RESUMO
Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation in preclinical models of breast cancer is associated with tumor growth and resistance to anticancer therapies, including paclitaxel. Effects of the pan-Class I PI3K inhibitor buparlisib (BKM120) appear synergistic with paclitaxel in preclinical and clinical models. Patients and methods: BELLE-4 was a 1:1 randomized, double-blind, placebo-controlled, adaptive phase II/III study investigating the combination of buparlisib or placebo with paclitaxel in women with human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer with no prior chemotherapy for advanced disease. Patients were stratified by PI3K pathway activation and hormone receptor status. The primary endpoint was progression-free survival (PFS) in the full and PI3K pathway-activated populations. An adaptive interim analysis was planned following the phase II part of the study, after ≥125 PFS events had occurred in the full population, to decide whether the study would enter phase III (in the full or PI3K pathway-activated population) or be stopped for futility. Results: As of August 2014, 416 patients were randomized to receive buparlisib (207) or placebo (209) with paclitaxel. At adaptive interim analysis, there was no improvement in PFS with buparlisib versus placebo in the full (median PFS 8.0 versus 9.2 months, hazard ratio [HR] 1.18), or PI3K pathway-activated population (median PFS 9.1 versus 9.2 months, HR 1.17). The study met protocol-specified criteria for futility in both populations, and phase III was not initiated. Median duration of study treatment exposure was 3.5 months in the buparlisib arm versus 4.6 months in the placebo arm. The most frequent adverse events with buparlisib plus paclitaxel (≥40% of patients) were diarrhea, alopecia, rash, nausea, and hyperglycemia. Conclusions: Addition of buparlisib to paclitaxel did not improve PFS in the full or PI3K pathway-activated study population. Consequently, the trial was stopped for futility at the end of phase II.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminopiridinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Método Duplo-Cego , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Morfolinas/administração & dosagem , Paclitaxel/administração & dosagem , Inibidores de Fosfoinositídeo-3 Quinase , Modelos de Riscos Proporcionais , Receptor ErbB-2/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
Lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 is thought to be restricted to lymph vessels and has been used as such to show that tumor lymphangiogenesis occurs on overexpression of lymphangiogenic factors in mouse tumor models. However, these studies have not yet been corroborated in human tumors. Here we show, first, that LYVE-1 is not exclusive to the lymph vessels. Indeed, LYVE-1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans. Surprisingly, LYVE-1 is absent from the angiogenic blood vessels of human liver tumors and only weakly present in the microcirculation of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. Second, we propose a novel approach to identify lymphatics in human and murine liver. By combining LYVE-1 and Prox 1 (a transcription factor) immunohistochemistry, we demonstrate that lymphatics are abundant in cirrhosis. In contrast, in human hepatocellular carcinoma and liver metastases, they are restricted to the tumor margin and surrounding liver. The absence of intratumor lymphatics in hepatocellular carcinomas and liver metastases may impair molecular and cellular transport in these tumors. Finally, the presence of LYVE-1 in liver sinusoidal endothelia suggests that LYVE-1 has functions beyond the lymph vascular system.
Assuntos
Carcinoma Hepatocelular/metabolismo , Glicoproteínas/biossíntese , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/irrigação sanguínea , Sistema Linfático/metabolismo , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundário , Regulação para Baixo , Endotélio Vascular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Proteínas Supressoras de Tumor , Proteínas de Transporte VesicularRESUMO
Recent studies in experimental animals have shown that combining antiangiogenic therapy with radiation can enhance tumor response. Whether this enhancement is mainly attributable to angiogenesis inhibition, endothelial cell radiosensitivity, tumor cell apoptosis, or a decrease in the number of hypoxic cells (improved oxygenation) is not known. We designed this study to discern the role of tumor oxygenation. We chose an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody (mAb) which has a known target, human VEGF. We also measured interstitial fluid pressure (IFP) to test the hypothesis that the decreased vascular permeability induced by the anti-VEGF mAb can lower IFP. The effect of anti-VEGF mAb on vascular density, partial oxygen tension (pO2), and apoptosis was also measured. Athymic NCr/Sed nu/nu mice bearing 6-mm xenograft of the human glioblastoma multiforme (U87), or colon adenocarcinoma (LS174T) were treated with anti-VEGF mAb injected i.p. on alternate days for a total of six injections at a dosage of 100 microg/injection/mouse. For combined anti-VEGF and radiation, single radiation doses were given under normal blood flow (20 and 30 Gy) or clamped hypoxic conditions (30 and 40 Gy) 24 h after the sixth injection of mAb. The inhibition of the growth of U87 and LS174T tumors by the anti-VEGF mAb was associated with a significant reduction in tumor vascular density and a relatively small increase in the number of apoptotic cells. Compared with size-matched controls, IFP decreased by 74% in LS174T, and 73% in U87 in mice treated with anti-VEGF mAb. After antibody treatment PO2 increased significantly in U87, but did not change in LS174T tumors. Combined treatment induced in U87 tumors a tumor-growth delay (TGD) which was greater than additive; in LS174T except for the 40-Gy hypoxic group, the effect was only additive. In both U87 and LS174T the TGD induced by the antibody was independent of oxygen levels in the tumor at the time of radiation. The fact that the increase in TGD occurred under both normoxic and hypoxic conditions suggests that anti-VEGF mAb treatment can compensate for the resistance to radiation induced by hypoxia.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/farmacologia , Neoplasias do Colo/terapia , Fatores de Crescimento Endotelial/imunologia , Glioblastoma/terapia , Linfocinas/imunologia , Oxigênio/metabolismo , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Hipóxia Celular , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Terapia Combinada , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/metabolismo , Espaço Extracelular/fisiologia , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Linfocinas/antagonistas & inibidores , Linfocinas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pressão Parcial , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: Cyclin D-cyclin-dependent kinase (CDK) 4/6-inhibitor of CDK4/6-retinoblastoma (Rb) pathway hyperactivation is associated with hormone receptor-positive (HR+) breast cancer (BC). This study assessed the biological activity of ribociclib (LEE011; CDK4/6 inhibitor) plus letrozole compared with single-agent letrozole in the presurgical setting. MATERIALS AND METHODS: Postmenopausal women (N = 14) with resectable, HR+, human epidermal growth factor receptor 2-negative (HER2-) early BC were randomized 1:1:1 to receive 2.5 mg/day letrozole alone (Arm 1), or with 400 or 600 mg/day ribociclib (Arm 2 or 3). Circulating tumor DNA and tumor biopsies were collected at baseline and, following 14 days of treatment, prior to or during surgery. The primary objective was to assess antiproliferative response per Ki67 levels in Arms 2 and 3 compared with Arm 1. Additional assessments included safety, pharmacokinetics, and genetic profiling. RESULTS: Mean decreases in the Ki67-positive cell fraction from baseline were: Arm 1 69% (range 38-100%; n = 2), Arm 2 96% (range 78-100%; n = 6), Arm 3 92% (range 75-100%; n = 3). Decreased phosphorylated Rb levels and CDK4, CDK6, CCND2, CCND3, and CCNE1 gene expression were observed following ribociclib treatment. Ribociclib and letrozole pharmacokinetic parameters were consistent with single-agent data. The ribociclib plus letrozole combination was well tolerated, with no Grade 3/4 adverse events over the treatment. CONCLUSION: The results suggest absence of a drug-drug interaction between ribociclib and letrozole and indicate ribociclib plus letrozole may reduce Ki67 expression in HR+, HER2- BC (NCT01919229).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , DNA de Neoplasias/sangue , Expressão Gênica/efeitos dos fármacos , Antígeno Ki-67/análise , Idoso , Aminopiridinas/administração & dosagem , Aminopiridinas/efeitos adversos , Aminopiridinas/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias da Mama/química , Neoplasias da Mama/genética , Neoplasias da Mama/cirurgia , Ciclina D2/genética , Ciclina D3/genética , Ciclina E/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Feminino , Humanos , Letrozol , Pessoa de Meia-Idade , Terapia Neoadjuvante , Nitrilas/administração & dosagem , Nitrilas/efeitos adversos , Nitrilas/farmacocinética , Proteínas Oncogênicas/genética , Purinas/administração & dosagem , Purinas/efeitos adversos , Purinas/farmacocinética , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Triazóis/administração & dosagem , Triazóis/efeitos adversos , Triazóis/farmacocinéticaRESUMO
Anandamide, an endogenous canabinoid substance, is hydrolyzed by an amidohydrolase activity present in rat brain and liver. We report that the bromoenol lactone, (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BTNP), is a potent inhibitor of this enzyme activity. BTNP prevented anandamide hydrolysis in rat brain microsomes with an IC50 of 0.8 +/- 0.3 microM. Kinetic and dialysis experiments indicated that this effect was non-competitive and irreversible. After chromatographic fractionation of the enzyme activity, BTNP was still effective, suggesting that it interacts directly with the enzyme. Anandamide hydrolysis was 12-fold greater in rat cortical neurons (1.94 +/- 0.1 pmol/min/mg protein) than in cortical astrocytes (0.16 +/- 0.01 pmol/min/mg protein) and, in either cell type, it was inhibited by BTNP (IC50 = 0.1 microM in neurons). These results suggest that BTNP may provide a useful lead for the development of novel inhibitors of anandamide hydrolysis.
Assuntos
Amidoidrolases/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Encéfalo/enzimologia , Naftalenos/farmacologia , Pironas/farmacologia , Animais , Astrócitos/enzimologia , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Hidrólise/efeitos dos fármacos , Ionóforos/farmacologia , Microssomos Hepáticos/enzimologia , Neurônios/enzimologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Alcamidas Poli-Insaturadas , Ratos , Ratos WistarAssuntos
Encéfalo/metabolismo , Lipídeos de Membrana/metabolismo , Neurônios/metabolismo , Fosfatidiletanolaminas/metabolismo , Receptores de Droga/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Cálcio/fisiologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Endocanabinoides , Ionomicina/farmacologia , Neurônios/efeitos dos fármacos , Fosfatidiletanolaminas/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Alcamidas Poli-Insaturadas , Ratos , Receptores de Canabinoides , Receptores de Droga/efeitos dos fármacos , Veratridina/farmacologiaRESUMO
It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.
Assuntos
Encéfalo/metabolismo , Canabinoides/metabolismo , Fosfatidiletanolaminas/metabolismo , Pró-Fármacos/metabolismo , Aciltransferases/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Endocanabinoides , Enzimas/metabolismo , Metabolismo dos Lipídeos , Masculino , Fosfolipídeos/metabolismo , Alcamidas Poli-Insaturadas , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
The presence of "mosaic" vessels in which both endothelial cells and tumor cells form the luminal surface has profound implications for metastasis, drug delivery, and antivascular therapy. Yet little is known of the frequency, and thus importance, of mosaic vessels in tumors. Using CD31 and CD105 to identify endothelial cells and endogenous green fluorescent protein labeling of tumor cells, we show that approximately 15% of perfused vessels of a colon carcinoma xenografted at two different sites in mice were mosaic vessels having focal regions where no CD31/CD105 immunoreactivity was detected and tumor cells appeared to contact the vessel lumen. These regions occupied approximately 25% of the perimeter of the mosaic vessels, or approximately 4% of the total vascular surface area in these colon carcinomas. In addition, we found similar numbers of mosaic vessels in human colon carcinoma biopsies. Our results are consistent with the observation that approximately 10(6) cells are shed daily per g of tumor. More importantly, our data offer a possible explanation for the antivascular effects of cytotoxic agents and suggest potential strategies for targeting the tumor vasculature.
Assuntos
Neoplasias do Colo/sangue , Neovascularização Patológica , Animais , Antígenos CD , Neoplasias do Colo/patologia , Endoglina , Humanos , Camundongos , Camundongos SCID , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Receptores de Superfície Celular , Células Tumorais Cultivadas , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
Deregulation of the G1/S checkpoint is a frequent event in the development of glioblastoma multiforme (GBM). Previous studies have shown more than 50% of primary GBM tumours contain either complete loss of the p16INK4a locus or amplification of the CDK4 gene. Moreover, many heterozygosity studies have shown deletion on human chromosome 19p13.2, where the p19INK4d gene has been localized. We examined the expression of p19INK4d and its two CDK substrates in a series of glioma-derived cell lines and tumours. No gene rearrangement or deletion was observed in the p19INK4d gene in these cell lines; however, expression of CDK4 and CDK6 was elevated relative to matched normal brain tissue in eight of 18 GBM tumours (44%). Furthermore, CDK6 expression level was increased in 12/14 glioblastomas, but undetectable in tumour samples of a previous lower grade tumour from the same patient. These data attest to the functional importance of both CDK4 and CDK6 in astrocytic tumourigenesis, particularly during the later stages of tumour progression.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Neoplasias do Sistema Nervoso Central/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Quinases Ciclina-Dependentes , Glioblastoma/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Western Blotting , Proteínas de Transporte/metabolismo , Neoplasias do Sistema Nervoso Central/metabolismo , Quinase 6 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19 , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Permanent glioma cell lines are invaluable tools in understanding the biology of glioblastomas. The present study reports the establishment of a clonal human cell line, GBM6840, derived from a biopsy of paediatric cerebellar glioblastoma multiforme. GBM6840 had a doubling time of 32 h and grew as a monolayer of large round cells that retained immunopositivity for glial fibrillary acidic protein and vimentin. Karyotypic analysis revealed a modal chromosome number of 68 and polysomies of chromosomes 3, 5 and 20, as well as the presence of 3-4 marker chromosomes. GBM6840 also showed anchorage-independent growth in soft agar and tumour formation in nude mice. The p16(CDKN2A) gene was transcriptionally silenced by hypermethylation, consistent with the lack of protein expression observed in the original tumour and cultured cells. Western blot analysis revealed normal protein expression of pRb and CDK4. It appears that p16 is the major component altered in the cell cycle pathway and may confer these cells unrestrained proliferation potential. Neither EGFR gene amplification nor over-expression of the protein was detected in the cultured cells. Over-expression of the p53 protein was observed in the majority of cells, despite undetectable mutation (exons 5-8) in the gene. One allele of the PTEN gene was found to be mutated during in vitro cultivation. Telomerase activity was demonstrated in the cultured cells but not in the original tumour, supporting the hypothesis that telomerase is required for the in vitro immortalization process.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Cerebelares , Glioblastoma , Proteínas Proto-Oncogênicas , Células Tumorais Cultivadas/citologia , Proteínas Supressoras de Tumor , Adolescente , Testes de Carcinogenicidade , Divisão Celular , Aberrações Cromossômicas , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/genética , Análise Mutacional de DNA , Receptores ErbB/análise , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cariotipagem , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Mutação Puntual , Proteína do Retinoblastoma/análise , Proteína do Retinoblastoma/genética , Telomerase/metabolismo , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/enzimologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The epidermal growth factor receptor (EGFR) is a protooncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased (P < 0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.
Assuntos
Elementos Antissenso (Genética)/farmacologia , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Glioblastoma/terapia , Anticorpos Monoclonais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proteína Glial Fibrilar Ácida/análise , Humanos , Antígeno Ki-67/análise , Transfecção , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/citologiaRESUMO
The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor-host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.