RESUMEN
Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Secuencia de Aminoácidos , Colágeno Tipo IV/metabolismo , Cisteína Endopeptidasas/química , Activación Enzimática , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Cinética , Laminina/metabolismo , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Proteínas de Neoplasias/química , Neoplasias/enzimología , Neoplasias/patología , Proteolisis , Especificidad por SustratoRESUMEN
Arsenic trioxide (As2O3) has recently been identified as an effective drug in different types of cancer therapy. It is a useful pharmacological agent in acute promyelocytic leukemia (APL) treatment, especially the form that is resistant to conventional chemotherapy with all-trans retinoic acid (ATRA). What is more, laboratory data suggest that As2O3 is also active when it comes to several solid tumor cell lines. However, the mechanism of action is not fully understood. As2O3 in high doses triggers apoptosis, while in lower concentrations it induces partial differentiation. The As2O3 mechanism of action involves effects on mitochondrial transmembrane potential which lead to apoptosis. It also acts on the activity of JNK kinase, glutathione, caspases, NF-ĸB nuclear factor or pro- and antiapoptotic proteins. This publication presents the current knowledge about the influence of arsenic trioxide in cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Óxidos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Trióxido de Arsénico , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Promielocítica Aguda/patología , MAP Quinasa Quinasa 4/efectos de los fármacos , MAP Quinasa Quinasa 4/metabolismo , Mitocondrias/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias/patologíaRESUMEN
Retinoids are useful pharmacological agents in therapy and prevention of cancer. All-trans retinoic acid (ATRA) is applied in chemoprevention and differentiation therapy of some cancers with particularly impressive results in the management of acute promyelocytic leukemia (APL). ATRA plays a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types. ATRA mediates these effects by regulating gene transcription. Nuclear retinoic acid receptors (RARs) are considered to be the mediators of most of the effects of ATRA on gene expression. We present a current state of knowledge on the effects of ATRA on cell growth and differentiation as well as describe RARs and their role in the cellular mechanism of ATRA action. A particular attention was paid to the effects of ATRA on proliferation and differentiation of cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/prevención & control , Tretinoina/farmacología , Antineoplásicos/uso terapéutico , Humanos , Tretinoina/uso terapéuticoRESUMEN
Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.
Asunto(s)
Ácido 1-Carboxiglutámico/análisis , Amnios/enzimología , Corion/enzimología , Cisteína Endopeptidasas/química , Melanoma/enzimología , Proteínas de Neoplasias/química , Ácido 1-Carboxiglutámico/inmunología , Anticuerpos Monoclonales/inmunología , Anticoagulantes/farmacología , Línea Celular Tumoral/enzimología , Cisteína Endopeptidasas/farmacología , Activación Enzimática/efectos de los fármacos , Factor X/efectos de los fármacos , Femenino , Humanos , Melanoma/patología , Proteínas de Neoplasias/farmacología , Embarazo , Warfarina/farmacologíaRESUMEN
Neoplastic cells produce procoagulants responsible for hypercoagulation states frequently observed in cancer patients. It is accepted that two major procoagulants from malignant tissue are tissue factor (TF) and a direct activator of coagulation factor X called cancer procoagulant (CP). Direct factor X-activating activity of cultured human malignant melanoma WM 115 cells has been analyzed in the cell extracts, whole cells and in the medium after the cell culture. The factor X-activating activity was detected in the malignant cell lysates but not in the cultured medium or intact malignant cells. The lysates contained no TF as determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using anti-TF monoclonal antibody. The enzymatic characteristics of the activity was typical for CP. The results suggest that cancer procoagulant is an intracellular protein.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factor X/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Tromboplastina/metabolismo , Animales , Línea Celular TumoralRESUMEN
Two common procoagulant activities associated with tumors are tissue factor and cancer procoagulant (CP), an activator of coagulation factor X. We have identified a convenient source of CP in transplanted Lobund-Wistar rat PA3 prostate tumors. CP activity was purified from 5 independent transplanted prostate tumors by column chromatography. The protein activated factor X in the absence of TF and factor VII. An antihuman CP antibody recognized rat CP in an ELISA and inactivated CP activity in a chromogenic assay. Lobund-Wistar prostate tumors may provide a convenient animal model useful in determining the role of CP in cancer development.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factor X/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Ensayo de Inmunoadsorción Enzimática , Masculino , Neoplasias de la Próstata/patología , Ratas , Ratas WistarRESUMEN
Cancer procoagulant (CP) is a cysteine protease produced by fetal and malignant tissues, activating in vitro blood coagulation factor X. It has been demonstrated that CP is able to stimulate blood platelet adhesion to fibrinogen and collagen. The pro-adhesive properties of CP could play an important role in metastatic spread of cancer as well as in primary tumor growth. Effects of anti-CP antibody on the growth of MCF-7 breast cancer cells and on the cells adhesion to vitronectin have been analyzed in vitro. Addition of polyclonal anti-CP antibody to MCF-7 cell culture resulted in 16-18% (P < 0.001) decrease in the cells viability as compared with the control (other antibody or no antibody in the culture). Preincubation of MCF-7 cells with anti-CP antibody reduced the cells adhesion to vitronectin. Further addition of purified CP (0.5-8 microg/ml) to the MCF-7 cells preincubated with anti-CP antibody resulted in complete recovery of adhesive properties of the cells. However, when high concentration (16 microg/ml) of CP was added to the sample, only partial recovery of the adhesive properties by the cells was observed. Results of the experiments support the hypothesis that CP is involved in the growth of cancer cells, but its pro-coagulative properties are of secondary importance. One of the possible mechanisms of the interactions between CP and malignant cell could be the regulation of the cell adhesion processes.
Asunto(s)
Anticuerpos/farmacología , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/inmunología , Inhibidores de Crecimiento/farmacología , Proteínas de Neoplasias/inmunología , Vitronectina/metabolismo , Adhesión Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Humanos , Vitronectina/inmunologíaRESUMEN
Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.