In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.
- Publikační typ
- časopisecké články MeSH
MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening (HTS) with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based HTS of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.
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- časopisecké články MeSH
