Elien De Thaye (1,2), An Vermeulen (1), Jan Van Bocxlaer (1), Pieter Colin (1)
(1) Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Ghent, Belgium. (2) Cancer Research Institute Ghent (CRIG), Ghent, Belgium.
Objectives: Circulating full-length and caspase-cleaved (cc) cytokeratin (CK) 18 are used in the study of apoptosis and chemotherapy-induced cell death. In patients with epithelial tumors, cytokeratins hold potential as biomarkers due to their epithelial specificity, abundance and cleavage by caspases. An in vitro cell-based assay was performed to investigate the potential of these two pharmacodynamic biomarkers in view of their use in personalized cancer treatment.
Methods: NIH:OVCAR-3, SK-OV-3, SK-OV-3LucIP1, PA-1 and Caov-3 ovarian cancer cells were exposed to increasing concentrations of paclitaxel (0 to 1000 nM) during 24 hours. Following exposure of all cell lines, levels of cleaved and intact CK18 were assessed in the culture medium, up to 5 days after washing away treatment, by specific ELISA assays (M30 Apoptosense® and M65 EpiDeath® ELISA, VLVbio, Sweden). In addition, cell survival was investigated using a MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, The Netherlands). 72 hours after washing away the treatment solutions, cell survival and ccCK18 release data were analysed using, respectively, a full inhibitory and stimulatory sigmoidal Emax model with NONMEM® (version 7.3.0, ICON, Hanover, MD, USA).
Results: In all cell lines, the 1 to 1000 nM paclitaxel concentrations were effective in inhibiting cell proliferation. The cumulative amount of released ccCK18 increased with increasing paclitaxel concentrations for all cell lines. The concentration-effect relationships for both cell survival and ccCK18 levels showed similar trends in terms of paclitaxel concentration needed to reach 50% of its effect (C50). Exposure-response modeling of the paclitaxel effects regarding cell survival resulted in estimated C50 values equal to 26.4 nM (SK-OV-3LucIP1; 23% RSE (relative standard error), 95% CI: 14.503 – 38.297), 10.4 nM (SK-OV-3; 31.3% RSE, 95% CI: 4.01 – 16.79), 9.06 nM (PA-1; 0.1% RSE, 95% CI: 9.039 – 9.081), 8.49 nM (NIH:OVCAR-3; 0.4% RSE, 95% CI: 8.425 – 8.555) and 1.91 nM (Caov-3; 15.2% RSE, 95% CI: 1.34 – 2.48). C50 parameters from the exposure-response modeling of the drug effects focusing on the released amount of ccCK18 were estimated to be 8.05 nM (SK-OV-3LucIP1; 5.4% RSE, 95% CI: 7.199 – 8.901), 6.41 nM (SK-OV-3; 17.6% RSE, 95% CI: 4.195 – 8.625), 3.66 nM (NIH:OVCAR-3; 9.9% RSE, 95% CI: 2.95 – 4.37), 1.15 nM (PA-1; 0.5% RSE, 95% CI: 1.139 – 1.161) and 0.834 nM (Caov-3; 1.2% RSE, 95% CI: 0.814 – 0.854). Regarding the cell survival data, C50 values increased from Caov-3, over NIH:OVCAR-3, PA-1 and SK-OV-3 to SK-OV-3LucIP1. Regarding the ccCK18 data, the same trend was observed, except for a switch between PA-1 and NIH:OVCAR-3. No pharmacodynamic analysis was performed based on the in vitro release data for total levels of CK18 as, except for the PA-1 cell line, it was difficult to observe clear differences in released amounts over drug concentration levels.
Conclusions: Based on this in vitro study, evidence of association was demonstrated between paclitaxel-induced cell toxicity and resulting ccCK18 levels based on the similar outcome in terms of C50 values across cell lines. This in vitro work illustrates the potential for using ccCK18 levels as a surrogate for cell survival.
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Reference: PAGE 27 (2018) Abstr 8542 [www.page-meeting.org/?abstract=8542]
Poster: Drug/Disease Modelling - Oncology