RESUMO
Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations.
Assuntos
Células/efeitos dos fármacos , Células/metabolismo , Esterases/metabolismo , Ésteres/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Biocatálise/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Células HeLa , Hipocampo/citologia , Humanos , Hidrólise/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , RatosRESUMO
Human neuroblastomas possess several mechanisms of self-defense that may confer an ability to resist apoptosis and contribute to the observed difficulty in treating these tumors in the clinical setting. These molecular alterations may include defects in proapoptotic genes as well as the overexpression of prosurvival factors, such as Akt among others. As a key regulator of the turnover of proteins that modulate the cell cycle and mechanisms of apoptosis, the proteasome could serve as an important target for the treatment of neuroblastoma. The present studies provide the first evidence that bortezomib, a newly approved inhibitor of proteasome function, inhibits phosphorylation of Akt, induces the translocation of proapoptotic Bid, and potently enhances the apoptosis of murine neuroblastoma tumor cells in vitro. Furthermore, in that inhibitors of the Akt pathway can sensitize otherwise resistant TBJ/Neuro-2a cells to apoptosis induced by IFN-gamma plus TNF-alpha, we hypothesized that bortezomib also could sensitize these cells to IFN-gamma plus TNF-alpha. We demonstrate for the first time that bortezomib not only up-regulates the expression of receptors for IFN-gamma and TNF-alpha on both TBJ neuroblastoma and EOMA endothelial cell lines, but also markedly enhances the sensitivity of these cells to apoptosis induced by IFN-gamma plus TNF-alpha in vitro. Furthermore, bortezomib enhances the in vivo antitumor efficacy of IFN-gamma/TNF-alpha-inducing cytokines, including both IL-2 and IL-12 in mice bearing well-established primary and/or metastatic TBJ neuroblastoma tumors. Collectively, these studies suggest that bortezomib could be used therapeutically to enhance the proapoptotic and overall antitumor activity of systemic cytokine therapy in children with advanced neuroblastoma.