Innovative substance 2250 as a highly promising anti-neoplastic agent in malignant pancreatic carcinoma - in vitro and in vivo

Abstract

Background: Former studies already revealed the anti-neoplastic properties of the anti-infective agent Taurolidine (TRD) against many tumor species in vitro and in vivo. Its anti-proliferative and cell death inducing capacity is largely due to its main derivative Taurultam (TRLT). In this study it could be demonstrated, that substance 2250 - a newly defined innovative structural analogue of TRLT - exhibits an anti-neoplastic effect on malignant pancreatic carcinoma in vitro and in vivo.

Methods: The anti-neoplastic potential of substance 2250 as well as its mode of action was demonstrated in extensive in vitro analysis, followed by successful and effective in vivo testings, using xenograft models derived from established pancreatic cancer cell lines as well as patient derived tissue.

Results: Our functional analysis regarding the role of oxidative stress (ROS) and caspase activated apoptosis showed, that ROS driven programmed cell death (PCD) is the major mechanisms induced by substance 2250 in pancreatic carcinoma. What is strongly relevant towards clinical practice is especially the observed inhibition of patient derived pancreatic cancer tumor growth in mice treated with this new substance in combination with its sharply higher metabolic stability.

Conclusion: These encouraging results provide new therapeutical opportunities in pancreatic cancer treatment and build the basis for further functional analysis as well as first clinical studies for this promising agent.

Keywords: Apoptosis; Cancer; Chemotherapy; Substance 2250; Taurolidine.

Figures

Fig. 1

Molecular structure of substances TRLT and 2250. Substance 2250, 1.4.5-oxathiazan-dioxid-4.4, is an oxathiazine derivative like TRLT with a moleculare weight of 137.15 g/mol

Fig. 2

Effects of 2250 in different malignant cell lines measured by MTT-assay. AsPC-1 (a), BxPC-3 (b), MiaPaca-2 (c), Panc-1 (d) and Panc TuI cells (e) were incubated with 2250 (100, 200, 500, 1000, 1500, 2000 μmol/l) and ddH2O (control) for 24 h and submitted to a MTT-assay. Values are means ± SEM of 6 replicates of three independent experiments with consecutive passages. Asterisk symbols indicate differences between control, which was adjusted to 100% and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, n.s. p > 0.05 (one-way ANOVA followed by Tukey’s post-hoc test)

Fig. 3

Effects of Taurolidine (TRD) on cell proliferation in different malignant cell lines measured by BrdU-assay. AsPC-1 (a), BxPC-3 (b), MiaPaca-2 (c), Panc-1 (d) and Panc TuI cells (e) were incubated with 2250 (100, 200, 500, 1000, 1500, 2000 μmol/l) and ddH2O (control) for 6 h and submitted to a BrdU-assay. Values are means ± SEM of 8 replicates of three independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control, which was adjusted to 100% and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, n.s. p > 0.05 (one-way ANOVA followed by Tukey’s post-hoc test)

Fig. 4

Effects of 1000 μmol/l and 1500 μmol/l 2250 on viability, apoptosis and necrosis in different malignant pancreatic cell lines measured by FACS analysis. AsPC-1 (a), BxPC-3 (b) and Panc-TuI (c) cells were incubated with 2250 (1000 and 1500 μmol/l) and ddH2O (control) for 24 h. The percentages of viable, apoptotic and necrotic cells were determined by FACS-analysis with Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4–6 independent experiments with three consecutive passages. Asterisk symbols on columns indicate differences between control and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, n.s. p > 0.05 (one-way ANOVA followed by Tukey’s post-hoc test)

Fig. 5

Effects of NAC on 2250 induced cell death in different malignant cell lines measured by MTT (a-e); impact of 2250 on the cellular level of ROS (f). AsPC-1 (a), BxPC-3 (b), MiaPaca-2 (c), Panc-1 (d) and Panc TuI (e) cells were incubated with either 2250 (500, 1000 μmol/l), NAC (5 mmol/l) or the combination of both agents (2250 500, 1000 μmol/l + NAC 5 mmol/l) and ddH2O as control for 24 h and submitted to a MTT-assay. The level of ROS was analyzed in untreated compared to 2250 treated cells, additional NAC treatment served as a neg. Control (f). Values are means ± SEM of 6 replicates of three independent experiments with consecutive passages. Asterisk symbols indicate differences between control, which was adjusted to 100% and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, (one-way ANOVA followed by Tukey’s post-hoc test)

Fig. 6

Effects of NAC on 2250 induced cell death in cell line AsPC-1 measured by FACS analysis. AsPC-1 cells were incubated with either 2250 (500, 1000 μmol/l), NAC (5 mmol/l) or the combination of both agents (2250 500, 1000 μmol/l + NAC 5 mmol/l) and ddH2O as control for 24 h. The percentage of viable (a), apoptotic (b) and necrotic (c) cells were determined by FACS analysis. Values are means ± SEM of 4–6 replicates of three independent experiments with consecutive passages. Asterisk symbols indicate differences between control, which was adjusted to 100% and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, n.s. p > 0.05 (one-way ANOVA followed by Tukey’s post-hoc test)

Fig. 7

calibration curve of metabolic half-life measurement. The calibration curve consisting of five different concentrations of substance 2250 served as a standard for the calculation of the metabolic half-life of substance 2250 in nude mice, which resulted in a value of 13.8 h. The according measured parameters are shown in Table 2

Fig. 8

Effects of 500 mg/kg*BW 2250 on the subcutaneous tumor growth in nude mice in vivo. Nude mice with tumors of MiaPaca-2 (a), Panc-TuI (c) or patient tissue (b, d) were incubated with 2250 (500 mg/kg*BW) and ddH2O (control) for up to 3 weeks. The tumor volume was measured on alternating days for up to 3 weeks. Asterisk symbols indicate differences between control and 2250 treatment. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, n.s. p >0.05 (one-way ANOVA followed by Tukey’s post-hoc test)