RESUMO
Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A-D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung fibrosis.
Assuntos
Raios gama/efeitos adversos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Fibrose Pulmonar/tratamento farmacológico , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Camundongos , Isoformas de Proteínas/metabolismo , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/fisiopatologia , Transdução de Sinais/efeitos da radiação , Irradiação Corporal TotalRESUMO
Purpose: KTN0158 is a novel anti-KIT antibody that potently inhibits wild-type and mutant KIT. This study evaluated the safety, biologic activity, and pharmacokinetic/pharmacodynamics profile of KTN0158 in dogs with spontaneous mast cell tumors (MCT) as a prelude to human clinical applications.Experimental Design: Cell proliferation, KIT phosphorylation, and mast cell degranulation were evaluated in vitro KTN0158 was administered to 4 research dogs to assess clinical effects and cutaneous mast cell numbers. Thirteen dogs with spontaneous MCT were enrolled into a prospective phase I dose-escalating open-label clinical study of KTN0158 evaluating 3 dose levels and 2 schedules and with weekly assessments for response and clinical toxicities.Results: KTN0158 was a potent inhibitor of human and dog KIT activation and blocked mast cell degranulation in vitro In dogs, KTN0158 was well tolerated and reduced cutaneous mast cell numbers in a dose-dependent manner. Clinical benefit of KTN0158 administration in dogs with MCT (n = 5 partial response; n = 7 stable disease) was observed regardless of KIT mutation status, and decreased KIT phosphorylation was demonstrated in tumor samples. Histopathology after study completion demonstrated an absence of neoplastic cells in the primary tumors and/or metastatic lymph nodes from 4 dogs. Reversible hematologic and biochemical adverse events were observed at doses of 10 and 30 mg/kg. The MTD was established as 10 mg/kg.Conclusions: KTN0158 inhibits KIT phosphorylation, demonstrates an acceptable safety profile in dogs, and provides objective responses in canine MCT patients with and without activating KIT mutations, supporting future clinical evaluation of KTN0158 in people. Clin Cancer Res; 23(10); 2565-74. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Doenças do Cão/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/imunologia , Animais , Anticorpos Monoclonais/imunologia , Proliferação de Células/efeitos dos fármacos , Doenças do Cão/genética , Doenças do Cão/imunologia , Cães , Feminino , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mutação , FosforilaçãoRESUMO
Head and neck squamous cell carcinoma (HNSCC) accounts for 3-5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Receptor ErbB-3/metabolismo , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neuregulina-1/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Receptor tyrosine kinase activation contributes to cell viability during cytotoxic therapy. The novel broad spectrum receptor tyrosine kinase inhibitor, SU11248, inhibits vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor, c-kit, and fetal liver tyrosine kinase 3. In this study, we maintained SU11248 plasma levels beyond the completion of radiotherapy to determine whether tumor regrowth can be delayed. The antiangiogenic effects of SU11248 were demonstrated using human umbilical vein endothelial cells in vitro. Apoptosis increased and clonogenic survival decreased when SU11248 was used in combination with radiation from 0 to 6 Gy on endothelial cells. In vivo tumor growth delay was increased in C57B6J mice with Lewis lung carcinoma or glioblastoma multiform (GL261) hind limb tumors. Mice were treated with daily i.p. injections (40 mg/kg) of SU11248 during 7 days of radiation treatment (21 Gy). Combined treatment with SU11248 and radiation significantly reduced tumor volume as compared with either treatment alone. Concomitant reduction in vasculature was confirmed using the dorsal vascular window model. The vascular length established using images taken from a consistent quadrant in the window show the combination therapy was more effective in destroying tumor vasculature than either treatment alone. SU11248 maintenance administration beyond the completion of radiotherapy results in prolongation of tumor control. In summary, SU11248 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. Moreover, inhibition of angiogenesis well beyond radiation therapy may be a promising treatment paradigm for refractory human neoplasms.
Assuntos
Indóis/farmacologia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/radioterapia , Neovascularização Patológica/tratamento farmacológico , Pirróis/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/radioterapia , Terapia Combinada , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/efeitos da radiação , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/irrigação sanguínea , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , SunitinibeRESUMO
A sound rationale exists for antibody targeting of the MET receptor tyrosine kinase, but therapeutic agents that can broadly block HGF ligand binding and exon 14-mutated or amplified MET to induce receptor degradation have yet to be reported. Here we report the identification of several MET monoclonal antibodies (mAb) that block MET-dependent signaling and tumor growth. In particular, the MET mAb KTN0073 and KTN0074 bind the Sema/PSI domain, at overlapping but distinct epitopes, preventing HGF interaction with MET and triggering receptor ubiquitination and degradation. Notably, both mAbs also triggered degradation of oncogenic MET exon 14 mutants, which propagate more durable MET signals due to a defect in receptor degradation. Mechanistic investigations showed that both mAbs engaged a pathway distinct from HGF-induced receptor degradation and protease-mediated shedding, independently of signaling driven by the exon 14-encoded sequences in the intracellular juxtamembrane region of the MET receptor. Grafting the mAb variable regions onto the IgG2 constant region dramatically enhanced the tumor inhibitory activities of KTN0073 but not KTN0074, suggesting a specific influence of antibody isotype of the epitopes for these two MET mAbs. Overall, our results highlight KTN0073 as a novel IgG2-based MET mAb that acts through exon 14-independent mechanisms to degrade the MET receptor, potentially offering a therapeutic tool to treat a broader range of human tumors where MET is exon 14 mutated or amplified. Cancer Res; 76(19); 5788-97. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoglobulina G/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Loss of the DNA-dependent protein kinase (DNA-PK) results in increased sensitivity to ionizing radiation due to inefficient repair of DNA double-strand breaks. Overexpression of DNA-PK in tumor cells conversely results in resistance to ionizing radiation. It is therefore possible that inhibition of DNA-PK will enhance the preferential killing of tumor cells by radiotherapy. Available inhibitors of DNA-PK, like wortmannin, are cytotoxic and stop the cell cycle because they inhibit phoshatidylinositol-3-kinases at 100-fold lower concentrations required to inhibit DNA-PK. In an effort to develop a specific DNA-PK inhibitor, we have characterized SU11752, from a three-substituted indolin-2-ones library. SU11752 and wortmannin were equally potent inhibitors of DNA-PK. In contrast, inhibition of the phoshatidylinositol-3-kinase p110gamma required 500-fold higher concentration of SU11752. Thus, SU11752 was a more selective inhibitor of DNA-PK than wortmannin. Inhibition kinetics and a direct assay for ATP binding showed that SU11752 inhibited DNA-PK by competing with ATP. SU11752 inhibited DNA double-strand break repair in cells and gave rise to a five-fold sensitization to ionizing radiation. At concentrations of SU11752 that inhibited DNA repair, cell cycle progression was still normal and ATM kinase activity was not inhibited. We conclude that SU11752 defines a new class of drugs that may serve as a starting point for the development of specific DNA-PK inhibitors.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA , Radiação IonizanteRESUMO
PURPOSE: The purpose of the following study was to investigate the safety and efficacy of the novel multitargeted indolinone receptor tyrosine kinase (RTK) inhibitor, SU11654, using a canine model of spontaneous tumors. This p.o. bioavailable compound exhibits potent inhibitory activity against members of the split kinase family of RTKs, including vascular endothelial growth factor receptor, platelet-derived growth factor receptor, Kit, and Flt-3, resulting in both direct antitumor and antiangiogenic activity. EXPERIMENTAL DESIGN: This was a Phase I trial in which successive cohorts of dogs with spontaneous tumors that had failed standard treatment regimens received escalating doses of SU11654 as oral therapy. Pharmacokinetics, toxicity, and tumor response were assessed. RESULTS: Fifty-seven dogs with a variety of cancers were enrolled; of these, 10 experienced progressive disease within the first 3 weeks. Measurable objective responses were observed in 16 dogs (including 6 complete responses), primarily in mast cell tumors (n = 11), mixed mammary carcinomas (n = 2), soft tissue sarcomas (n = 2), and multiple myeloma (n = 1), for an overall response rate of 28% (16 of 57). Stable disease of sufficient duration to be considered clinically meaningful (>10 weeks) was seen in an additional 15 dogs, for a resultant overall biological activity of 54% (31 of 57). CONCLUSIONS: This study provides the first evidence that p.o. administered kinase inhibitors can exhibit activity against a variety of spontaneous malignancies. Given the similarities of canine and human cancers with regard to tumor biology and the presence of analogous RTK dysregulation, it is likely that such agents will demonstrate comparable antineoplastic activity in people.
Assuntos
Doenças do Cão/tratamento farmacológico , Indóis/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/veterinária , Pirróis/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Indóis/administração & dosagem , Concentração Inibidora 50 , Masculino , Modelos Químicos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Pirróis/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Nus , Modelos Químicos , Transplante de Neoplasias , Fosforilação , Sunitinibe , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Troglitazone, a thiazolidinedione containing compound, was widely used to treat non-insulin dependent-diabetes. Unfortunately, troglitazone was associated with a sporadic liver toxicity that led to a cessation of its use clinically. Here we show that troglitazone induces a rapid and dose-dependent drop of mitochondrial membrane potential in liver HepG2 cells. The decrease in mitochondrial membrane potential induced by 100 microM troglitazone was completed after 5 min and similar in magnitude to that caused by carbonyl cyanide m-chloro phenylhydrazone. The troglitazone-induced loss of mitochondrial membrane potential preceded changes in cell permeability and cell count. In addition, troglitazone-induced a rise of intracellular calcium, subsequent to the drop in mitochondrial membrane potential, which was blocked by EGTA and the Na+/Ca2+ exchange inhibitor bepridil. Finally, application of 100 microM troglitazone for 24h to HepG2 cells resulted in activation of caspase 3. The results of this study shed light on the molecular mechanisms by which troglitazone can cause cytotoxicity.
Assuntos
Cromanos/farmacologia , Hipoglicemiantes/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Caspase 3 , Caspases/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Interpretação de Imagem Assistida por Computador , Indicadores e Reagentes , Potenciais da Membrana/efeitos dos fármacos , Microscopia de Fluorescência , Fixação de Tecidos , TroglitazonaRESUMO
Interaction between vascular endothelial growth factor (VEGF) and its cognate receptors, KDR/Flk-1 and Flt-1, of vascular endothelial cells is expected to induce an angiogenesis "switch" in tumors and other angiogenesis-associated diseases. SU5416, a selective inhibitor of the KDR/Flk-1 tyrosine kinase, is known to be a potent inhibitor of tumor angiogenesis. In this study, we first observed that SU5416 inhibited Flt-1 tyrosine kinase activity at similar doses, in addition to inhibiting KDR/Flk-1 tyrosine kinase activity in response to VEGF. SU5416 inhibited cell migration of human vascular endothelial cells expressing both Flt-1 and KDR in response to VEGF and also inhibited the cell migration in response to placenta growth factor (PIGF), a specific ligand for Flt-1. Chemotaxis of monocytes expressing only Flt-1 was also inhibited by SU5416 in a dose-dependent manner. Moreover, SU5416 was found to inhibit tyrosine kinase of Flt-1 in response to PIGF in vitro. And angiogenesis induced by PIGF in a Matrigel plug assay was inhibited by administration of SU5416. The antiangiogenic effects by this VEGF receptor-targeting compound appeared to be mediated through interference not only with KDR/Flk-1 but also with Flt-1. Cell migration of vascular endothelial cells and monocytic cells through Flt-1, thus, might play a key role in VEGF-induced tumor angiogenesis in concert with KDR/Flk-1.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Indóis/farmacologia , Pirróis/farmacologia , Células 3T3 , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia , Colágeno/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Endotélio Vascular/citologia , Humanos , Immunoblotting , Laminina/farmacologia , Masculino , Camundongos , Monócitos/metabolismo , Cadeias Pesadas de Miosina , Neovascularização Patológica , Miosina não Muscular Tipo IIB , Fosforilação , Fator de Crescimento Placentário , Proteínas da Gravidez/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/farmacologia , Transfecção , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The hepatocyte growth factor/scatter factor (HGF/SF) receptor, Met, mediates various cellular responses on activation with its ligand, including proliferation, survival, motility, invasion, and tubular morphogenesis. Met expression is frequently up-regulated in sarcomas and carcinomas. Experimental evidence suggests that Met activation correlates with poor clinical outcome and the likelihood of metastasis. Therefore, inhibitors of Met tyrosine kinase may be useful for the treatment of a wide variety of cancers that have spread from the primary site. We have discovered potent and selective pyrrole-indolinone Met kinase inhibitors and characterized them for their ability to inhibit HGF/SF-induced cellular responses in vitro. These compounds inhibit HGF/SF-induced receptor phosphorylation in a dose-dependent manner. They also inhibit the HGF/SF-induced motility and invasion of epithelial and carcinoma cells. Therefore, these compounds represent a class of prototype small molecules that selectively inhibit the Met kinase and could lead to identification of compounds with potential therapeutic utility in treatment of cancers.
Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Invasividade Neoplásica , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas c-met/química , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Protein kinases play a critical regulatory role in essentially every aspect of cell biology. Of the 518 known kinases, the most successful class for drug targeting is the receptor tyrosine kinase (RTK) family consisting of 58 distinct and diverse members. RTKs regulate a broad range of cellular functions, including proliferation, differentiation, survival, and apoptosis and have been intensively studied in development and cancer. Targeting of RTKs has resulted in many marketed small molecule and antibody-based drugs in a number of different solid tumors and hematological malignancies, and more recently in inflammatory diseases such as idiopathic pulmonary fibrosis. In this review, we discuss some of the RTKs in cancer in which drugs targeting the ErbB family (EGFR, HER2, and ErbB3) and KIT have had meaningful clinical benefit to cancer patients, RTKs' emerging role in regulating innate immunity, and the potential to explore targeting RTKs outside of oncology.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Inflamação/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/farmacologia , Feminino , Humanos , Inflamação/tratamento farmacológico , Masculino , Neoplasias/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
The effect of combining SU5416 with fractionated radiotherapy or with low molecular weight (LMW) heparin (dalteparin) was studied in U87 human glioblastoma xenografts in nude mice. SU5416 is antiangiogenic by a specific inhibition of the vascular endothelial growth factor receptor 2 (VEGFR-2), and heparins are assumed to bind VEGF. Both SU5416 (100 mg/kg every second day in 5 days) and 3 Gyx5 produced moderate, yet significant, growth inhibition. Tumors treated with concomitant irradiation and short-term SU5416 maintained a lower growth rate during regrowth than the other treatment groups (P=.007). Dalteparin (1000 IE/kg subcutaneously once a day) had no growth-inhibitory effect on its own, but when this LMW heparin was added to the SU5416 schedule, a significantly enhanced growth inhibition was obtained. VEGF protein content in tumors was not significantly altered by SU5416, but a significant decrease in VEGF levels was found in tumors treated with concomitant dalteparin and SU5416 compared with controls (P=.03). We conclude that: 1) an additive growth-inhibitory effect is obtained by combining SU5416 and fractionated radiotherapy; and 2) LMW heparin (dalteparin), in combination with SU5416, decreases the level of VEGF in tumors and increases the growth-inhibitory effect of SU5416.
Assuntos
Fracionamento da Dose de Radiação , Inibidores Enzimáticos/farmacologia , Fibrinolíticos/química , Heparina/química , Indóis/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Animais , Divisão Celular , Linhagem Celular Tumoral , Dalteparina/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Transplante de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Recent studies have demonstrated radiosensitization by inhibiting receptor tyrosine kinases (RTKs). Irradiation activates RTKs and their downstream prosurvival molecule, Akt. In this study, we investigated the mechanism by which SU6668, an inhibitor of RTKs involved in angiogenic pathways, enhances effects of irradiation. METHODS AND MATERIALS: Western blots were used to determine Akt phosphorylation. Clonogenic assays were performed to determine endothelial survival after combination of SU6668 and irradiation. This combination therapy was also tested in mouse models with Lewis lung carcinoma or glioblastoma multiforme (GL261) for inhibition of tumor growth and tumor vasculature by examining tumor volume, tumor vascular window, and blood flow. RESULTS: We found that SU6668 inhibited the Akt activation inducible by irradiation. Clonogenic survival of endothelial cells was decreased after the combined therapy compared with radiotherapy alone. In vivo studies demonstrated reduction of tumor vasculature and blood flow. In addition, 21 Gy in 7 fractions given concurrently with SU6668 resulted in tumor growth delay compared to either treatment alone. CONCLUSION: These data suggest that the combination therapy was more effective in destroying tumor vasculature than either treatment alone. SU6668 augments tumor-suppressive effects of radiotherapy in Lewis lung carcinoma and GL261 xenographs, possibly through reducing the survival of tumor endothelium.
Assuntos
Endotélio Vascular/efeitos da radiação , Indóis/uso terapêutico , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Pirróis/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/radioterapia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxindóis , Fosforilação , Propionatos , Proteínas Proto-Oncogênicas c-aktRESUMO
PURPOSE: Picoplatin is a new generation platinum designed to overcome platinum resistance. The goal of this study was to assess picoplatin anti-tumor activity and measure various cellular parameters in small-cell lung cancer (SCLC) cells resistant to cell killing by cisplatin and carboplatin. METHODS: We developed several platinum-resistant SCLC cell lines to evaluate picoplatin activity and drug resistance mechanisms in vitro. Drug cytotoxicity was measured by MTS assay. Total cellular platinum accumulation was measured by inductively coupled plasma mass spectrometry (ICP-MS). Whole genome gene expression profiling was carried out by microarray analysis. RESULTS: Picoplatin retained significant cytotoxic activity in platinum-resistant SCLC lines compared to cisplatin and carboplatin. Cellular picoplatin accumulation in platinum-resistant and parental cells was high relative to levels of cellular platinum found in the same cell lines after cisplatin or carboplatin treatment. Gene expression analyses revealed substantial differences in gene expression and highlighted specific annotation clusters in carboplatin-resistant cells. In addition, a similar gene expression pattern was observed in picoplatin-treated carboplatin-resistant and parental cells. CONCLUSIONS: Our study demonstrates that picoplatin can overcome carboplatin and cisplatin resistance. The results suggest decreased platinum accumulation as a potential mechanism of platinum resistance in SCLC cells, provide candidate markers (e.g. several genes in the Hox, glutathione biosynthetic process, and MAGE families) that may serve as signatures for platinum resistance, support distinct effects of picoplatin on SCLC cells compared to other platinums, and provide a rationale to develop picoplatin for the treatment of recurrent SCLC following initial therapy with cisplatin or carboplatin.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Linhagem Celular Tumoral , Análise por Conglomerados , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Neoplasias Pulmonares , Carcinoma de Pequenas Células do PulmãoRESUMO
Tumor cells can grow in an anchorage-independent manner. This is mediated in part through survival signals that bypass normal growth restraints controlled by integrin cell surface receptors. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that associates with integrins and modulates various cellular processes including growth, survival, and migration. As increased FAK expression and tyrosine phosphorylation are associated with tumor progression, inhibitors of FAK are being tested for anti-tumor effects. Here, we analyze PND-1186, a substituted pyridine reversible inhibitor of FAK activity with a 50% inhibitory concentration (IC50) of 1.5 nM in vitro. PND-1186 has an IC50 of ~100 nM in breast carcinoma cells as determined by anti-phospho-specific immunoblotting to FAK Tyr-397. PND-1186 did not alter cSrc or p130Cas tyrosine phosphorylation in adherent cells, yet functioned to restrain cell movement. Notably, 1.0 µM PND-1186 (>5-fold above IC50) had limited effects on cell proliferation. However, under non-adherent conditions as spheroids and as colonies in soft agar, 0.1 µM PND-1186 blocked FAK and p130Cas tyrosine phosphorylation, promoted caspase-3 activation, and triggered cell apoptosis. PND-1186 inhibited 4T1 breast carcinoma subcutaneous tumor growth correlated with elevated tumor cell apoptosis and caspase 3 activation. Addition of PND-1186 to the drinking water of mice was well tolerated and inhibited ascites- and peritoneal membrane-associated ovarian carcinoma tumor growth associated with the inhibition of FAK Tyr-397 phosphorylation. Our results with low-level PND-1186 treatment support the conclusion that FAK activity selectively promotes tumor cell survival in three-dimensional environments.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Aminopiridinas/química , Animais , Antineoplásicos/química , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Tirosina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidoresRESUMO
Tumor metastasis is a leading cause of cancer-related death. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase recruited to integrin-mediated matrix attachment sites where FAK activity is implicated in the control of cell survival, migration, and invasion. Although genetic studies support the importance of FAK activity in promoting tumor progression, it remains unclear whether pharmacological FAK inhibition prevents tumor metastasis. Here, we show that the FAK inhibitor PND-1186 blocks FAK Tyr-397 phosphorylation in vivo and exhibits anti-tumor efficacy in orthotopic breast carcinoma mouse tumor models. PND-1186 (100 mg/kg intraperitoneal, i.p.) showed promising pharmacokinetics (PK) and inhibited tumor FAK Tyr-397 phosphorylation for 12 hours. Oral administration of 150 mg/kg PND-1186 gave a more sustained PK profile verses i.p., and when given twice daily, PND-1186 significantly inhibited sygeneic murine 4T1 orthotopic breast carcinoma tumor growth and spontaneous metastasis to lungs. Moreover, low-level 0.5 mg/ml PND-1186 ad libitum administration in drinking water prevented oncogenic KRAS- and BRAF-stimulated MDA-MB-231 breast carcinoma tumor growth and metastasis with inhibition of tumoral FAK and p130Cas phosphorylation. Although PND-1186 was not cytotoxic to cells in adherent culture, tumors from animals receiving PND-1186 exhibited increased TUNEL staining, decreased leukocyte infiltrate and reduced tumor-associated splenomegaly. In vitro, PND-1186 reduced tumor necrosis factor-a triggered interleukin-6 cytokine expression, indicating that FAK inhibition may impact tumor progression via effects on both tumor and stromal cells. As oral administration of PND-1186 also decreased experimental tumor metastasis, PND-1186 may therefore be useful clinically to curb breast tumor progression.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Administração Oral , Aminopiridinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Substrato Associada a Crk/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Fosforilação/efeitos dos fármacos , Tirosina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The angiogenic vascular endothelial growth factor (VEGF) is believed to play a critical role in endothelial cell proliferation, differentiation, and sprouting. Small molecules that selectively inhibit the VEGF receptor-associated tyrosine kinase activities of Flk-1 (KDR) and Flt-1 have been developed. These agents, a prototype being SU5416, have effects on the proliferation of cultured endothelial cells, constrain angiogenesis in vivo, and have been proposed as antitumor drugs. Although SU5416 inhibits in vivo angiogenesis, it is not clear which of the complex processes leading to angiogenesis are impacted by VEGF receptor-associated tyrosine kinase inhibition. We utilized SU5416 and a microvascular endothelial cell line derived from mouse heart (SMHEC4) to specifically examine the role of VEGF receptor-associated tyrosine kinase activity on in vitro models of angiogenesis. We characterized spheroid formation and sprouting, a new model of angiogenesis, in this stable cell line. SU5416 inhibits (approximately 50%) VEGF (50 ng/ml) stimulated and basal DNA synthesis of SMHEC4 cultured in monolayer. SU5416 does not prevent the aggregation and organization of SMHEC4 into tri-dimensional spheroids. CD31, a marker of differentiated endothelial cells, is negligibly expressed in monolayer cultures but highly expressed in SMHEC4 spheroids. The content and biochemical characteristics of spheroidal CD31 are unaltered by SU5416. SU5416 also does not prevent the spontaneous and rapid (approximately 3-h) alignment into cords by SMHEC4 on Matrigel. These two models suggest that the organization and differentiation of endothelial cells is independent of VEGF receptor-associated tyrosine kinase signaling. SMHEC4 spheroids embedded in collagen gels spontaneously and rapidly (approximately 6 h) sprout capillary-like projections and subsequently (1-2 days) form complex self-anastomosing networks. In addition, VEGF (50 ng/ml) markedly stimulates sprouting of capillary-like projections from SMHEC4 spheroids. Both the spontaneous and the VEGF-stimulated sprouting are nearly eliminated by SU5416. This demonstrates that VEGF receptor-associated tyrosine kinase activity is essential to the formation of capillary-like structures from SMHEC4 spheroids. Overall, these observations demonstrate that (a) the spheroid sprouting model is appropriate for the study of angiogenesis since it appears to recapitulate many of its steps and (b) SU5416 can inhibit endothelial cell proliferation and sprouting without impacting the organization and differentiation of endothelial cells.
Assuntos
Inibidores da Angiogênese/farmacologia , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/patologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Linfocinas/metabolismo , Neovascularização Patológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Colágeno/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Proteínas da Matriz Extracelular/biossíntese , Fator 2 de Crescimento de Fibroblastos/metabolismo , Laminina/farmacologia , Camundongos , Cadeias Pesadas de Miosina , Miosina não Muscular Tipo IIB , Proteoglicanas/farmacologia , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular , Cordão Umbilical/citologia , Cordão Umbilical/patologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Protein tyrosine phosphatases (PTPs) are important in the regulation of signal transduction processes. Certain enzymes of this class are considered as potential therapeutic targets in the treatment of a variety of diseases such as diabetes, inflammation, and cancer. However, many PTP inhibitors identified to date are peptide-based and contain a highly charged phosphate-mimicking component. These compounds usually lack membrane permeability and this limits their utility in the inhibition of intracellular phosphatases. In the present study, we have used structure-based design and modeling techniques to explore catalytic-site directed, reversible inhibitors of PTPs. Employing a non-charged phosphate mimic and non-peptidyl structural components, we have successfully designed and synthesized a novel series of trifluoromethyl sulfonyl and trifluoromethyl sulfonamido compounds as PTP inhibitors. This is the first time that an uncharged phosphate mimic is reported in the literature for general, reversible, and substrate-competitive inhibition of PTPs. It is an important discovery because the finding may provide a paradigm for the development of phosphatase inhibitors that enter cells and modify signal transduction.