RESUMO
Tracking the activity of signalling pathways is a fundamental method for basic science, as well as in cancer- and pharmaceutical research. The developmental pathways Wnt, Hedgehog and Notch are frequently deregulated in cancers and represent a valuable target for the discovery of novel anticancer compounds. Here we present reporter systems for tracking activity of these pathways by using specific promoter elements driving the expression of either sensitive luciferases or fluorescent proteins. A high level of sensitivity was obtained using the luciferase reporter genes for firefly (FLuc), secreted Gaussia (GLuc) and synthetic NanoLuc (NLuc). As fluorescent reporter proteins, mTurqouise2, tdTomato and iRFP720 were chosen. Specificity of pathway activity was validated by co-transfection with pathway activating genes, showing significant response to induction. In addition, multi-gene plasmids were cloned, allowing the detection of all three pathways by one vector. By using the multi-gene vector 3P-Luc (wnt-NLuc, hedgehog-FLuc, Notch-GLuc), we could unambiguously demonstrate the crosstalk between pathways, while excluding cross reactivity of luciferase substrates. First studies with synthetic compounds confirmed the applicability of the system for future drug screening approaches.
Assuntos
Genes Reporter , Proteínas Hedgehog/metabolismo , Plasmídeos/genética , Receptores Notch/metabolismo , Linhagem Celular , Clonagem Molecular , Células HEK293 , Células HeLa , Proteínas Hedgehog/genética , Humanos , Luciferases/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Receptores Notch/genética , Transdução de SinaisRESUMO
Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin α6) is frequently upregulated in metastasizing, highly aggressive tumors. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.
