Synergistischer Effekt von Taurolidin und rhTRAIL bei der Apoptose-Induktion in HCT15 Coloncarcinom Zellen

Abstract

Background: Induction of apoptosis in tumor cells by TRAIL (TNF related apoptosis inducing ligand) is a promising therapeutic option in oncology, although toxicity and resistance against TRAIL are limiting factors. Taurolidine (TRD) is an anti-neoplastic agent with low toxicity and thus a potential candidate for combined therapy with TRAIL. The aim of this study was to evaluate the combined treatment of TRAIL and TRD in the HCT15 human colonic carcinoma cell-line. Material and Methods: HCT15 cells were cultured and incubated with increasing concentrations of recombinant human TRAIL (50 to 500 ng/ml) or TRD (50 to1000 µmol/l) to evaluate the dose dependent effects of both substances concerning apoptosis and necrosis. Thereafter, cells were incubated in a second experiment with TRAIL (50 and 250 ng/ml) or TRD (100 and 1000 µmol/l) alone as well as with combinations of both agents in different concentrations. At different time points (3 to 36 h), cell viability, apoptosis and necrosis were quantified by FACS analysis with propidium iodide and Annexin V staining. Results are expressed as means ± SEM, statistical analysis was carried out by ANOVA, pair comparison by Tukey-test. P values ≤ 0.05 were considered as statistically significant. Results: Incubation with TRD resulted in a dose dependent cell death induction with maximum effects of 100 µmol/l and 1000 µmol/l after 24 h and 36 h resulting in a reduction of viable cells from 60 % to 17–33 %. 250 ng/ml and 500 ng/ml TRAIL led to a decrease of viable cells from 70 % to 6–7 % as early as 6h with a partial recovery of viable cells to 13 % after 36 h. Combined treatment of TRD (100 µmol/l) and TRAIL (50 ng/ml) caused a sustained induction of apoptosis after 24 h and 36 h showing a significant synergistic effect of both substances, clearly exceeding a simply additive effect. After 24 h incubation with TRD (100 µmol/l) and TRAIL (50 ng/ml), only 2.1 % (± 0.5 %) of the cells were viable compared to 43.9 % (± 3.0 %) for TRD 100 µmol/l (p ≤ 0.001) and 17.7 % (± 2.0 %) for TRAIL 50 ng/m (p ≤ 00.05) alone. Similar results were obtained after 36h. Conclusion: We showed for the first time a synergistic effect of human recombinant TRAIL and Taurolidin on apoptosis induction of human colon carcinoma cells in vitro. Combined treatment with TRAIL and Taurolidin resulted in sustained cell death which was superior to single agent application. Combination of TRAIL with the non-toxic Taurolidin offers a novel therapeutic rationale in oncological therapy.