One-third of top-selling drugs are derived from natural products. When only a fraction of the bioactive natural products diversity has been explored, huge opportunities still remain for discovering novel leads for the development of new drugs. Clear cell renal cell carcinoma (ccRCC) is a highly vascular tumour arising from epithelial elements. Mutations in the Von Hippel-Lindau (VHL) gene are responsible for VHL disease and arise in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer. Renal carcinoma cell lines with naturally occurring VHL mutations (RCC4 VA) and their genetically matched wild-type VHL (RCC4 VHL) counterparts were seeded onto 96-well plates and allowed to attach overnight. Fungal extracts were tested on both cell lines. Clinically useful antitumor agents were used as positive controls and as reference points to establish the efficacy and selectivity of the new compounds. Renal cell carcinoma cell lines expressing VHL or not were treated with Carboxyfluorescein succinimidyl ester (CFSE). The day after cell inoculation, extracts were added and during the following days of incubation, fluorescence intensity was measured as a surrogate marker for cell viability. The most promising extracts selectively inhibited growth of pVHL-defi- cient cells but not of wild-type VHL cells. We used High Content Bio-imaging, a complete cellular imaging workflow that integrates instruments and software to acquire and analyze images, to evaluate their effect. Cell imaging can reveal effects that would be overlooked by other cell assay approaches. This target-based whole cell screen is a new strategy, which ensures cell permeability and target selectivity especially in natural product screening where natural product purification is a labour of extensive work. This approach permitted a dynamic study where fluorescence was measured without affecting cell viability and enabling a better detection of cytotoxic effects such as autophagy, senescence or late apoptosis.
Cite this paper
Cautain, B. , Pedro, N. , Escalona, M. , Tormo, J. , Genilloud, O. and Vicente, F. (2013) HCS strategy targeting dysregulation of the VHL/HIF pathway for drug discovery. Advances in Bioscience and Biotechnology, 4, 398-405. doi: 10.4236/abb.2013.43053.
 Carlson, E.E. (2010) Natural products as chemical probes. ACS Chemical Biology, 5, 639-653.
 Hu Lonser, R.R., Glenn, G.M., Walther, M., Chew, E.Y., Libutti, S.K., Linehan, W.M., et al. (2003) Von Hippel- Linda disease. Lancet, 361, 2059-2067.
 Iliopoulos, O., Levy, A.P., Jiang, C., Kaelin Jr., W.G. and Goldberg, M.A. (1996) Negative regulation of hypoxia inducible genes by the von Hippel-Lindau protein. Proceedings of the National Academy of Science of the United States of America, 93, 10595-10599.
 Iliopoulos, O., Kibel, A., Gray, S. and Kaelin Jr., W.G., (1995) Tumour suppression by the human von Hippel- Lindau gene product. Nature Medicine, 1, 822-826.
 Zanella, F., Rosado, A., Blanco, F., Henderson, B.R., Carnero, A. and Link, W. (2007) An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays. ASSAY and Drug Development Technologies, 5, 333-341.
 Haney, S.A., LaPan, P., Pan, J. and Zhang, J. (2006) High-content screening moves to the front of the line. Drug Discovery Today, 11, 889-894.
 Zanella, F., Lorens, J.B. and Link, W. (2010) High content screening: Seeing is believing. Trends in Biotechnology, 28, 237-245. doi:10.1016/j.tibtech.2010.02.005
 Liptrot, C. (2001) High content screening—From cells to data to knowledge. Drug Discovery Today, 6, 832-834.
 Sutphin, P.D., Chan, D.A., Li, J.M., Turcotte, S., Krieg, A.J. and Giaccia, A.J. (2007) Targeting the loss of the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells. Cancer Research, 67, 5896-5905.
 Turcotte, S., Sutphin, P.D. and Giaccia, A.J. (2008) Targeted therapy for the loss of von Hippel-Lindau in renal cell carcinoma: A novel molecule that induces autophagic cell death. Autophagy, 4, 944-946.
 Dolma, S., Lessnick, S.L., Hahn, W.C. and Stockwell, B.R. (2003) Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell, 3, 285-296.
 Giaccia, A., Siim, B.G. and Johnson, R.S. (2003) HIF-1 as a target for drug development. Nature Reviews Drug Discovery, 2, 803-811. doi:10.1038/nrd1199
 Kaelin Jr., W.G. (2005) The concept of synthetic lethality in the context of anticancer therapy. Nature Reviews Cancer, 5, 689-698. doi:10.1038/nrc1691
 Sutphin, P.D., Chan, D.A. and Giaccia, A.J. (2004) Dead cells don’t form tumors: HIF-dependent cytotoxins. Cell Cycle, 3, 160-163. doi:10.4161/cc.3.2.617
 Gnarra, J.R., Lerman, M.I., Zbar, B. and Linehan, W.M. (1995) Genetics of renal-cell carcinoma and evidence for a critical role for von Hippel-Lindau in renal tumorigenesis. Seminars in Oncology, 22, 3-8.
 Gossage, L. and Eisen, T. (2010) Alterations in VHL as potential biomarkers in renal-cell carcinoma. Nature Reviews Clinical Oncology, 7, 277-288.
 Milella, M. and Felici, A. (2011) Biology of metastatic renal cell carcinoma. Journal of Cancer, 2, 369-373.
 Turcotte, S., Chan, D.A., Sutphin, P.D., Hay, M.P., Denny, W.A. and Giaccia, A.J. (2008) A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy. Cancer Cell, 14, 90-102.
 Cowey, C.L., Sonpavde, G. and Hutson, T.E. (2010) New advancements and developments in treatment of renal cell carcinoma: Focus on pazopanib. Journal of OncoTargets and Therapy, 3, 147-155. doi:10.2147/OTT.S12480
 Xu, C.F., Reck, B.H., Xue, Z., Huang, L., Baker, K.L., Chen, M., et al. (2010) Pazopanib-induced hyperbilirubinemia is associated with Gilbert’s syndrome UGT1A1 polymorphism. British Journal of Cancer, 102, 1371- 1377. doi:10.1038/sj.bjc.6605653
 Linehan, W.M., Bratslavsky, G., Pinto, P.A., Schmidt, L.S., Neckers, L., Bottaro, D.P., et al. (2010) Molecular diagnosis and therapy of kidney cancer. Annual Review of Medicine, 61, 329-343.
 Motzer, R.J., Hutson, T.E., Tomczak, P., Michaelson, M.D., Bukowski, R.M., Oudard, S., et al. (2009) Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. Journal of Clinical Oncology, 27, 3584-3590.
 Yang, J.C., Haworth, L., Sherry, R.M., Hwu, P., Schwartzentruber, D.J., Topalian, S.L., et al. (2003) A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. New England Journal of Medicine, 349, 427-434.
 Wilhelm, S.M., Adnane, L., Newell, P., Villanueva, A., Llovet, J.M. and Lynch, M. (2008) Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Molecular Cancer Therapeutics, 7, 3129-3140.
 Escudier, B., Eisen, T., Stadler, W.M., Szczylik, C., Oudard, S., Staehler, M., et al. (2009) Sorafenib for treatment of renal cell carcinoma: Final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. Journal of Clinical Oncology, 27, 3312-3318. doi:10.1200/JCO.2008.19.5511
 Hudson, C.C., Liu, M., Chiang, G.G., Otterness, D.M., Loomis, D.C., Kaper, F., et al. (2002) Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Molecular and Cellular Biology, 22, 7004-7014.
 Thomas, G.V., Tran, C., Mellinghoff, I.K., Welsbie, D.S., Chan, E., Fueger, B., et al. (2006) Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nature Medicine, 12, 122-127.
 Motzer, R.J., Escudier, B., Oudard, S., Hutson, T.E., Porta, C., Bracarda, S., et al. (2008) Efficacy of everolimus in advanced renal cell carcinoma: A double-blind, randomised, placebo-controlled phase III trial. Lancet, 372, 449-456. doi:10.1016/S0140-6736(08)61039-9
 Robinson, J.P. (1998) Current protocols in cytometry. John Wiley and Sons, Inc., New York, 9.11.1-9.11.9.