ABSTRACT
Photoactivatable molecules enable ablation of malignant cells under the control of light, yet current agents can be ineffective at early stages of disease when target cells are similar to healthy surrounding tissues. In this work, we describe a chemical platform based on amino-substituted benzoselenadiazoles to build photoactivatable probes that mimic native metabolites as indicators of disease onset and progression. Through a series of synthetic derivatives, we have identified the key chemical groups in the benzoselenadiazole scaffold responsible for its photodynamic activity, and subsequently designed photosensitive metabolic warheads to target cells associated with various diseases, including bacterial infections and cancer. We demonstrate that versatile benzoselenadiazole metabolites can selectively kill pathogenic cells - but not healthy cells - with high precision after exposure to non-toxic visible light, reducing any potential side effects in vivo. This chemical platform provides powerful tools to exploit cellular metabolic signatures for safer therapeutic and surgical approaches.
Subject(s)
Bacterial Infections/drug therapy , Fluorescent Dyes/administration & dosage , Glioblastoma/drug therapy , Organoselenium Compounds/administration & dosage , Photochemotherapy/methods , Animals , Coculture Techniques , Fluorescent Dyes/adverse effects , Fluorescent Dyes/chemistry , Fluorescent Dyes/radiation effects , Glioblastoma/pathology , Humans , Intravital Microscopy , Light , Microbial Sensitivity Tests , Microscopy, Confocal , Microscopy, Fluorescence , Organoselenium Compounds/adverse effects , Organoselenium Compounds/chemistry , Organoselenium Compounds/radiation effects , Spheroids, Cellular , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60-100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway.
Subject(s)
Caveolae/metabolism , Protein Serine-Threonine Kinases/genetics , Signal Transduction/physiology , Zebrafish Proteins/genetics , Zebrafish/physiology , Animals , HEK293 Cells , Hippo Signaling Pathway , Humans , Protein Serine-Threonine Kinases/metabolism , Serine-Threonine Kinase 3 , Zebrafish/genetics , Zebrafish Proteins/metabolismABSTRACT
Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system(1) in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis(2,3). Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.
