La fusión entre un anticuerpo anti-HER2 y C5adesArg aumenta su internalización y disminuye la fosforilación intracelular en células tumorales que sobreexpresan HER2 // The fusion of C5adesArg to an anti-HER2 antibody increases its internalization and reduces the antibody-induced tyrosine phosphorylation in a HER2-overexpressing cell line
Resumen
Resumen
Los anticuerpos monoclonales dirigidos contra HER2 han sido utilizados con fines terapéuticos por más de 20 años. En el presente trabajo se exploraron algunos de los mecanismos involucrados en la funcionalidad de células de la línea de cáncer de mama SKBR-3, que sobre-expresa HER2, tratadas con un anticuerpo recombinante anti-HER2 fusionado a la molécula C5adesArg. Para ello, se estudiaron la internalización del complejo anticuerpo/receptor, los niveles de fosforilación intracelular de tirosina y la expresión de la fosfatasa PTEN (del Inglés Phosphatase and Tesin Homologue, homólogo de tensina y fosfatasa), mediante la técnica de microscopía de fluorescencia. De acuerdo a los resultados obtenidos, la capacidad de unión del anticuerpo antiHER2/C5adesArg pareciera ser mayor, lo que podría estar mediado por la carga catiónica de la molécula C5adesArg. Este incremento aparente no se asoció con un incremento concomitante de la fosforilación intracelular de tirosina, sugiriendo que la molécula C5adesArg pudiese interferir con la activación de rutas y mecanismos alternos de supervivencia/rescate celular, normalmente inducidos por el tratamiento con el anticuerpo sin fusionar. La tendencia a una expresión
sostenida de PTEN en el contexto del tratamiento con anti-HER2/C5adesArg
sugiere que la molécula C5adesArg pudiera contribuir a la inhibición de la fosforilación detirosina desencadenada por el anticuerpo sin fusionar. Estas observaciones aportan evidencias para un mejor entendimiento de los mecanismos de muerte inducidos por anti-HER2/C5adesArg en esta línea celular.
Abstract
Therapeutic antibodies against HER2, such as trastuzumab, have been used in the treatment of breast cancer for over 20 years. In this work, we explored some of the cellular mechanisms involved in the lethal effect of an anti-HER2/C5adesArg fusion antibody on a human breast cancer cell line that over-expresses HER2 (SKBR-3). To this end, the internalization of the HER2/Anti-HER2 complex, tyrosine phosphorylation levels, and PTEN (Phosphatase and Tesin Homologue)expression were measured using fluorescence microscopy. We observed an increased trend in the binding of the anti-HER2/C5adesArg antibody to the cell surface. Also, overall tyrosine phosphorylation levels appeared increased in cells treated with the non-fused version of the antibody, as opposed to what was observed in cells treated with the anti-HER2/C5adesArg version. Additionally, our results point to a diminished expression of PTEN in cells treated with the anti-HER2 (non-fused) antibody that did not occur in cells treated with anti-HER2/C5adesArg. Therefore, we presume that the anti-HER2/C5adesArg binding capacity is higher, which could be mediated by the cationic nature of the C5adesArg molecule. This increase does not seem to be associated to a concomitant increase in intracellular tyrosine phosphorylation, suggesting that C5adesArg could also interfere with the activation of survival/recue pathways triggered by the antibody alone. The observed sustained expression of PTEN could contribute to the inhibition of the intracellular phosphorylation induced by the antibody alone. These observations provide further evidence for a better understanding of the mechanisms involved in the lethality of anti-HER2/C5adesArg on this cell line
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