RESUMEN
(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.
Asunto(s)
Biomarcadores de Tumor/análisis , Antígeno Carcinoembrionario/genética , Neoplasias del Colon/sangre , Queratina-19/genética , Células Neoplásicas Circulantes/patología , Estaurosporina/farmacología , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Antígeno Carcinoembrionario/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Inhibidores Enzimáticos/farmacología , Femenino , Fluorouracilo/farmacología , Humanos , Immunoblotting , Separación Inmunomagnética , Queratina-19/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Preliminary studies, performed in our laboratory, showed that staurosporine (ST), a protein-kinase (PK) inhibitor, increases the expression of the carcinoembryonic antigen (CEA) in a human colon cancer cell line. The present study explores the cellular and molecular effects of ST on the CEA expression in breast cancer MCF-7 line and in a number of colon cancer cell lines characterized by the different basal levels of the antigen, including two cloned sublines (i.e. C22.20 and C6.6, expressing low and high CEA levels, respectively). In all cases, increase of the CEA expression was observed at drug concentrations devoid of marked cytostatic effects (e.g. 5 nM) and was accompanied by the enhanced CEA shedding in the supernatant. Moreover, the increase of the CEA levels both occurred in the cell membranes and in the cytosolic compartments and appeared to be the result of the enhanced CEA gene transcription. Similar results have been previously obtained with interferon-gamma. However, ST treatment, different from interferon-gamma, did not up-regulate the level of the HLA class I molecules. A preliminary investigation also showed that other PKC inhibitors did not substantially modulate the CEA expression. Therefore, the biochemical mechanism underlying the effect of ST should not be correlated with that involved in the PKC inhibition. The present study suggests that ST and, presumably, its analogs used in the cancer treatment could enhance the CEA expression on neoplastic cells in patients affected by the CEA-positive malignancies. This appears to be of potential clinical interest for the development of new immunotherapeutic or diagnostic approaches based on the pharmacological modulation of this antigenic marker.