an effective therapeutic intervention.
Assuntos
Caquexia/metabolismo , Neoplasias Renais/metabolismo , Síndromes Paraneoplásicas/metabolismo , Animais , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/genética , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Citocinas/biossíntese , Citocinas/genética , Dinoprostona/genética , Dinoprostona/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hipercalcemia/tratamento farmacológico , Hipercalcemia/etiologia , Hipercalcemia/genética , Hipercalcemia/metabolismo , Neoplasias Renais/complicações , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Lactonas/farmacologia , Lactonas/uso terapêutico , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Síndromes Paraneoplásicas/tratamento farmacológico , Síndromes Paraneoplásicas/etiologia , Síndromes Paraneoplásicas/genética , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Transplante HeterólogoRESUMO
The concept of individualized cancer chemotherapy emerged three decades ago from the observation that a small fraction of cells in primary tumors can form colonies in soft agar similar to stem cells of the hematopoietic system. In a series of retrospective and prospective clinical studies, clonogenic tumor growth and effects of anticancer agents on the putative cancer stem cells were assessed as predictive factors. The results of these trials showed that clonogenic growth is associated with poor outcome and drug resistance. Recent breakthroughs enabling isolation and the molecular classification of cancer stem cells have renewed interest in cancer stem cells as a therapeutic target. Here, we provide a current overview of cancer stem cell biology and highlight possibilities for targeted intervention with existing and novel experimental anticancer agents.
Assuntos
Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/patologia , Estudos Prospectivos , Estudos Retrospectivos , Ensaio Tumoral de Célula-TroncoRESUMO
Interference with telomerase and telomere maintenance is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single-stranded 3' overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends. We report here the effects of a 3,6,9-trisubstituted acridine compound, BRACO-19, on telomerase function in vitro and in vivo. The biological activity of BRACO-19 was evaluated in the human uterus carcinoma cell line UXF1138L, which has very short telomeres (2.7 kb). In vitro, nuclear human telomerase reverse transcriptase (hTERT) expression was drastically decreased after 24 hours, induction of cellular senescence and complete cessation of growth was seen after 15 days, paralleled by telomere shortening of ca. 0.4 kb. In vivo, BRACO-19 was highly active as a single agent against early-stage (68 mm(3)) tumors in a s.c. growing xenograft model established from UXF1138L cells, if given chronically at 2 mg per kg per day i.p. BRACO-19 produced growth inhibition of 96% compared with controls accompanied by partial regressions (P < 0.018). Immunostaining of xenograft tissues showed that this response was paralleled by loss of nuclear hTERT protein expression and an increase in atypical mitoses indicative of telomere dysfunction. Cytoplasmic hTERT expression and its colocalization with ubiquitin was observed suggesting that hTERT is bound to ubiquitin and targeted for enhanced degradation upon BRACO-19 treatment. This is in accord with a model of induced displacement of telomerase from the telomere. The in vitro and in vivo data presented here is consistent with the G-quadruplex binding ligand BRACO-19 producing an anticancer effect by inhibiting the capping and catalytic functions of telomerase.
Assuntos
Acridinas/farmacologia , DNA/metabolismo , Telomerase/antagonistas & inibidores , Telômero/efeitos dos fármacos , Neoplasias Uterinas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/efeitos dos fármacos , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Feminino , Quadruplex G , Guanina/metabolismo , Humanos , Camundongos , Camundongos Nus , Telomerase/biossíntese , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Ubiquitina/metabolismo , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.
Assuntos
Neoplasias da Mama/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Prognóstico , Modelos de Riscos Proporcionais , Inibidores de Proteassoma , Receptores de Estrogênio/biossíntese , Transfecção , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
It has been hypothesized that defects in DNA-mismatch repair are associated with smoking in certain types of transformed non-Hodgkin lymphoma (NHL). We have analyzed biopsy samples from two indolent B-cell lymphomas, follicular lymphoma (FL) and chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL), that have transformed to diffuse-large B-cell lymphoma (DLBCL). We correlated the presence or absence of DNA-mismatch repair enzymes by immunostaining as well as the p53 status to smoking history. Of all patients (n = 30), 37% showed negative immunostaining of MLH1, 16% showed negative immunostaining of MSH2 and 63% had p53 mutations and/or protein expression. Eighteen out of 20 transformed follicular lymphomas and seven out of 10 CLL/SLL that have transformed to DLBCL (Richter's syndrome) were informative for smoking histories. We found that the relative risk of negative immunostaining for either MLH1 or MSH2 was 2.2 times higher in smokers than non-smokers (relative risk = 2.2041, 95% confidence interval: 0.89714, 5.41491). No direct correlation was found between smoking and the mutations in the p53 gene. These results suggest that cigarette smoking may play a role in the development of transformed lymphomas through defective mismatch repair.