RESUMEN
OBJECTIVE: Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been identified as a potential biomarker in diseases such as cancer, cardiovascular diseases and diabetes. Since TIMP-1 resides in most tissues and bodily fluids, we evaluated the potential of using saliva to obtain reproducible TIMP-1 measurements in a non-invasive manner. MATERIAL AND METHODS: Samples of unstimulated and stimulated whole saliva and saliva collected from individual glands were analysed for TIMP-1 content. A TIMP-1 ELISA was validated for use in saliva testing and the most optimal sampling and handling procedures for reproducible measurements identified. Western blotting and MALDI-TOF mass spectrometry were used for confirmatory analyses. RESULTS: The TIMP-1 ELISA was found suitable for saliva measurements. All saliva secretions contained TIMP-1, but in different concentrations ranging from 2.81 ng/mL in submandibular/sublingual saliva to 173.88 ng/mL in parotid saliva. TIMP-1 concentrations were influenced to a varying degree by fluctuations in flow. We found the lowest output in submandibular/sublingual saliva stimulated with 0.5% citric acid (3.56 ng/min) and highest output in chewing-stimulated whole saliva (267.01 ng/min). CONCLUSION: This study shows that saliva contains authentic TIMP-1, the concentration of which was found to depend on gland type and salivary flow. Stimulated whole saliva is suggested as a reliable and easily accessible source for TIMP-1 determinations in bodily fluids.
Asunto(s)
Glándula Parótida/metabolismo , Saliva/química , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Femenino , Humanos , Masculino , Masticación , Glándula Parótida/química , Glándulas Salivales Menores/metabolismoRESUMEN
TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP-1 may hold important clinical information, and either alone or in combination with measurement of full-length TIMP-1 they may improve the prognostic and/or predictive value of TIMP-1 analyses.
Asunto(s)
Empalme Alternativo , Biomarcadores de Tumor/biosíntesis , Neoplasias del Colon/metabolismo , Proteínas de Neoplasias/biosíntesis , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Neoplasias de la Mama/metabolismo , Neoplasias del Colon/diagnóstico , Exones , Femenino , Células HL-60 , Humanos , Masculino , PronósticoRESUMEN
Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of four inhibitors of the matrix metalloproteinases, which are capable of degrading most components of the extracellular matrix. However, in recent years, TIMP-1 has been recognised as a multifunctional protein, playing a complex role in cancer. In this regard, several studies have demonstrated an antiapoptotic effect of TIMP-1 in a number of different cell types. Since chemotherapy works by inducing apoptosis in cancer cells, we raised the hypothesis that TIMP-1 promotes resistance against chemotherapeutic drugs. In order to investigate this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells and their genetically identical wild-type controls. For future studies, this cell system can be used to uncover the mechanisms and signalling pathways involved in the TIMP-1-mediated inhibition of apoptosis as well as to investigate the possibility of using TIMP-1 inhibitors to optimise the effect of conventional chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/genética , Animales , Apoptosis/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Células Cultivadas , Citarabina/farmacología , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Femenino , Expresión Génica/genética , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/deficiencia , Vincristina/farmacologíaRESUMEN
Whether patients diagnosed with primary breast cancer are offered adjuvant systemic therapy following surgical removal of the tumor is based on prognosis. Prognosis is estimated in every patient using established prognostic variables. Unfortunately, when using the currently available prognostic parameters a significant proportion of patients are over-treated. Thus, in order to improve stratification of breast cancer patients, additional prognostic factors need to be identified. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of the promising candidates for new prognostic markers in breast cancer, as a number of studies have demonstrated an association between high tumor-tissue levels of TIMP-1 mRNA as well as TIMP-1 protein and a poor prognosis of breast cancer patients. TIMP-1 is a member of the TIMP family, currently comprising four members (TIMP-1-4), and its main function is inhibition of the activity of various matrix metalloproteinases (MMPs). The association between high levels of protease inhibitor and poor prognosis may be somewhat surprising, as proteolytic activity plays a pivotal role in cancer cell invasion and metastasis. However, the recent discovery of other biological functions of TIMP-1 such as growth-stimulating functions, as well as anti-apoptotic and pro-angiogenetic effects, may in part explain this paradox. The purpose of this review is to give an update on the current status of TIMP-1 in breast cancer, emphasizing the prognostic utility of the inhibitor. In addition, the suggested tumor-stimulatory roles of TIMP-1 will be outlined.
Asunto(s)
Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Biomarcadores de Tumor/genética , Femenino , Humanos , Pronóstico , ARN Mensajero/análisis , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Recent studies strongly suggest an active involvement of the c-Jun N-terminal kinase (JNK) signaling pathway in tumor necrosis factor (TNF)-induced apoptosis. The direct evidence for the role of JNK and its isoforms has been missing and the mechanism of how JNK actually could facilitate this process has remained unclear. In this study, we show that Jnk2-/- primary mouse embryonic fibroblasts (pMEFs) exhibit resistance towards TNF-induced apoptosis as compared to corresponding wild-type and Jnk1-/- pMEFs. JNK2-deficient pMEFs could be resensitized to TNF via retroviral transduction of any of the four different JNK2 splicing variants. Jnk2-/- pMEFs displayed deficient and delayed effector caspase activation as well as impaired cytosolic cystein cathepsin activity: processes that both were needed for efficient TNF-induced apoptosis in pMEFs. Our work demonstrates that JNK has a central role in the promotion of TNF-induced apoptosis in pMEFs, and that the JNK2 isoform can regulate both mitochondrial and lysosomal death pathways in these cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Catepsinas/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Empalme Alternativo , Animales , Caspasas/análisis , Catepsina B/metabolismo , Catepsinas/análisis , Supervivencia Celular , Citocromos c/metabolismo , Citosol/enzimología , ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Feto/citología , Feto/metabolismo , Fibroblastos/citología , Eliminación de Gen , Variación Genética , Lisosomas/metabolismo , Ratones , Microscopía Confocal , Mitocondrias/metabolismo , Modelos Biológicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Retroviridae/genética , Coloración y EtiquetadoRESUMEN
Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.
Asunto(s)
Apoptosis/efectos de los fármacos , Catepsina B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Inhibidores de Caspasas , Caspasas/metabolismo , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Cistatinas/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Activación Enzimática/efectos de los fármacos , Fibroblastos , Fibrosarcoma/enzimología , Fibrosarcoma/patología , Genes Dominantes/genética , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/metabolismo , Ratones , FN-kappa B/metabolismo , Oligonucleótidos Antisentido/genética , Fosfatidilserinas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.
Asunto(s)
Proteínas 14-3-3/metabolismo , Apoptosis/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Dedos de Zinc , Proteínas 14-3-3/genética , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Apoptosis/fisiología , Sitios de Unión , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 8 , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular Tumoral , ADN Complementario/genética , Proteínas de Unión al ADN , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Nucleares , Oligopéptidos/biosíntesis , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Factor 2 Asociado a Receptor de TNF/biosíntesis , Transfección , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, and its two analogues, EB 1089 and CB 1093, are novel putative anticancer agents with an interesting profile of induction of growth inhibition, differentiation, and apoptosis in tumor cells. To study the signaling pathways mediating these events, we used two human breast cancer cell lines: MCF-7 cells, expressing a wild-type p53 tumor suppressor protein, and T47D cells, lacking a functional p53. Vitamin D compounds induced a growth arrest followed by apoptosis in both cell lines at concentrations ranging from 1 to 100 nM, indicating that p53 is not necessary for growth-inhibitory effects induced by vitamin D compounds. Surprisingly, apoptosis induced by these compounds occurred also independently of known caspases. Inhibition of caspase activation by overexpression of a cowpox-derived caspase inhibitor CrmA or by addition of inhibitory peptides acetyl-Asp-Glu-Val-Asp-aldehyde (200 microM), acetyl-Ile-Glu-Thr-Asp-aldehyde (50 microM), and Z-Val-Ala-D,L-Asp-fluoromethylketone (1 microM) showed no effect on the induction of growth arrest or apoptosis by vitamin D compounds under assay conditions in which apoptosis induced by TNF or staurosporine was effectively inhibited. Moreover, overexpression of caspase-3 in MCF-7 cells had no sensitizing effect to vitamin D compounds, and neither caspase-3-like protease activity nor cleavage of a caspase substrate poly(ADP)ribose polymerase was detected in lysates from apoptotic cells following the treatment with these compounds. Contrary to CrmA, overexpression of an antiapoptotic protein Bcl-2 in MCF-7 cells conferred a nearly complete protection from apoptosis induced by vitamin D compounds. Taken together, these data indicate that vitamin D compounds induce apoptosis via a novel caspase- and p53-independent pathway that can be inhibited by Bcl-2. This may prove useful in the treatment of tumors that are resistant to therapeutic agents that are dependent on the activation of p53 and/or caspases.