RESUMO
We induced thrombosis of blood vessels in solid tumors in mice by a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA, targeting aminopeptidase N (CD13) and the integrin alpha(v)beta(3) (CD51/CD61) on tumor vascular endothelium. The designed fusion protein tTF-NGR retained its thrombogenic activity as demonstrated by coagulation assays. In vivo studies in mice bearing established human adenocarcinoma (A549), melanoma (M21), and fibrosarcoma (HT1080) revealed that systemic administration of tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels as shown by histologic analysis. tTF-NGR, but not untargeted tTF, induced significant tumor growth retardation or regression in all 3 types of solid tumors. Thrombosis induction in tumor vessels by tTF-NGR was also shown by contrast enhanced magnetic resonance imaging (MRI). In the human fibrosarcoma xenograft model, MRI revealed a significant reduction of tumor perfusion by administration of tTF-NGR. Clinical first-in-man application of low dosages of this targeted coagulation factor revealed good tolerability and decreased tumor perfusion as measured by MRI. Targeted thrombosis in the tumor vasculature induced by tTF-NGR may be a promising strategy for the treatment of cancer.
Assuntos
Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Tromboplastina/antagonistas & inibidores , Adulto , Inibidores da Angiogênese/uso terapêutico , Animais , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Embolia/induzido quimicamente , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias/irrigação sanguínea , Oligopeptídeos/metabolismo , Terapia de Salvação , Tromboplastina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In acute myeloid leukemia (AML), receptor tyrosine kinase ligands promote growth and survival and contribute to AML-associated marrow neoangiogenesis. We have tested simultaneous inhibition of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptor signaling by novel indolinone derivatives using 14 myeloid, including 11 human leukemic, cell lines. Compounds inhibited colony formation of all cell lines in a dose-dependent fashion. Inhibitory concentrations for 50% of the colony formation/survival (IC50) for BIBF1000 were <100 nmol/L for 3 of 11, Assuntos
Indóis/farmacologia
, Leucemia Mieloide/tratamento farmacológico
, Inibidores de Proteínas Quinases/farmacologia
, Receptores Proteína Tirosina Quinases/antagonistas & inibidores
, Doença Aguda
, Apoptose/efeitos dos fármacos
, Processos de Crescimento Celular/efeitos dos fármacos
, Linhagem Celular Tumoral
, Citarabina/farmacologia
, Humanos
, Leucemia Mieloide/sangue
, Leucemia Mieloide/patologia
, Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores
, Proteínas Quinases Ativadas por Mitógeno/metabolismo
, Fosforilação/efeitos dos fármacos
, Receptores Proteína Tirosina Quinases/metabolismo
, Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores
, Receptores de Fatores de Crescimento de Fibroblastos/sangue
, Receptores de Fatores de Crescimento de Fibroblastos/metabolismo
, Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores
, Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo
, Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
, Receptores de Fatores de Crescimento do Endotélio Vascular/sangue
, Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo
, Transdução de Sinais/efeitos dos fármacos
RESUMO
RhKGF is developed for prevention and treatment of chemotherapy- and radiation-induced epithelial damage in cancer patients. Little information exists on growth modulation of malignant cells by KGF. We have tested the anchorage-independent clonal growth of 35 human tumor and 22 lymphoma and leukemia cell lines in semisolid media with or without rhKGF. Growth of the majority of cell lines was not significantly influenced by rhKGF. Growth of 5 cell lines (2 lung, 1 stomach, 1 colorectal, 1 breast) was significantly stimulated by rhKGF. The effect was dose-dependent with significant stimulation at concentrations >1-10 ng/ml. Growth modulation was amplified by serum reduction and abolished by anti-hKGF antibodies. Responding cell lines expressed KGF receptor RNA and showed specific KGF-binding. To determine whether KGF-induced higher colony numbers represented cell divisions with resulting differentiation and growth arrest or self-renewal we performed experiments on serial plating efficacy of colony-derived tumor cells with or without continued presence of rhKGF. Results revealed KGF stimulation of colony formation as representing increase of cellular self-renewal. To test possible interaction of rhKGF with activity of cytotoxic drugs in clinical protocols, we have used combination protocols with 5-fluorouracil (5-FU). Results showed that rhKGF incubation before, after (or both) addition of 5-FU did not diminish the sensitivity of tumor cells to 5-FU cytotoxicity under serum concentrations relevant for the clinical situation. In conclusion, some epithelial tumor cells have receptors for KGF signaling for clonal growth. When considered in the planning of treatment protocols, this effect is unlikely to compromise chemotherapy sensitivity.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Fluoruracila/farmacologia , Leucemia/patologia , Linfoma/patologia , Neoplasias Epiteliais e Glandulares/patologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator 7 de Crescimento de Fibroblastos , Humanos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Adhesion molecules are involved in cell-cell and cell-matrix interactions and may be informative to characterize intercellular mechanisms of invasion and metastasis. This study was performed to characterize radiation-induced changes in the adhesion molecule profile of Ewing tumor subpopulations on a single cell level. MATERIALS AND METHODS: In the present study, two Ewing tumors were characterized in vitro 4, 24 and 72 hours after radiation with 5 Gy and in vivo in a xenograft model 4, 6 and 15 days after radiation with 30 Gy, together with non-irradiated controls, by five parameter flow cytometry. Directly fluorescence-conjugated antibodies that were directed against adhesion molecules (LFA-1 (CD11a), HCAM (CD44), VLA-2 (CD49b), ICAM-1 (CD54), NCAM (CD56), LECAM-1 (CD62L) and CD86) were used. Annexin V and 7-AAD were used to characterize radiation-induced apoptosis. RESULTS: Tumor cell subpopulations were identified by the expression of adhesion molecules, apoptotic markers and DNA content. Heterogeneous changes of the adhesion molecule profile were identified on tumor cell subpopulations after radiation. The expression of CD11a and CD62L correlated with the expression of apoptosis-associated markers. CONCLUSION: The changes of flow cytometric profile under radiation may potentially correlate with a changed metastatic potential of tumor cell subpopulations.
Assuntos
Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/efeitos da radiação , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/radioterapia , Animais , Antígenos CD/biossíntese , Antígenos CD/efeitos da radiação , Apoptose/fisiologia , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Citometria de Fluxo , Técnica Direta de Fluorescência para Anticorpo , Humanos , Camundongos , Camundongos Nus , Sarcoma de Ewing/patologia , Fatores de Tempo , Regulação para Cima/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: To characterize the interaction of tumour necrosis factor alpha (TNF-alpha) and ionising radiation in six sarcoma, lymphoma and carcinoma cell lines. MATERIALS AND METHODS: Cells were characterized regarding annexin V/propiduim iodine affinity and caspase-3 status after application of TNF-alpha, radiation, or combined treatment. RESULTS: Three cell lines showed similar results with additive effects of TNF-alpha and radiation in both assays. The other three cell lines significantly differed regarding the detection of apoptotic cells between treatment conditions (radiation and/or TNF application) and between the two apoptosis assays. CONCLUSION: The interaction between TNF-alpha and radiation differs between tumour entities and cannot be described by using only one parameter.
Assuntos
Apoptose/efeitos da radiação , Carcinoma/radioterapia , Linfoma/radioterapia , Sarcoma/radioterapia , Fator de Necrose Tumoral alfa/farmacologia , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sarcoma/patologiaRESUMO
tTF-NGR consists of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), a ligand of the surface protein aminopeptidase N (CD13). After deamidation of the NGR-peptide moiety, the fusion protein is also a ligand for integrin αvß3 (CD51/CD61). Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR showed binding to specific binding sites on endothelial cells in vitro as shown by flow cytometry. Subcutaneous injection of tTF-NGR into athymic mice bearing human HT1080 fibrosarcoma tumors induced tumor growth retardation and delay. Contrast enhanced ultrasound detected a decrease in tumor blood flow in vivo after application of tTF-NGR. Histological analysis of the tumors revealed vascular disruption due to blood pooling and thrombotic occlusion of tumor vessels. Furthermore, a lack of resistance was shown by re-exposure of tumor-bearing mice to tTF-NGR after regrowth following a first cycle of treatment. However, after subcutaneous (s.c.) push injection with therapeutic doses (1-5 mg/kg bw) side effects have been observed, such as skin bleeding and reduced performance. Since lethality started within the therapeutic dose range (LD10 approximately 2 mg/kg bw) no safe therapeutic window could be found. Limiting toxicity was represented by thrombo-embolic events in major organ systems as demonstrated by histology. Thus, subcutaneous injection of tTF-NGR represents an active, but toxic application procedure and compares unfavourably to intravenous infusion.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Infarto/induzido quimicamente , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Tromboplastina/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/efeitos adversos , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Oligopeptídeos/efeitos adversos , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/química , Tromboplastina/efeitos adversos , Tromboplastina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Flow cytometry was applied to test for platelet-activating-factor receptor (PAF-R) presence on the membranes of acute myeloid leukemia (AML) cells. We have used six human AML cell lines and freshly taken density gradient separated blasts from the bone marrow of ten AML patients covering the majority of French-American-British (FAB) subtypes. Additionally, we have used one histiocytic lymphoma cell line and mature human granulocytes/monocytes as controls. Our results indicate lack of membrane PAF-R on AML of all FAB subtypes tested. This was particularly true for the more mature and differentiated subtypes M4 and M5, including monocytic cell elements, and the promyelocytic M3 AML. In contrast, membrane PAF-R could be easily detected in a histiocytic lymphoma cell line and mature granulocytes/monocytes from peripheral blood used as positive controls. In conclusion, this observation precludes the use of membrane PAF-R as an immunophenotypic marker for AML classification or detection of minimal residual disease.