Computational modelling of LY303511 and TRAIL-induced apoptosis suggests dynamic regulation of cFLIP

Abstract

Motivation: TRAIL has been widely studied for the ability to kill cancer cells selectively, but its clinical usefulness has been hindered by the development of resistance. Multiple compounds have been identified that sensitize cancer cells to TRAIL-induced apoptosis. The drug LY303511 (LY30), combined with TRAIL, caused synergistic (greater than additive) killing of multiple cancer cell lines. We used mathematical modelling and ordinary differential equations to represent how LY30 and TRAIL individually affect HeLa cells, and to predict how the combined treatment achieves synergy.

Results: Model-based predictions were compared with in vitro experiments. The combination treatment model was successful at mimicking the synergistic levels of cell death caused by LY30 and TRAIL combined. However, there were significant failures of the model to mimic upstream activation at early time points, particularly the slope of caspase-8 activation. This flaw in the model led us to perform additional measurements of early caspase-8 activation. Surprisingly, caspase-8 exhibited a transient decrease in activity after LY30 treatment, prior to strong activation. cFLIP, an inhibitor of caspase-8 activation, was up-regulated briefly after 30 min of LY30 treatment, followed by a significant down-regulation over prolonged exposure. A further model suggested that LY30-induced fluctuation of cFLIP might result from tilting the ratio of two key species of reactive oxygen species (ROS), superoxide and hydrogen peroxide. Computational modelling extracted novel biological implications from measured dynamics, identified time intervals with unexplained effects, and clarified the non-monotonic effects of the drug LY30 on cFLIP during cancer cell apoptosis.

Fig. 1.

Schematic diagram of TRAIL-induced apoptosis. The dashed arrows indicate catalytic effects. Solid arrows indicate that the species at the base is consumed or translocated. For example, the oligomerization of mitochondrial Bax leads to formation of a pore in the mitochondrial outer membrane, which allows release of cytochrome c and Smac into the cytosol. Inhibitory relationships are denoted by a bar with crossbrace. (Details of inhibition are provided in Supplementary Tables S1.1–S1.3) Synthesis and degradation are not shown

Fig. 2.

Schematic of how LY30 affects TRAIL-induced apoptosis. The receptors alone would have slower reaction rates than the primed receptors. Parameter values are listed in the Supplementary Material

Fig. 3.

Simulated and observed cell viability. Cell viability was measured by crystal violet assay at 24 h after treatment with LY30 and/or TRAIL (repeated three times and normalized to untreated control)

Fig. 4.

Comparison of simulated caspase-8 activity versus experimental measurements for caspase-8 activation by LY30 and TRAIL. Solid lines represent averaged results of 10 000 Monte Carlo simulations. Caspase-8 activity is plotted as relative fold-change versus untreated, meaning that untreated cells (time = 0) have activity 1.0. Black squares show the published fold-change of protein activity relative to untreated control. Supplementary Material 1.6 describes the conversion from simulated levels of absolute caspase-8 activity, into estimates of relative fold-change of measured activity, to account for cross-talk between multiple caspase isoforms

Fig. 5.

Caspase-8 activity measurements in Hela cells after different durations of LY30 treatment. Cells were treated with LY30 for 30, 60, 120, 180 or 240 min, or untreated (0 min). LY30 treatment caused a significant decrease of caspase-8 activity at 2 h, according to a one-sample t-test with Bonferroni correction

Fig. 6.

Western blot analysis of cFLIP in Hela after different durations of LY30 treatment. (a) Western blot of time dynamics of cFLIP after LY30 treatment; (b) Quantified fold-change of cFLIP protein levels after 30 min of LY30 treatment. cFLIP band intensities (with three biological replications) were normalized to β-actin intensity (loading control) before comparing with untreated to obtain relative fold-change

Fig. 7.

Hypothetical model for LY30 to cause non-monotonic regulation of cFLIP via and H₂O₂. (a) Simplified diagram of LY30’s influence on cFLIP. This model is roughly divided into two phases. In the earlier phase, is produced after LY30 treatment, and the increased will block the degradation of cFLIP, thus inducing its up-regulation. In the later phase, H₂O₂ is produced by conversion of , and inhibits the production of cFLIP, lowering its concentration. (b) Simulations of cFLIP, cFLIP_mRNA and degraded cFLIP (cFLIP_Deg) over time, as predicted by the model in Figure 7a. The model is fully specified in Supplementary Tables

Fig. 8.

Western blots of cFLIP in Hela after LY30 treatment in the presence of ROS scavengers. (a) Western blot of cFLIP after 30 min LY30 treatment in the absence/presence of Tiron. Hela is pre-incubated with Tiron 1 h before adding LY30. (b) Western blot of cFLIP after 6 h LY30 treatment in the absence/presence of catalase