Larson B, Banks P, Pierre N, Graham S, Degorce F, Georgiou S
BioTek Instruments, Inc., Vermont, USA. Cisbio Bioassays, Codolet, France. Enzo Life Sciences, Farmingdale, New York, USA.
HTRF 5th Symposium, Avignon, France
According to the Centers of Disease Control and Prevention (CDC), each year about 20,000 women in the United States are diagnosed with ovarian cancer1. 90% of these cancers are classifi ed as epithelial and are believed to arise from the surface(epithelium) of the ovary and are termed Epithelial Ovarian Cancer (EOC). While prognosis is good with early diagnosis, in disease stages I/II, symptoms are non-specifi c and diffi cult to trace. The majority of ovarian cancers are diagnosed at late stage III/IV, where symptoms become more evident. Unfortunately at this point, prognosis is poor. A common manifestation of EOC at this late stage of its progression is a build-up of fl uid in the abdominal cavity (ascites). It has been shown that high levels of IL-6 are present in patients with ascites2. The origin of the high expression of IL-6 is linked to the receptor Epidermal Growth Factor Receptor (EGFR). EGFR is expressed in up to 70% of EOCs, and its altered expression is associated with late stage disease and poor prognosis3. EGFR, a member of ErbB family of receptor tyrosine kinases, activates multiple signaling cascades including the activation of NFkB, which is known to activate the transcription of infl ammation-related proteins such as IL-64. In a recent publication, it was shown that EGFR ligand binding induces the expression of IL-6 via the NFkB pathway in advancedstage epithelial ovarian cancer5. In this application note we demonstrate an in vitro microplate assay that can monitor IL-6 secretion from plated SKOV-3 ovarian carcinoma cells induced through EGFR ligand binding and NFkB activation. The assay workfl ow involved a two plate protocol where cells are plated and EGFR ligand-activated. IL-6 measurements are made in a separate microplate by transferring a portion of the cell supernatant. We also showed that IL-6 secretion can be inhibited at the level of either EGFR or NFkB using known inhibitors. Inhibitors that are potentially toxic to the plated cells can be assessed through digital widefi eld fl uorescence microscopy using fl uorescent probes. This provides a quantitative determination of whether IL-6 suppression is caused by the inhibition of receptor/transcription factor activation or through cell toxicity. All microplate measurements were made on the Cytation3 Cell Imaging Multi-Mode Reader.