RESUMEN
PURPOSE: Erythropoiesis-stimulating agents (ESAs) are recommended for treating chemotherapy-induced anemia in breast cancer patients. Reduced survival rates in ESAs-treated patients have been reported, possibly due to thromboembolic complications, however the exact mechanism remains obscure. The principal activator of blood coagulation in cancer is tissue factor (TF). There are data that erythropoietin receptor (EPO-R) is expressed in tumor cells. The purpose of this study was to evaluate the expression of EPO-R and TF in loco in breast cancer. METHODS: The expression of EPO-R and TF was investigated in 24 invasive breast carcinoma specimens. Immunohistochemical (IHC) methodologies according to ABC technique and double-staining IHC procedure were employed utilizing antibodies against EPO-R and TF. RESULTS: Expression of EPO-R and TF was demonstrated in the tumor cells in all breast cancer specimens. No staining for EPO-R and TF was visualized in normal breast tissue. Double staining studies revealed co-expression of both EPO-R and TF in breast cancer cells and endothelial cells. CONCLUSIONS: EPO-R and TF expression and their coexpression in breast cancer cells suggest a possibility that EPO-R might be responsible for some adverse effects and reduced survival observed in ESAs-treated breast cancer patients with anemia, possibly due to the interaction with TF. Further experimental studies are warranted to determine the role of both EPO-R and TF in the treatment with ESAs of breast cancer patients with chemotherapy-induced anemia.
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Neoplasias de la Mama/química , Receptores de Eritropoyetina/análisis , Tromboplastina/análisis , Anemia/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Femenino , Humanos , Inmunohistoquímica , Tromboembolia/etiologíaRESUMEN
We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.
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Carbocianinas/análisis , Factor VIIa/análisis , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Imagen Óptica/métodos , Tromboplastina/inmunología , Clorometilcetonas de Aminoácidos/química , Animales , Carbocianinas/química , Células Cultivadas , Factor VIIa/química , Xenoinjertos/inmunología , Humanos , Ratones , Neoplasias/inmunología , Neoplasias/patología , Paclitaxel/químicaRESUMEN
In gastric cancer, hemostatic system components contribute to cancer progression, as activation of factor X (FX) was observed. The protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex inhibits factor Xa proteolytic activity. The purpose of this study was to determine the distribution of ZPI and PZ in relation to FX, and prothrombin fragment (F1 + 2), a standard marker for blood coagulation activation, in human gastric cancer tissue. ABC procedures and a double staining method employed polyclonal antibodies against PZ, FX, and F1 + 2 and a monoclonal antibody against ZPI. In situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either PZ or ZPI mRNAs. FX and components of PZ/ZPI coagulation inhibitory system were observed in cancer cells. F1 + 2 was observed in gastric cancer cells as well. Double staining studies revealed FX/PZ, FX/ZPI, and PZ/ZPI co-localization on gastric cancer cells. ISH studies demonstrated the presence of PZ mRNA and ZPI mRNA in gastric cancer cells indicating induced synthesis of these proteins. The co-localization of PZ/ZPI and FX in gastric cancer cells indicates in loco that these proteins may play a role in anticoagulant events at the tumor tissue.
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Adenocarcinoma/genética , Proteínas Sanguíneas/genética , Factor Xa/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Serpinas/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Coagulación Sanguínea , Proteínas Sanguíneas/metabolismo , Progresión de la Enfermedad , Factor Xa/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Protrombina/genética , Protrombina/metabolismo , ARN Mensajero/metabolismo , Serpinas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs). TF plays a central role in cancer angiogenesis, growth, and metastasis and, as such, is a target for therapy and drug delivery. TF is the cognate receptor of factor VIIa (fVIIa). We have coupled PTX (paclitaxel, also named Taxol) with a tripeptide, phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) and conjugated it with fVIIa. The key aim of the work is to evaluate the antiangiogenic effects of PTX-FFRck-fVIIa against a PTX-resistant breast cancer cell line. Matrigel mixed with VEGF and MDA-231 was injected subcutaneously into the flank of athymic nude mice. Animals were treated by tail vein injection of the PTX-FFRck-fVIIa conjugate, unconjugated PTX, or PBS. The PTX-FFRck-fVIIa conjugate significantly reduces microvessel density in matrigel (p < 0.01-0.05) compared to PBS and unconjugated PTX. The breast cancer lung metastasis model in athymic nude mice was developed by intravenous injection of MDA-231 cells expressing luciferase. Animals were similarly treated intravenously with the PTX-FFRck-fVIIa conjugate or PBS. The conjugate significantly inhibits lung metastasis as compared to the control, highlighting its potential to antagonize angiogenesis in metastatic carcinoma. In conclusion, PTX conjugated to fVIIa is a promising therapeutic approach for improving selective drug delivery and inhibiting angiogenesis.
RESUMEN
INTRODUCTION: Several hemostatic system components, including factor X (FX), contribute to cancer progression. The Protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex directly inhibits factor Xa proteolytic activity. The aim of this study was to determine the antigenic distribution of ZPI and PZ, in relation to FX, as well as indicators of blood coagulation activation (F1+2 and fibrin) in human colon cancer tissue. MATERIALS & METHODS: Studies were performed on human colon cancer fragments. Immunohistochemical (IHC) ABC procedures and double staining method employed polyclonal antibodies against PZ, FX, F1+2 and monoclonal antibodies against ZPI and fibrin. In-situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either FX, PZ or ZPI mRNAs. RESULTS: Expression of FX, PZ and ZPI in association with colon cancer cells was observed by IHC. Moreover, the presence of both F1+2 and fibrin in association with colon cancer cells was found, which indicates that blood coagulation activation proceeds extravascularly at the tumor site. Furthermore, expression of FX and PZ was visualized in association with endothelial cells. In turn, colon cancer-associated macrophages were characterized by FX , PZ and ZPI presence. The double staining studies revealed strong FX/PZ, FX/ZPI, as well as PZ/ZPI co-localization on colon cancer cells. ISH studies revealed the presence of FX mRNA, PZ mRNA and ZPI mRNA in colon cancer cells indicating induced synthesis of these proteins. CONCLUSIONS: The localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. Strong co-localization of PZ/ZPI and FX in cancer cells, and the presence of the mRNAs encoding the proteins, suggests their role in the tumor's biology. However, the presence of F1+2 and fibrin at the colon cancer site also suggests that the regulation of FXa by the PZ/ZPI complex at this site is incomplete.
Asunto(s)
Factores de Coagulación Sanguínea/metabolismo , Proteínas Sanguíneas/metabolismo , Neoplasias del Colon/metabolismo , Hemostasis , Serpinas/metabolismo , Factor X , Humanos , Células Tumorales CultivadasRESUMEN
INTRODUCTION: NSCLC progression is often associated with VTE. Activation of factor X is an important step in blood coagulation activation in cancer patients. PZ)/ZPI contribute to direct factor Xa inhibition, and ZPI - attenuates factors IXa and XIa activity. The role of the PZ/ZPI in NSCLC is obscure. The aim of the study was to localize ZPI and PZ in NSCLC tissue in relation to factors X, IX and XI, as well as indicators of blood coagulation activation: prothrombin fragment F1+2 (F1+2) and fibrin. MATERIAL & METHODS: Immunohistochemical studies were performed on surgical NSCLC specimens employing antibodies against ZPI, PZ, coagulation factors X, IX, XI, as well as fibrinogen, F1+2 and fibrin. A semiquantitative analysis (acc. to immunoreactive score-IRS) was conducted. RESULTS: Medium expression of ZPI(IRS=6.5), together with weak expression of PZ(IRS=4), was observed in cancer cells. Strong or medium staining for factors IX, X, and XI(IRS=8-9) was revealed in cancer cells. Fibrinogen(IRS=10) and fibrin(IRS=8) were demonstrated in tumor stroma and cancer cells. F1+2(IRS=10) was localized in NSCLC cells. Endothelial cells (ECs) and tumor infiltrating macrophages (TAMs) were characterized by a positive staining for ZPI and PZ. CONCLUSIONS: ZPI and PZ expression in NSCLC cells, ECs and TAMs may suggest a role for PZ/ZPI in the anticoagulant mechanisms at the tumor site. The presence of F1+2 and fibrin, along with a disproportional expression of ZPI and PZ, might point to impaired function of the coagulation inhibitory system in NSCLC tissue.
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Factores de Coagulación Sanguínea/metabolismo , Proteínas Sanguíneas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Serpinas/metabolismo , Humanos , Células Tumorales CultivadasAsunto(s)
Proteínas Sanguíneas , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama , ARN Mensajero/biosíntesis , Proteínas Sanguíneas/biosíntesis , Proteínas Sanguíneas/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Técnicas de Laboratorio Clínico , Factor Xa/genética , Factor Xa/metabolismo , Femenino , Humanos , Hibridación in Situ , ARN Mensajero/análisis , Serpinas/genética , Serpinas/metabolismoRESUMEN
BACKGROUND: Colon cancer (CC) is frequently complicated by thromboembolic episodes. Thrombin plays a role in angiogenesis and among others induces the synthesis of vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2). The aim of this study was to assess the expression of prothrombin fragment F1+2 (F1+2), a byproduct in thrombin generation (indicating the presence of thrombin), in relation to the presence of VEGFR-2-bound VEGF (VEGF:VEGFR-2), as an indicator of VEGFR-2 activation in human CC tissue. MATERIALS AND METHODS: Immunohistochemical ABC and double staining studies were performed using antibodies against F1+2 and VEGF:VEGFR-2 in 59 specimens obtained from CC patients. RESULTS: Medium and high expression of both F1+2 and VEGF:VEGF2 in association with CC cells and endothelial cells was demonstrated. Moreover, coexpression of F1+2 and VEGF:VEGFR-2 was observed in the cells. CONCLUSION: The results may suggest a possible functional interaction between thrombin and VEGF-R2 stimulation in human CC in vivo.
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Neoplasias del Colon/metabolismo , Fragmentos de Péptidos/metabolismo , Protrombina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adulto , Neoplasias del Colon/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Transporte de ProteínasRESUMEN
Activation of blood coagulation, a phenomenon frequently observed in breast and colon cancer patients, contributes to tumour progression. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF), while tissue factor pathway inhibitor (TFPI) is the main inhibitor of the TF-dependent pathway of blood coagulation. Previous immunohistochemical studies revealed no expression of TFPI in human cancer cells. The aim of the study was to evaluate the expression of TFPI protein and mRNA in breast and colon cancer tissues. A total of 108 cancer tissues (from primary tumours and metastatic lymph nodes) were obtained from 87 patients during surgical treatment. Immunohistochemical studies using a polyclonal anti-TFPI antibody were performed including a semiquantitative analysis. The in situ hybridisation method employed single-stranded DNA oligonucleotide (probe sequence: 5'Biotin-CCACCATACTTGAAACGTTCACACT-Biotin3') directed against TFPI mRNA. Strong or medium expression of TFPI protein was observed in cancer cell bodies in all breast cancers and in most (39/66 cases) colon cancers examined. Weaker expression of TFPI was detected in cancer cells localised in lymph node metastatic foci of breast cancer. Endothelial cells were also TFPI-positive. TFPI mRNA was demonstrated in all cases of breast and in approximately 80% cases of colon cancer cells. TFPI mRNA and protein are present in association with colon and breast cancer cells, suggesting that the protein may play a role in cancer biology. The presence of TFPI in association with breast cancer cells localised in regional lymph nodes may indicate its role in lymphatic spread.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Neoplasias del Colon/química , Lipoproteínas/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/secundario , Neoplasias del Colon/genética , Neoplasias del Colon/secundario , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Lipoproteínas/genética , Metástasis Linfática , Masculino , Estadificación de Neoplasias , ARN Mensajero/análisisRESUMEN
AIM: To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells. METHODS: Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS: Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin. CONCLUSION: These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.
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Fibroblastos/metabolismo , Hígado/metabolismo , Proteína C/metabolismo , Antígenos CD/metabolismo , Células Cultivadas , Colágeno/metabolismo , Receptor de Proteína C Endotelial , Fibroblastos/efectos de los fármacos , Humanos , Proteína C/farmacología , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Trombomodulina/metabolismoRESUMEN
INTRODUCTION: Colorectal cancer (CRC) is often complicated by thromboembolic episodes. It has been recognized that blood coagulation proteins play a role in cancer progression. An important inhibitory mechanism is provided by the protein C (PC) system consisting of PC, protein S (PS) and thrombomodulin (TM). Recently, novel biological activities have been ascribed to the PC system that do not relate to their hemostatic functions, eg. in angiogenesis, apoptosis and inflammation. OBJECTIVES: The purpose of the study was to elucidate the solid phase interactions between CRC tissue and components of the PC system that may contribute to tumor progression. MATERIAL AND METHODS: CRC tissues were obtained at surgical resection during treatment of 66 patients. Immunohistochemical studies were performed using polyclonal antibodies against PC, PS and TM. A semiquantitative analysis of the protein expression was also performed. RESULTS: Weak expression of PC was observed in cancer cells of two-thirds of the specimens examined, while in 3/66 cases there was no staining for PC in cancer cells. One fourth of CRCs exhibited strong expression of PC. The presence of PS was demonstrated in 64/66 cases of CRC. However, its expression was irregular in terms of intensity of staining and percentage of cancer cells exhibiting protein expression. Weak expression of TM was demonstrated in two thirds of the cases examined, while a strong TM staining was revealed in one third of colon cancers. CONCLUSION: Heterogeneous expression of the PC system components in CRC tissue may point to their biological activity modulating tumor growth.
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Anticoagulantes/metabolismo , Neoplasias Colorrectales/metabolismo , Proteína C/metabolismo , Proteína S/metabolismo , Trombomodulina/metabolismo , Adulto , Neoplasias Colorrectales/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
Bovine aprotinin, a reversible inhibitor of plasmin and kallikrein, has been clinically approved for over two decades to prevent perioperative blood loss during cardiac surgery. However, because of postoperative renal dysfunction in thousands of these patients, aprotinin was voluntarily withdrawn from the market. Our earlier studies indicated that a R24K mutant of the first Kunitz-type domain of human tissue factor pathway inhibitor-2 (R24K KD1) exhibited plasmin inhibitory activity equivalent to aprotinin in vitro. In this study, we compared the effects on renal function after infusion of aprotinin and recombinant R24K KD1 in chronically instrumented, conscious rats. Aprotinin-infused rats exhibited statistically significant decreases in glomerular filtration rate and effective renal plasma flow relative to rats infused with phosphate-buffered saline (PBS) or R24K KD1 dissolved in PBS. In addition, aprotinin-treated rats exhibited marked increases in serum creatinine, blood urea nitrogen, urinary protein, and effective renal vascular resistance, whereas these renal parameters remained essentially unchanged in vehicle and R24K KD1-treated rats for a one-week period. Moreover, with use of a highly sensitive apoptosis detection assay, a significant increase in the rate of early and late apoptotic events in renal tubule cells occurred in aprotinin-treated rats relative to R24K KD1-treated rats. In addition, histological examination of the rat kidney revealed markedly higher levels of protein reabsorption droplets in the aprotinin-infused rats. Our data collectively provide suggestive evidence that R24K KD1 does not induce the renal dysfunction associated with aprotinin, and may be an effective clinical alternative to aprotinin as an antifibrinolytic agent in cardiac surgery.
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Aprotinina/efectos adversos , Glicoproteínas/efectos adversos , Riñón/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Aprotinina/administración & dosificación , Aprotinina/genética , Bovinos , Escherichia coli/genética , Femenino , Glicoproteínas/administración & dosificación , Glicoproteínas/genética , Humanos , Riñón/patología , Pruebas de Función Renal , Microscopía Confocal , Mutación , Ratas , Ratas Long-Evans , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/genética , TransfecciónRESUMEN
Tissue factor pathway inhibitor-2 (TFPI-2) is a serine proteinase inhibitor that induces caspase-mediated apoptosis when offered to a variety of tumor cells. In order to investigate the mechanism of TFPI-2-induced apoptosis, we initially studied the uptake and trafficking of TFPI-2 by HT-1080 cells. Exogenously offered TFPI-2 was rapidly internalized and distributed in both the cytosolic and nuclear fractions. Nuclear localization of TFPI-2 was also detected in a variety of endothelial cells constitutively expressing TFPI-2. Nuclear localization of TFPI-2 required a NLS sequence located in its Lys/Arg-rich C-terminal tail comprising residues 191-211, as a TFPI-2 construct lacking the C-terminal tail failed to localize to the nucleus. Complexes of TFPI-2 and importin-alpha were co-immunoprecipitated from cell lysates of HT-1080 cells either offered or overexpressing this protein, providing evidence that TFPI-2 was shuttled to the nucleus by the importin system. Our results provide the initial description of TFPI-2 internalization and translocation to the nucleus in a number of cells.
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Núcleo Celular/metabolismo , Glicoproteínas/metabolismo , Carioferinas/metabolismo , Aorta , Apoptosis , Capilares/metabolismo , Línea Celular , Línea Celular Tumoral , Endotelio Vascular/metabolismo , Fibrosarcoma , Glicoproteínas/genética , Humanos , Riñón/embriología , Cinética , Microscopía Confocal , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Piel/irrigación sanguínea , TransfecciónRESUMEN
Human TFPI-2 is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor. We previously demonstrated that a human fibrosarcoma cell line, HT-1080, does not express TFPI-2, but genetic restoration of TFPI-2 expression in these cells markedly inhibited their growth and metastasis in vivo. In the present study, either full-length recombinant TFPI-2, or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide and acridine orange staining, fluorescence activated cell sorting, immunoblotting and gene expression profiling. R24K KD1 induced apoptosis in 69% of HT-1080 cells in a 48 h period compared to 39% for TFPI-2, while a KD1 preparation lacking a reactive site arginine/lysine residue (R24Q KD1) produced only an 18% apoptosis rate, suggesting that the observed apoptosis was related to proteinase inhibition. Immunoblotting experiments indicated increased caspase 3 and 9 activation, up-regulation of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2 protein. Finally, microarray analyses of R24K KD1-treated cells indicated elevated expression of several pro-apoptotic genes and under-expression of anti-apoptotic genes. Collectively, our results demonstrate that treatment of HT-1080 cells exogenously with either TFPI-2 or R24K KD1 activates caspase-mediated, pro-apoptotic signaling pathways resulting in apoptosis.
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Apoptosis/efectos de los fármacos , Caspasas/fisiología , Glicoproteínas/fisiología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Línea Celular Tumoral , Neoplasias del Colon/patología , Regulación hacia Abajo , Fibrosarcoma/patología , Citometría de Flujo , Glicoproteínas/genética , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/patología , Proteína X Asociada a bcl-2/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.
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Antineoplásicos/administración & dosificación , Compuestos de Bencilideno/administración & dosificación , Factor VIIa/administración & dosificación , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Piperidonas/administración & dosificación , Tromboplastina/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Endocitosis , Humanos , Ratones , Neoplasias Experimentales/irrigación sanguínea , Conejos , Tromboplastina/análisis , Tromboplastina/fisiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignancy is associated with alterations in the hemostatic system that present as thromboembolic or bleeding complications. Antineoplastic treatment further escalates blood coagulation and fibrinolytic abnormalities. Moreover, hemostatic system inhibitors play a role in tissue maintenance or, contrarily, contribute to cancer progression. The inhibitors regulate migration, proliferation, apoptosis, angiogenesis, and distant metastases formation, as well as interfere with host defense system mechanisms. They exhibit different functions depending on tumor type, histologic grade, and clinical stage of the disease. The activity of coagulation inhibitors underlies the pathomechanisms of some complications resulting from therapeutic procedures, such as radiation injury to normal tissues. Because coagulation activation is widely recognized to influence cancer growth and distant dissemination, numerous attempts were made to introduce various forms of coagulation inhibitors to antineoplastic treatment. This review summarizes up-to-date information on preclinical and clinical benefits and pitfalls of hemostatic system inhibitors administration in cancer, with special emphasis on tumor biology and prophylaxis and treatment of various complications observed in the course of malignant disease.
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Anticoagulantes/uso terapéutico , Fibrinolíticos/uso terapéutico , Hemostasis , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Anticoagulantes/sangre , Anticoagulantes/farmacología , Fibrinolíticos/sangre , Fibrinolíticos/farmacología , Humanos , Neoplasias/complicacionesRESUMEN
Tissue factor pathway inhibitor-2 (TFPI-2), a member of the Kunitz-type serine proteinase inhibitor family, is a structural homologue of tissue factor pathway inhibitor (TFPI). The expression of TFPI-2 in tumors is inversely related to an increasing degree of malignancy, which may suggest a role for TFPI-2 in the maintenance of tumor stability and inhibition of the growth of neoplasms. TFPI-2 inhibits the tissue factor/factor VIIa (TF/VIIa) complex and a wide variety of serine proteinases including plasmin, plasma kallikrein, factor XIa, trypsin, and chymotrypsin. Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers and cancer cell lines was widely documented to be responsible for diminished expression of mRNA encoding TFPI-2 and decreased or inhibited synthesis of TFPI-2 protein during cancer progression. Furthermore, an aberrantly spliced variant of TFPI-2 mRNA (designated asTFPI-2) was detected, which represents an untranslated form of TFPI-2. The levels of asTFPI-2 were very low or undetectable in normal cells but markedly upregulated in neoplastic tissue. TFPI-2 functions in the maintenance of the stability of the tumor environment and inhibits invasiveness and growth of neoplasms, as well as metastases formation. TFPI-2 has also been shown to induce apoptosis and inhibit angiogenesis, which may contribute significantly to tumor growth inhibition. Restoration of TFPI-2 expression in tumor tissue inhibits invasion, tumor growth, and metastasis, which creates a novel possibility of cancer patient treatment. However, more information is still needed to define the precise role of TFPI-2 in human tumor biology.
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Glicoproteínas/genética , Glicoproteínas/metabolismo , Neoplasias/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Apoptosis , Cromosomas Humanos Par 7/genética , Progresión de la Enfermedad , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Glicoproteínas/química , Humanos , Neoplasias/genética , Neoplasias/patología , Neovascularización Patológica , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genéticaRESUMEN
BACKGROUND: Previous studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter. In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2). RESULTS: Nucleotide sequence analyses indicated that asTFPI-2 consists of complete exons II and V, fused with several nucleotides derived from exons III and IV, as well as six nucleotides derived from intron C. 5'- and 3'-RACE analyses of total RNA amplified exclusively the wild-type TFPI-2 transcript, indicating that asTFPI-2 lacks either a 5'-untranslated region (UTR) or a 3'-poly (A)+ tail. Quantitative real-time RT-PCR analyses revealed that several human tumor cells contain 4 to 50-fold more copies of asTFPI-2 in comparison to normal cells. In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells. CONCLUSION: Our studies reveal the existence of a novel, aberrantly-spliced TFPI-2 transcript predominantly expressed in tumor cells and provides suggestive evidence for an additional mechanism for tumor cells to down-regulate TFPI-2 protein expression enhancing their ability to degrade the extracellular matrix.
Asunto(s)
Glicoproteínas/genética , Proteínas de Neoplasias/genética , Empalme del ARN , Regiones no Traducidas 5'/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Dactinomicina/farmacología , Exones/genética , Glicoproteínas/biosíntesis , Glicoproteínas/química , Semivida , Humanos , Intrones/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Especificidad de Órganos , Poli A/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba , Vísceras/metabolismoRESUMEN
OBJECTIVE: The purpose of this study is to investigate the expression and regulation of type-2 tissue factor pathway inhibitor (TFPI-2) in endothelial cells, as well as the regulation of human endothelial cell (EC) function by TFPI-2. METHODS AND RESULTS: Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that vascular endothelial growth factor (VEGF) induced both time- and dose-dependent increase in TFPI-2 mRNA and protein expression in endothelial cells. TFPI-2 mRNA expression was also significantly upregulated by IL-1beta, and modestly increased by both tumor necrosis factor (TNF)-alpha and fibroblast growth factor (FGF)-2, but not placental growth factor (PlGF). VEGF upregulation of TFPI-2 was dramatically reduced by inhibition of the MEK pathway. Administration of TFPI-2 protein suppressed both VEGF and FGF-2 stimulation of EC proliferation in a dose-dependent manner. A recombinant preparation of the first Kunitz-type domain of TFPI-2 (KD1) did not suppress growth factor stimulation of EC proliferation, suggesting a mechanism distinct from the proteinase inhibitory activity of TFPI-2. Exogenously added TFPI-2 protein suppressed VEGF-induced EC migration in 2 different assays. Recombinant wt-KD1 or the R24K mutant of KD1, but not the R24Q mutant, dramatically suppressed VEGF-induced EC migration. TFPI-2 protein, but not recombinant KD1, blocked VEGF-induced activation of both Akt and ERK1/2 in ECs. At higher doses, TFPI-2 protein blocked VEGFR2 activation. CONCLUSIONS: Our data suggest that VEGF-upregulation of TFPI-2 expression in endothelial cells may represent a mechanism for negative feedback regulation and modulation of its pro-angiogenic action on endothelial cells. TFPI-2, or derivatives of TFPI-2, may be novel therapeutics for treatment of angiogenic disease processes.
Asunto(s)
Proliferación Celular , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Glicoproteínas/genética , Humanos , Interleucina-1beta/farmacología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
ADAMTS1 is an extracellular metalloproteinase known to participate in a variety of biological processes that includes inflammation, angiogenesis, and development of the urogenital system. Many of its functions rely on its catalytic activity, which thus far has been limited to the cleavage of the matrix proteoglycans aggrecan and versican. However, it is likely that other substrates exist. Using a yeast two-hybrid screen, we identified the Kunitz-type inhibitor, tissue factor pathway inhibitor-2 (TFPI-2), as a binding partner of ADAMTS1. The interaction was confirmed by several biochemical and cell-based assays. In addition, our studies revealed alterations in the pattern of TFPI-2-secreted isoforms and in its extracellular location caused by the specific action of ADAMTS1. Interestingly, we found that TFPI-2 is a novel substrate of ADAMTS1. The cleavage removes a protease-sensitive C-terminal region in TFPI-2, altering its binding properties. The proposed role of TFPI-2 as a maintenance factor of extracellular remodeling suggests the indirect function of ADAMTS1 as an additional homeostatic player by its ability to alter the extracellular location of TFPI-2 and, therefore, to disrupt the remodeling machinery, a phenomenon directly associated to pathologies such as atherosclerosis and tumor progression.