Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
RESEARCH
Open Access
Comparative analysis of cell death induction by
Taurolidine in different malignant human cancer
cell lines
Ansgar M Chromik1*†, Adrien Daigeler2†, Daniel Bulut3, Annegret Flier1, Christina May1, Kamran Harati1,
Jan Roschinsky1, Dominique Sülberg1, Peter R Ritter4, Ulrich Mittelkötter1, Stephan A Hahn5, Waldemar Uhl1
Abstract
Background: Taurolidine (TRD) represents an anti-infective substance with anti-neoplastic activity in many
malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by
TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by
TRD simultaneously in different malignant cell lines.
Materials and methods: Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma,
AsPC-1/pancreas and BxPC-3/pancreas) were incubated with increasing concentrations of TRD (100 μM, 250 μM
and 1000 μM) for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis
(Propidiumiodide/AnnexinV staining). Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the
radical scavenger N-Acetylcystein (NAC) and the Gluthation depleting agent BSO to examine the contribution of
caspase activation and reactive oxygen species in TRD induced cell death.
Results: All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic
agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and
BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in
TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly
among the cell lines.
Conclusion: This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across
several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell
lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide
multifaceted cell death mechanisms leading to a cell line specific diversity.
Background
Taurolidine (TRD), a substance derived from the aminosulfoacid Taurin, was originally used in peritonitis and
catheter related blood stream infections due to its antimicrobial and anti-inflammatory properties [1-3]. Over
the last years, TRD has also been shown to exert antineoplastic activity in vitro as well as in vivo [4]. TRD
induces cell death in a variety of malignant cell lines
derived from colon carcinoma [5,6], squamous cell
* Correspondence: a.chromik@klinikum-bochum.de
† Contributed equally
1
Department of Visceral and General Surgery, St Josef Hospital, RuhrUniversity Bochum, Germany
esophageal carcinoma [7] glioblastoma [8,9], melanoma
[10,11], mesothelioma [12,13] and sarcoma [14,15].
Furthermore, first reports about systemic application of
TRD in patients with gastric carcinoma and glioblastoma revealed promising results with almost absent toxicity [16-18]. Favorable pharmacokinetics and safety
profile of TRD render this compound to a promising
agent in oncology [19].
However, mechanisms underlying induction of cell
death by TRD are not yet fully elucidated. Among different types of programmed cell death (PCD) [20,21],
the classical apoptotic cell death has been described for
TRD including the intrinsic mitochondrial [9,12,22-24]
© 2010 Chromik et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
as well as the extrinsic death receptor associated pathway [6,7,14,24-26]. Furthermore, there seems to be a
dose dependency regarding the relative contribution to
apoptotic and necrotic cell death [6,7,9,26,27]. There is
an ongoing discussion about the involvement of caspase
activity to TRD induced PCD. Some studies revealed
enhanced caspase activity or even reversibility of TRD
induced cell death by caspase-inhibition [12,13,15,22,28]
whereas other denied any relevant contribution to TRD
induced PCD [9,24]. As a result, additional caspase independent forms of PCD have been suggested like autophagy or necrosis [9]. Furthermore, there is growing
evidence from recent publications, that generation of
reactive oxygen species (ROS) plays an important role in
TRD induced PCD [9,13,24,29]. However, the majority
of information about TRD effects is provided from studies with one single cell line or several cell lines of one
single malignancy. Methodical diversity often makes it
difficult to compare results from individual cell lines
and experiments. There is a lack of a comprehensive
and comparative view across several cell lines of different malignancies. Furthermore, no human pancreatic
cancer cell line has been evaluated for taurolidine susceptibility so far. The aim of this study was therefore, to
perform a comparative analysis of cell death induction
by TRD simultaneously in several cell lines of different
malignancies including pancreatic cancer - focussing on
dose dependency and relative contribution of apoptosis
and necrosis to TRD induced cell death. Furthermore,
the role of caspase activity and ROS were assessed functionally by applying specific inhibitors.
Materials and methods
Cell lines and culture conditions
Five different human neoplastic cancer cell lines were
used for this experiment: HT29 colon carcinoma (CLS
Cell Lines Service, Eppelheim, Germany), Chang Liver
(HeLa contaminant, CLS Cell Lines Service, Eppelheim,
Germany), HT1080 fibrosarcoma (ATCC - LGC Standards GmbH, Wesel, Germany), AsPC-1 pancreas carcinoma (CLS Cell Lines Service, Eppelheim, Germany)
and BxPC-3 pancreas carcinoma (ATCC - LGC Standards GmbH, Wesel, Germany). Chang Liver cells were
maintained with Dulbecco’s Modified Eagle Medium
(DMEM) - Hams’s F12, whereas HT1080 cells were cultured in modified Eagle’s medium (MEM). The remaining cell lines (HT29, AsPC-1, BxPC-3) were maintained
in RPMI 1640 (Biowest, Nuaille, France). All cultures
were supplemented with 10% fetal bovine serum, supplemented with penicillin (100 U/ml), streptomycin (100
μg/ml) and 2 mM L-Glutamine (Biowest, Nuaille,
France). AsPC-1 and HT1080 cells were further supplemented with 1 mM Sodium Pyruvate. Cells were grown
Page 2 of 16
as subconfluent monolayer and cultured in 25 cm2 flasks
at 37°C and 5% CO2 in a humidified atmosphere.
Reagents
TRD (Taurolin®) ultrapure powder (kindly provided by
Geistlich Pharma AG, Wolhusen, Switzerland) was dissolved in a 5% Povidon solution (K16 Povidon, generously provided by Geistlich Pharma AG, Wolhusen,
Switzerland) and sterile filtered to achieve the respective
TRD concentrations. A 5% Povidon solution in equal
volume served as a control for TRD treatment. Recombinant human TRAIL (Bender MedSystems, Vienna,
Austria) was dissolved in distilled water according to the
manufacturer’s instructions. N-acetylcysteine (NAC)
(Sigma-Aldrich, Munich, Germany) and DL-buthionin(S,R)-sulfoximine (BSO) (Sigma-Aldrich, Munich, Germany) were dissolved in distilled water according to the
manufacturer’s instructions. The Caspase Inhibitor zVAD-FMK (z-VAD) (Alexis Biochemicals, Enzo Live
Sciences, Lörrach, Germany) was applied according to
the manufacturer’s instructions.
Dose-effect relationship of TRD
Cells were seeded to a density of 3 × 106 cells/well in 6well plates (growth area 9.6 cm2/well) and incubated for
18-24 hours under the above mentioned culture conditions to obtain a subconfluent monolayer. Subsequently,
cells were washed and incubated for another 2 hours
before reagents were added to the culture medium. To
examine the dose-effect relationship of TRD in different
malignant cell lines, cells were incubated with increasing
concentrations of TRD (100, 250, and 1000 μM) and 5%
Povidon as control for 6 h and 24 h. All experiments
were repeated with at least 4 consecutive passages.
Flow Cytometry Analysis and cell morphology
At the indicated incubation time, floating cells were collected together with the supernatant and adherent cells
were harvested by trypsinization. Cells were sedimented
by centrifugation, resuspended and fixed in 195 μl binding buffer (Bender MedSystems, Vienna, Austria). Cell
density in the cell suspension was adjusted to 2 × 103
cells/μl. Subsequently, 5 μl Annexin V-FITC (BD Biosciences, Heidelberg, Germany) was added to the cell
suspension followed by gently vortexing and incubation
for 10 min at room temperature in the dark. Thereafter,
the cell suspension was centrifuged followed by resuspension in 190 μl binding buffer before 10 μl Propidiumiodide (Bender MedSystems, Vienna, Austria) was
added.
Cells were analyzed immediately using a FACS (fluoresence activated cell sorting) flow cytometer (FACS Calibur BD Biosciences, Heidelberg, Germany) for Annexin
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
V-FITC and Propidiumiodide binding. For each measurement, 20.000 cells were counted. Dot plots and histograms were analyzed by CellQuest Pro software (BD
Biosciences, Heidelberg, Germany). Annexin V positive
cells were considered apoptotic; Annexin V and PI positive cells were identified as necrotic. Annexin V and PI
negative cells were termed viable. Morphology of adherent cells and cells suspended in culture medium was
studied and documented using a phase contrast microscope, Zeiss Axiovert 25 (Karl Zeiss, Jena, Germany).
Each image was acquired at a magnification of × 20
with a spot digital camera from Zeiss.
Page 3 of 16
co-incubated with TRAIL, a known inductor of caspase
dependent cell death, together with z-VAD.
Statistical analysis
Results of FACS-analysis for percentage of viable, apoptotic and necrotic cells are expressed as means ± SEM
of at least four independent experiments with consecutive passages. Comparison between experimental groups
was performed using one-way ANOVA with Tukey’s
post-hoc text. P-values ≤ 0.05 were considered as statistically significant and indicated in the figures as follows:
*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Contribution of reactive oxygen species to TRD induced
cell death
Results
To evaluate the contribution of reactive oxygen species
(ROS) to TRD induced cell death, cells were co-incubated with TRD together with either the radical scavenger N-acetylcysteine (NAC) (5 mM) or the glutathione
depleting agent DL-buthionin-(S,R)-sulfoximine (BSO)
(1 mM). BSO is a selective and irreversible inhibitor of
g-glutamylcysteine synthase representing the rate-limiting biosynthetic step in glutathion snyhtesis [30,31]. In
HT29, Chang Liver, HT1080 and BxPC-3 cells, TRD
concentration for co-incubation was 250 μM, since
there was a significant reduction of viable cells and a
significant apoptotic effect in these cell lines after incubation with 250 μM as a single agent. In AsPC-1 cells,
1000 μM TRD was selected representing the only TRD
dose with significant cell death induction in this particular cell line. After 6 h and 24 h, cells were analyzed by
FACS for Annexin V and PI to define the relative contribution of apoptotic and necrotic cell death as
described above. Results from co-incubation experiments were compared with untreated controls (Povidon
5%) and the respective single substances (TRD, NAC or
BSO). Protection was considered as ‘complete’ when coincubation with either NAC or BSO completely abrogated the TRD induced reduction of viable cells leading
to a cell viability which was not significantly different
from untreated controls. By contrast, protection was
considered as ‘partial’ when co-incubation with either
NAC or BSO decreased significantly the TRD induced
reduction of viable cells without reaching the cell viability of untreated controls.
FACS analysis for Annexin V-FITC and Propidiumiodide revealed that treatment with TRD resulted in a significant reduction of viable cells compared to control
treatment with Povidon 5% as early as 6 h incubation
and more pronounced after 24 h (fig. 1, fig. 2, additional
file 1).
Reversibility of TRD induced cell death by caspase
inhibition
To determine the contribution of caspase activity to
TRD induced cell death, cells were co-incubated with
TRD (1000 μM for AsPC-1 and 250 μM HT29, Chang
Liver, HT1080 and BxPC-3) and the pan-caspase
inhibitor z-VAD-fmk (2 μM) for 24 h and analyzed by
FACS analysis. As positive control, cells were also
TRD induces cell death in all cell lines
TRD induced cell death is characterized by a cell line
specific contribution of apoptosis and necrosis
After 24 hours incubation, FACS analysis revealed an
inhomogeneous and complex dose response effect
among cell lines. In HT29 and Chang Liver cells, maximal effects on cell viability were achieved by treatment
with 250 μM TRD leading to 66.2% ± 5.6% and 33.2% ±
1.0% viable cells in HT29 (fig. 1a) and Chang Liver cells
(fig. 1d), respectively. In HT29 cells, this effect was due
to a significant rise in apoptotic cells (fig. 1b), whereas
Chang liver cells responded with significant increase in
both apoptotic and necrotic cells (fig. 1e+f). In HT1080
fibrosarcoma cells, the strongest reduction of cell viability was observed after 100 μM TRD leading to 26.8% ±
3.7% viable cells (fig. 1g), mainly due to a pronounced
apoptotic effect (fig. 1h). In contrast, both pancreatic
cancer cell lines, AsPC-1 and BxPC-3, showed the highest response after 24 h upon treatment with 1000 μM
TRD, resulting in 36.8% ± 5.2% (AsPC-1, fig. 2a) and
25.7% ± 4.3% (BxPC-3, fig. 2d) viable cells. Interestingly,
this reduction of cell viability was reflected by an exclusive enhancement of necrosis without any significant
effect on apoptosis. The observed proportions of necrotic cells for AsPC-1 and BxPC-3 were the highest
observed in this study (fig. 2c+f) (table 1). The results
for 6 hours incubation are provided in additional file 1
and summarized in table 1.
TRD shows specific patterns of dose response effects
among different cell lines
Dose response effects after 24 h were neither straight
proportional nor uniform among different cell lines. The
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 4 of 16
Figure 1 Effects of Taurolidine on viability, apoptosis and necrosis in HT29, Chang Liver and HT1080 cells. HT29 (a-c), Chang Liver (d-f)
and HT1080 cells (g-i) were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The
percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and
Propidiumiodide. Values are means ± SEM of 5 (HT29), 4 (Chang Liver) and 9 (HT1080) independent experiments with consecutive passages.
Asterisk symbols on columns indicate differences between control and TRD treatment. Asterisk symbols on brackets indicate differences between
TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
only cell line with an obvious proportional dose effect
was BxPC-3. In this cell line, all TRD concentrations
(100 μM, 250 μM and 1000 μM) caused a significant
reduction of viable cells compared to control treatment
with significant differences between increasing TRD
concentrations (fig. 2d). The other pancreatic cancer cell
line, AsPC-1, displayed at least some characteristics of a
proportional dose effect. The reduction of viable cells
with increasing TRD concentrations became statistically
significant for 1000 μM TRD, as illustrated in fig. 2a.
Two cell lines were characterized by an V-shaped dose
response pattern after 24 h. HT29 and Chang Liver cells
had the maximal reduction of viable cells after incubation with 250 μM TRD, which represents the intermediate concentration between 100 μM and 1000 μM TRD
(fig. 1a+d). Unlike all other cell lines, HT1080 cells
demonstrated an anti-proportional dose response with
the highest reduction of viable cells by 100 μM TRD.
Both following concentrations - 250 μM and 1000 μM
TRD - were also capable of a significant reduction of
cell viability - but not as strongly as 100 μM TRD
(fig.1g) (table 1). Representative FACS dot plots for
Chang Liver, HT1080 and BxPC-3 cells are presented in
figure 3 - indicating the different patterns of dose
response among these cell lines (fig. 3).
The radical scavenger N-acetylcysteine (NAC) and the
glutathione depleting agent L-S, R-Buthionine sulfoximine
(BSO) show cell line specific and divergent effects on TRD
induced cell death
In HT29 colon carcinoma cells, co-incubation of
TRD with NAC for 24 h led to a complete protection of
TRD induced cell death. NAC completely abrogated the
TRD induced reduction of viable cells leading to a cell
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 5 of 16
Figure 2 Effects of Taurolidine on viability, apoptosis and necrosis in AsPC-1 and BxPC-3 cells. AsPC-1 (a-c) and BxPC-3 cells (d-f) were
incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d),
apoptotic (b, d) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4
independent experiments with consecutive passages. Asterisk symbols on columns indicate differences between control and TRD treatment.
Asterisk symbols on brackets indicate differences between TRD groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 6 of 16
Table 1 Effect of increasing Taurolidine concentrations on viable, apoptotic and necrotic cells in different cell lines
HT29
Chang Liver
HT1080
AsPC-1
BxPC-3
Reduction of viable cells after 6 h
TRD 250
TRD 1000
TRD 1000
TRD 100
TRD 1000
TRD 1000
TRD 250
Increase of apoptotic cells after 6 h
TRD 250
TRD 1000
TRD 250
TRD 1000
TRD 100
TRD 1000
TRD 1000
TRD 250
Increase of necrotic cells after 6 h
Ø
TRD 1000
TRD 1000
TRD 1000
TRD 1000
Reduction of viable cells after 24 h
TRD 250
TRD 1000
TRD 250
TRD 100
TRD 1000
TRD 100
TRD 250
TRD 1000
TRD 1000
TRD 1000
TRD 250
TRD 100
Increase of apoptotic cells after 24 h
TRD 250
TRD 1000
TRD 250
TRD 100
TRD 1000
TRD 100
TRD 250
TRD 1000
Ø
TRD 250
Increase of necrotic cells after 24 h
TRD 1000
TRD 250
TRD 100
TRD 1000
TRD 250
TRD 100
TRD 1000
TRD 1000
TRD 1000
TRD 250
Pattern of dose response (viable cells) after 24 h (FACS anaylsis)
V-shaped
V-Shaped
Anti-Prop.
Prop.
Prop.
FACS analysis
Effect of increasing Taurolidin (TRD) concentrations (100 μM, 250 μM and 1000 μM) in different cell lines measured by FACS analysis (Annexin V/Propidium
Iodide). TRD concentrations in μM with significant differences in viable, apoptotic or necrotic cells compared to untreated controls.
TRD = Taurolidin, Prop. = proportional, Anti-Prop. = anti-proportional
Ø = no significant effect
Bold print = TRD concentration (in μM) with the highest reduction of viable cells after 6 h and 24 h.
Figure 3 Representative dot plots obtained by FACS-anaylsis after incubation of different cell lines with Taurolidine. Chang Liver,
HT1080 and BxPC-3 cells were incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and with Povidon 5% (control) for 24 h. FACSanalysis was performed for Annexin V-FITC (x-axis) and Propidiumiodide (y-axis). Lower left quadrant: Annexin V and propidium iodide negative
(viable), lower right quadrant: Annexin V positive and propidium iodide negative (apoptotic), upper right quadrant: Annexin V and propidium
iodide positive (necrotic).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
viability which was not different from untreated controls
(fig. 4a). This effect was related to a significant reduction of apoptotic cells compared to TRD alone (fig. 4b).
Consistent with this finding, co-incubation with the glutathione depleting compound BSO for 24 h led to a significant enhancement of TRD induced cell death
which was caused by a significant increase in necrosis
(fig. 5a+c) (table 2). However, BSO itself also reduced
cell viability significantly through pronounced necrosis
(fig. 5a+c) (table 2).
In AsPC-1 cells, NAC co-incubation was characterized
by a strong reduction of necrosis compared to TRD
alone (fig. 6c). Together with a small - but significant increase in apoptotic cells (fig. 6b) this effect led to a
significant increase in viable cells compared to TRD
alone (fig. 6a). However, there was no complete recovery
Page 7 of 16
in the proportion of viable cells compared to untreated
controls (fig. 6a). For that reason the effect could only
be designated as partial protection (table 2). In line with
the protective effects of NAC, co-incubation with BSO
resulted in a significant increase of necrotic cells compared to TRD alone (fig. 7c) leading to a deleterious
effect on cell viability after (fig. 7a). It is important to
note, that BSO as a single agent had no significant effect
on cell viability, apoptosis and necrosis in this particular
cell line (fig. 7a-c).
The second pancreatic cancer cell line, BxPC3, showed
some similarities with AsPC-1 cells regarding the
response to NAC and BSO co-incubation (fig. 6+7;d-f).
A partial protective effect of NAC co-incubation could
be demonstrated leading to a significant increase in
viable cells compared to TRD alone without full
Figure 4 Effects of N-acetylcysteine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells. HT29 (a-c), Chang Liver
(d-f) and HT1080 cells (g-i) were incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM) or the
combination of both agents (TRD 250 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic
(b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4
(HT29 and Chang Liver) and 12 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences
between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 8 of 16
Figure 5 Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells. HT29 (a-c),
Chang Liver (d-f) and HT1080 cells (g-i) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM),
Taurolidine (TRD) (250 μM) or the combination of both agents (TRD 250 μM + BSO 1 mM) and with Povidon 5% (control) for 24 h. The
percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells (c, f, i) were determined by FACS-analysis for Annexin V-FITC and
Propidiumiodide. Values are means ± SEM of 9 (HT29 and HT1080) and 4 (Chang Liver) independent experiments with consecutive passages.
Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
recovery compared to untreated controls (fig. 6d). This
partial recovery by NAC was again related to a reduction of necrotic cells compared to TRD alone (fig. 6f)
(table 2). Unlike AsPC-1 cells, BxPC-3 cells responded
to BSO as a single agent with a significant reduction of
viable cells compared to untreated controls (fig. 7d+f).
Nevertheless, there was again a significant deleterious
effect of BSO co-incubation with TRD on cell viability
compared to TRD or BSO alone (fig. 7d), which was
related to a strong enhancement of apoptosis (fig. 7e).
Chang Liver cells responded least to NAC and BSO
co-incubation (fig. 4+5; d-f). However after 24 h, a partial protection by NAC co-incubation could be encountered leading to a significant increase in viable cells
compared to TRD alone without complete recovery
compared to control treatment (fig. 4d). The partial
protective effect was characterized by a significant
decrease in apoptotic cells compared to TRD alone (fig.
4e+f). Co-incubation with BSO did not result in any significant effect on cell viability, apoptosis and necrosis
compared to TRD alone (fig. 6d-f) (table 2).
Compared to all other cell lines, HT1080 cells were
characterized by a unique and occasionally completely
contrary response to radical scavenging by NAC (fig.
4g-i). NAC co-incubation did not result in cell rescue
but led to further significant reduction of viable cells
compared to TRD alone (fig. 4g). This deleterious effect
of NAC was mirrored by significantly enhanced apoptosis and necrosis compared to TRD alone (fig. 4h+i). Coincubation with BSO did not result in any significant
effect on cell viability, apoptosis and necrosis compared
to TRD alone (fig. 5g-i).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 9 of 16
Table 2 Effect of N-Acetylcystein, DL-buthionin-(S,R)-sulfoximine or z-VAD co-incubation with Taurolidine in different
cell lines
HT29
Chang Liver
HT1080
AsPC-1
BxPC-3
Viable:
Ø
Ø
Ø
CoProt
Ø
Apo/Nec:
Apo⇓
Ø
Nec⇓
Nec⇓
Ø
NAC+TRD 24 h
Viable:
Apo/Nec:
CoProt
Apo⇓
PaProt.
Apo⇓
Del
Apo⇑ Nec⇑
PaProt
Nec⇓ Apo⇑
PaProt
Nec⇓
BSO alone 6 h
Viable:
Ø
Ø
Ø
Ø
Ø
Apo/Nec
Ø
Ø
Ø
Ø
Ø
Viable:
Ø
Ø
Ø
Ø
Del
Apo/Nec:
Ø
Ø
Nec⇓
Nec⇑ Apo⇓
Nec⇑
BSO alone 24 h
Viable:
Del
Ø
Ø
Ø
Del
Apo/Nec:
Nec⇑
Ø
Ø
Ø
Nec⇑
BSO+TRD 24 h
Viable:
Del
Ø
Ø
Del
Del
Apo/Nec:
Nec⇑
Ø
Ø
Nec⇑
Apo⇑ Nec⇓
Viable:
CoProt
PaProt
PaProt
Ø
Ø
Apo/Nec:
Apo⇓
Ø
Nec⇓
Nec⇑
Nec⇓
NAC+TRD 6 h
BSO+TRD 6 h
z-VAD+ TRD 24 h
Effect of N-Acetylcystein (NAC), DL-buthionin-(S,R)-sulfoximine (BSO) or z-VAD co-incubation with Taurolidin (TRD) in different cell lines measured by FACS
analysis (Annexin V/Propidium Iodide).
NAC = N-Acetylcysteine
BSO = DL-buthionin-(S,R)-sulfoximine
TRD = Taurolidine
Viable = viable cells
Apo = apoptotic cells
Nec = necrotit cells
Ø = no significant effect
⇓ = significant decrease
⇑ = significant increase
CoProt. = complete protection
PaProt. = partial protection
Del. = deleterious
The results for 6 hours co-incubation with NAC and
BSO are provided in additional file 2 and 3, respectively
and summarized in table 2.
The reversibility of TRD induced cell death by caspase
inhibition is divergent and cell line specific
Overall, there was no effect on cell viability, apoptosis
or necrosis of z-VAD alone in any of the five cell lines.
HT29 was the only cell line with a complete protection
of TRD induced cell death by z-VAD co-incubation
and thus a complete reversibility of TRD induced cell
death (fig. 8a). The relatively mild reduction of viable
cells by TRD to 69.6% ± 0.3% was significantly abrogated by z-VAD co-incubation and not different from
untreated controls (fig. 8a). The protective effect was
associated with a significant decrease of apoptotic cells
(fig. 8b) without any detectable effect on necrosis
(fig. 8c).
In Chang Liver and HT1080 cells, the TRD induced
cell death was only partially reversible by z-VAD dependent caspase inhibition. The rescue effect of z-VAD coincubation did not lead to the same cell viability like
untreated controls. In Chang Liver cells, the protective
effect of z-VAD co-incubation compared to TRD alone
was relatively small (45.7% ± 1.8% vs. 37.4% ± 2.6%)
although it reached statistical significance (fig. 8d). This
partial rescue effect of z-VAD was paralleled by a small
and non-significant reduction of both apoptotic and
necrotic cells (fig. 8e+f). HT1080 cells responded similar
to z-VAD co-incubation with a partial protective effect
characterized by a significantly increased cell viability
compared to TRD alone but not compared to untreated
(fig. 8g). The partial protection by z-VAD was mainly
achieved by a significant reduction of necrosis (fig. 8i).
Both pancreatic cancer cell-lines, AsPC-1 and BxPC-3
did not show any detectable effect on cell viability after
z-VAD co-incubation. In AsPC-1 cells, TRD 1000 μM
induced reduction of viable cells could not be reversed
by z-VAD co-incubation (fig. 9a). In contrast, z-VAD
co-incubation resulted in a significant increase in necrotic cells (fig. 9c). In BxPC-3 cells, the TRD induced
reduction of viable cells could not significantly be
reversed by z-VAD co-incubation (fig. 9d) although
there was a significant decrease in necrotic cells following z-VAD co-incubation compared to TRD alone (fig.
9f) (table 2).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 10 of 16
Figure 6 Effects of N-acetylcysteine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells. AsPC-1 (a-c) and BxPC-3 cells (d-f) were
incubated with either the radical scavenger N-acetylcysteine (NAC) (5 mM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the
combination of both agents (TRD 250 μM/1000 μM + NAC 5 mM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d),
apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 4
independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups. *** p ≤ 0.001,
** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 11 of 16
Figure 7 Effects of DL-buthionin-(S,R)-sulfoximine on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells. AsPC-1 (a-c) and BxPC-3
cells (d-f) were incubated with either the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine(BSO) (1 mM), Taurolidine (TRD) (250 μM for
BxPC-3 and 1000 μM for AsPC-1) or the combination of both agents (TRD 250 μM/1000 μM + BSO 1 mM) and with Povidon 5% (control) for 24
h. The percentages of viable (a, d), apoptotic (b, e) and necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and
Propidiumiodide. Values are means ± SEM of 4 independent experiments with consecutive passages. Asterisk symbols on brackets indicate
differences between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 12 of 16
Figure 8 Effects of caspase-inhibition on Taurolidine induced cell death in HT29, Chang Liver and HT1080 cells. HT29 (a-c), Chang Liver
(d-f) and HT1080 cells (g-i) were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD
250 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d, g), apoptotic (b, e, h) and necrotic cells
(c, f, i) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 5 (HT29), 6 (Chang Liver) and
4 (HT1080) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups.
*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Discussion
Although the anti-neoplastic effects of TRD have been
extensivley analyzed in vitro by proliferation assays like
BrdU or MTT [12-14,27,28,32], only few studies have
exploited the potential of FACS analysis to differentiate
in a quantitative manner between apoptotic and necrotic
cell death [13,26,33,34]. Furthermore, all available studies were performed on single cell lines or on different
cell lines of one particular malignancy. There is a lack
of a comparative analysis of TRD effects in cell lines of
different malignancies including pancreatic cancer.
Therefore, in the first part of this study we sought to
determine dose-response characteristics and relative
contribution of apoptosis and necrosis of TRD induced
cell death simultaneously in 5 cell lines from 4 malignancies. Surprisingly, dose response effects of TRD were
not homogenous among the 5 cell lines. In fact, we
found three different patterns of dose response: proportional, V-shaped and anti-proportional dose effects. The
two pancreatic cancer cell lines BxPC-3 and AsPC-1
which have never been tested before, were characterized
by a proportional dose effect. Increasing concentrations
of TRD led to increasing cell death after 6 and 24
hours. The proportional dose effect pattern of TRD has
been described in the majority of available studies either
by proliferation assays [11-13,28,32,35], FACS analysis
[7] or other cell viability/toxicity assays [9,27,36]. BxPC3 cells displayed also a dose dependency regarding the
relative contribution of necrotic and apoptotic cell
death. The response on cell viability upon incubation
with TRD 250 μM for 24 hours was characterized by a
mixed apoptotic and necrotic effect whereas TRD 1000
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Page 13 of 16
Figure 9 Effects of caspase-inhibition on Taurolidine induced cell death in AsPC-1 and BxPC-3 cells. AsPC-1 (a-c) and BxPC-3 cells (d-f)
were incubated with either z-VAD.fmk (1 μM), Taurolidine (TRD) (250 μM for BxPC-3 and 1000 μM for AsPC-1) or the combination of both
agents (TRD 250 μM/1000 μM + zVAD.fmk 1 μM) and with Povidon 5% (control) for 24 h. The percentages of viable (a, d), apoptotic (b, e) and
necrotic cells (c, f) were determined by FACS-analysis for Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 3 (AsPC-1) and 6
(BxPC-3) independent experiments with consecutive passages. Asterisk symbols on brackets indicate differences between treatment groups.
*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
μM was characterized by an exclusive and pronounced
necrotic effect. This phenomenon became even more
obvious in AsPC-1 cells, were TRD 1000 μM led to a
strong necrotic effect. The observed dose dependency of
apoptotic and necrotic cell death is consistent with previous studies by others [27] as well as by our group
[6,26,34]. The V-shaped dose effect was found in HT29
cells as well as in Chang Liver cells and was characterized by a dose response with maximal effects on cell viability and apoptosis with the intermediated
concentration of TRD 250 μM whereas the highest
(TRD 1000 μM) and lowest (TRD 100 μM) concentrations were less effective. This V-shaped dose effect has
been described only once by our group [34]. However,
to our surprise HT1080 cells presented in the current
study with a anti-proportional dose effect with decreasing effects on cell viability and apoptosis upon treatment
for 24 h with increasing TRD concentrations. We can
only speculate about the reason for this inverse proportionality. Our assays were repeated with nine consecutive passages, thus excluding biological assay variability
as a possible explanation for this unusual finding.
The second part of the study comprised the evaluation
of the contribution of reactive oxygen species (ROS) to
TRD induced PCD by co-incubation experiments with
either the radical scavenger N-acetylcysteine (NAC) or
the glutathione depleting agent DL-buthionin-(S,R)-sulfoximine (BSO). Previous studies have presented first
evidence for involvement of TRD mediated ROS production [9,13,36]. Furthermore, cell death induced by
TRD has been shown to be reversible by application of
radical scavengers like NAC [9,12,13,36] and to be
enhanced by inhibitors of ROS detoxification like BSO
[9]. In our study, all cell lines except HT1080 fibrosarcoma cells responded to NAC co-incubation with an
attenuation of TRD induced cell death. However, the
magnitude of protection was divergent among cell lines
ranging from partial protection (Chang Liver, AsPC-1,
BxPC-3) to complete protection (HT29). To our surprise and in contrast to the available literature, HT1080
cells presented a completely contrary response to radical
scavenging by NAC leading to enhancement rather than
attenuation of TRD induced cell death. The biological
cause behind this unexpected response pattern is currently unknown. However, ROS can be regarded as a
“double edged sword” in terms of anti-neoplastic activity
[37]. Excessive ROS generation in tumor cells and subsequent activation of PCD is a well known therapeutic
principle of many chemotherapeutics e.g. anthracyclines,
platinum or arsenic [37-40]. On the other hand, ROS
can promote tumor cell proliferation and survival under
certain circumstances [37,41] and anti-oxidant therapeutics may provide anti-neoplastic activity by inhibiting
Page 14 of 16
ROS production [37]. In conclusion, generation of ROS
and activation of subsequent pathways does explain
TRD induced cell death in many, but obviously not in
every cell line or malignancy. ROS generation is rather
unlikely to be the universal key mechanism of TRD
induced PCD in all cell lines.
The second major cell death associated pathway analyzed in this study was the caspase pathway by applying
the pan caspase inhibitor z-VAD. Activation of the caspase pathway by TRD has been reported in several
malignant cell lines [12,13,15,22]. Concordant with the
divergent and cell line specific results of our ROS
experiments - we encountered an inhomogeneous
response to co-treatment with z-VAD among our 5 cell
lines. Z-VAD was capable of protecting tumor cells
from TRD induced cell death only in HT29 (complete
protection), Chang Liver and HT1080 cells (partial protection), but both pancreatic cancer cell lines AsPC-1
and BxPC-3 were not protected at all. Comparable
divergent findings about the contribution of caspase
activity to TRD induced cell death have recently been
reported by others [9,15,28,36] suggesting both caspase
dependent and independent pathways [12]. During the
last years, it became clear that PCD can occur independently of caspase activation which is no longer regarded
as a mandatory feature of PCD [20,42,43]. Interestingly,
AIF (apoptosis inducing factor) representing a key protein in caspase independent PCD has recently been
shown to be involved in TRD induced cell death [9,36].
However, no study has provided a comparative analysis
of caspase inhibition and TRD simultaneously in different cell lines.
The herein observed divergent response in cell lines of
different malignancies towards inhibition of TRD
induced cell death by z-VAD as well as by NAC leads to
the assumption, that there is a cell line specificity
regarding involvement of caspases and ROS following
TRD treatment. Further studies are necessary to elucidate the different types of programmed cell death following TRD treatment.
Conclusions
This is the first study providing a simultaneous evaluation of TRD induced cell death across several cell lines
of different malignancies. TRD is characterized by cell
line specific dose response effects and dose response
patterns. However, all cell lines were susceptible to TRD
induced cell death without any resistance. Functional
analysis for involvement of ROS driven cell death and
caspase activation revealed substantial cancer cell type
specific differences for both routes of cell death. Thus,
TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
Additional file 1: Effects of Taurolidine on viability, apoptosis and
necrosis in HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 cells
after 6 h. HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 cells were
incubated with Taurolidine (TRD) (100 μM, 250 μM and 1000 μM) and
with Povidon 5% (control) for 6 h. The percentages of viable (vital),
apoptotic (apo) and necrotic cells (necr) were determined by FACSanalysis for Annexin V-FITC and Propidiumiodide. Values are means ±
SEM of 5 (HT29), 4 (Chang Liver, AsPC-1 and BxPC-3) and 9 (HT1080)
independent experiments with consecutive passages. Asterisk symbols
on columns indicate differences between control and TRD treatment.
Asterisk symbols on brackets indicate differences between TRD groups.
*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1756-9966-2921-S1.JPEG ]
Additional file 2: Effects of N-acetylcysteine on Taurolidine induced
cell death in HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 cells
after 6 h. HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 cells were
incubated with either the radical scavenger N-acetylcysteine (NAC) (5
mM), Taurolidine (TRD) (250 μM) or the combination of both agents (TRD
250 μM + NAC 5 mM) and with Povidon 5% (control) for 6 h. The
percentages of viable (vital), apoptotic (apo) and necrotic cells (necr)
were determined by FACS-analysis for Annexin V-FITC and
Propidiumiodide. Values are means ± SEM of 4 (HT29, Chang Liver, AsPC1 and BxPC-3) and 12 (HT1080) independent experiments with
consecutive passages. Asterisk symbols on brackets indicate differences
between treatment groups. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 (oneway ANOVA).
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1756-9966-2921-S2.JPEG ]
Additional file 3: Effects of DL-buthionin-(S,R)-sulfoximine on
Taurolidine induced cell death in HT29, Chang Liver, HT1080, AsPC1 and BxPC-3 cells after 6 h. HT29, Chang Liver, HT1080, AsPC-1 and
BxPC-3 cells were incubated with either the glutathione depleting agent
DL-buthionin-(S,R)-sulfoximine (BSO) (1 mM), Taurolidine (TRD) (250 μM)
or the combination of both agents (TRD 250 μM + BSO 1 mM) and with
Povidon 5% (control) for 6 h. The percentages of viable (vital), apoptotic
(apo) and necrotic cells (necr) were determined by FACS-analysis for
Annexin V-FITC and Propidiumiodide. Values are means ± SEM of 9
(HT29 and HT1080) and 4 (Chang Liver, AsPC-1 and BxPC-3) independent
experiments with consecutive passages. Asterisk symbols on brackets
indicate differences between treatment groups. *** p ≤ 0.001,
** p ≤ 0.01, * p ≤ 0.05 (one-way ANOVA).
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1756-9966-2921-S3.JPEG ]
Abbreviations
BSO: DL-buthionin-(S,R)-sulfoximine; PCD: Programmed cell death; NAC: NAcetylcysteine; ROS: Reactive oxygen species; TRAIL: Tumor Necrosis Factor
Related Apoptosis Inducing Ligand; TRD: Taurolidine
Acknowledgements
The authors thank Prof Dr W.E. Schmidt (Department of Internal Medicine I,
St Josef Hospital, Ruhr-University of Bochum) as well as Prof Dr A. Mügge
(Department of Internal Medicine II, St Josef Hospital, Ruhr-University of
Bochum) for generously supporting cell culture experiments and FACS
analysis. Furthermore, they thank Ilka Werner, Kirsten Mros and Rainer Lebert
(Gastrointestinal Research Laboratory, St. Josef Hospital, Ruhr-University of
Bochum) for technical assistance. This study was supported by FoRUM AZ
F472-2005 and FoRUM AZ F544-2006 from the Ruhr-University Bochum,
Germany.
Author details
1
Department of Visceral and General Surgery, St Josef Hospital, RuhrUniversity Bochum, Germany. 2Department of Plastic Surgery, Burn Centre,
Hand Centre, Sarcoma Reference Centre, BG University Hospital
Page 15 of 16
Bergmannsheil GmbH, Ruhr-University Bochum, Germany. 3Department of
Medicine II, St Josef Hospital, Ruhr-University Bochum, Germany.
4
Department of Medicine I, St Josef Hospital, Ruhr-University Bochum,
Germany. 5Department of Molecular Gastrointestinal Oncology, RuhrUniversity Bochum, Germany.
Authors’ contributions
AMC and AD conceived of the study and its design, coordinated the
experiments, carried out the statistical analysis and drafted the manuscript.
AF supervised the cell culture experiments and carried out the inhibitor
experiments. DB was responsible for adjusting the FACS analysis and helped
to draft the manuscript. CM, KH and JR carried out the cell culture
experiments. DS helped with the statistical analysis and revised manuscript.
PR, UM, SH and WU participated in the design and coordination of the
study and revised the manuscript. All authors have read and approved the
final manuscript.
Competing interests
AMC received financial support by Geistlich Pharma (Suisse) for laboratory
experiments. All other authors declare that they have no competing
interests.
Received: 16 January 2010 Accepted: 7 March 2010
Published: 7 March 2010
References
1. Baker DM, Jones JA, Nguyen-Van-Tam JS, Lloyd JH, Morris DL, Bourke JB,
Steele RJ, Hardcastle JD: Taurolidine peritoneal lavage as prophylaxis
against infection after elective colorectal surgery. Br J Surg 1994,
81:1054-1056.
2. Simon A, Ammann RA, Wiszniewsky G, Bode U, Fleischhack G, Besuden MM:
Taurolidine-citrate lock solution (TauroLock) significantly reduces CVADassociated grampositive infections in pediatric cancer patients. BMC
Infect Dis 2008, 8:102.
3. Koldehoff M, Zakrzewski JL: Taurolidine is effective in the treatment of
central venous catheter-related bloodstream infections in cancer
patients. Int J Antimicrob Agents 2004, 24:491-495.
4. Jacobi CA, Menenakos C, Braumann C: Taurolidine–a new drug with antitumor and anti-angiogenic effects. Anticancer Drugs 2005, 16:917-921.
5. Braumann C, Schoenbeck M, Menenakos C, Kilian M, Jacobi CA: Effects of
increasing doses of a bolus injection and an intravenous long-term
therapy of taurolidine on subcutaneous (metastatic) tumor growth in
rats. Clin Exp Metastasis 2005, 22:77-83.
6. Chromik AM, Daigeler A, Hilgert C, Bulut D, Geisler A, Liu V, Otte JM, Uhl W,
Mittelkotter U: Synergistic effects in apoptosis induction by taurolidine
and TRAIL in HCT-15 colon carcinoma cells. J Invest Surg 2007, 20:339-348.
7. Daigeler A, Chromik AM, Geisler A, Bulut D, Hilgert C, Krieg A, KleinHitpass L, Lehnhardt M, Uhl W, Mittelkötter U: Synergistic apoptotic effects
of taurolidine and TRAIL on squamous carcinoma cells of the
esophagus. Int J Oncol 2008, 32:1205-1220.
8. Stendel R, Stoltenburg-Didinger G, Al Keikh CL, Wattrodt M, Brock M: The
effect of taurolidine on brain tumor cells. Anticancer Res 2002, 22:809-814.
9. Stendel R, Biefer HR, Dekany GM, Kubota H, Munz C, Wang S, Mohler H,
Yonekawa Y, Frei K: The antibacterial substance taurolidine exhibits antineoplastic action based on a mixed type of programmed cell death.
Autophagy 2009, 5:194-210.
10. Braumann C, Jacobi CA, Rogalla S, Menenakos C, Fuehrer K, Trefzer U,
Hofmann M: The tumor suppressive reagent taurolidine inhibits growth
of malignant melanoma–a mouse model. J Surg Res 2007, 143:372-378.
11. Sun BS, Wang JH, Liu LL, Gong SL, Redmond HP: Taurolidine induces
apoptosis of murine melanoma cells in vitro and in vivo by modulation
of the Bcl-2 family proteins. J Surg Oncol 2007, 96:241-248.
12. Opitz I, Sigrist B, Hillinger S, Lardinois D, Stahel R, Weder W, HopkinsDonaldson S: Taurolidine and povidone-iodine induce different types of
cell death in malignant pleural mesothelioma. Lung Cancer 2007,
56:327-336.
13. Aceto N, Bertino P, Barbone D, Tassi G, Manzo L, Porta C, Mutti L,
Gaudino G: Taurolidine and oxidative stress: a rationale for local
treatment of mesothelioma. Eur Respir J 2009, 34:1399-1407.
14. Daigeler A, Brenzel C, Bulut D, Geisler A, Hilgert C, Lehnhardt M,
Steinau HU, Flier A, Steinstraesser L, Klein-Hitpass L, et al: TRAIL and
Chromik et al. Journal of Experimental & Clinical Cancer Research 2010, 29:21
http://www.jeccr.com/content/29/1/21
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Taurolidine induce apoptosis and decrease proliferation in human
fibrosarcoma. J Exp Clin Cancer Res 2008, 27:82.
Walters DK, Muff R, Langsam B, Gruber P, Born W, Fuchs B: Taurolidine: a
novel anti-neoplastic agent induces apoptosis of osteosarcoma cell lines.
Invest New Drugs 2007, 25:305-312.
Braumann C, Winkler G, Rogalla P, Menenakos C, Jacobi CA: Prevention of
disease progression in a patient with a gastric cancer-re-recurrence.
Outcome after intravenous treatment with the novel antineoplastic
agent taurolidine. Report of a case. World J Surg Oncol 2006, 4:34.
Stendel R, Picht T, Schilling A, Heidenreich J, Loddenkemper C, Janisch W,
Brock M: Treatment of glioblastoma with intravenous taurolidine. First
clinical experience. Anticancer Res 2004, 24:1143-1147.
Stendel R, Scheurer L, Schlatterer K, Stalder U, Pfirrmann RW, Fiss I,
Mohler H, Bigler L: Pharmacokinetics of taurolidine following repeated
intravenous infusions measured by HPLC-ESI-MS/MS of the derivatives
taurultame and taurinamide in glioblastoma patients. Clin Pharmacokinet
2007, 46:513-524.
Gong L, Greenberg HE, Perhach JL, Waldman SA, Kraft WK: The
pharmacokinetics of taurolidine metabolites in healthy volunteers. J Clin
Pharmacol 2007, 47:697-703.
Hotchkiss RS, Strasser A, McDunn JE, Swanson PE: Cell death. N Engl J Med
2009, 361:1570-1583.
Hail N Jr, Carter BZ, Konopleva M, Andreeff M: Apoptosis effector
mechanisms: a requiem performed in different keys. Apoptosis 2006,
11:889-904.
Darnowski JW, Goulette FA, Cousens LP, Chatterjee D, Calabresi P:
Mechanistic and antineoplastic evaluation of taurolidine in the DU145
model of human prostate cancer. Cancer Chemother Pharmacol 2004,
54:249-258.
Han Z, Ribbizi I, Pantazis P, Wyche J, Darnowski J, Calabresi P: The
antibacterial drug taurolidine induces apoptosis by a mitochondrial
cytochrome c-dependent mechanism. Anticancer Res 2002, 22:1959-1964.
Rodak R, Kubota H, Ishihara H, Eugster HP, Konu D, Mohler H, Yonekawa Y,
Frei K: Induction of reactive oxygen intermediates-dependent
programmed cell death in human malignant ex vivo glioma cells and
inhibition of the vascular endothelial growth factor production by
taurolidine. J Neurosurg 2005, 102:1055-1068.
Stendel R, Scheurer L, Stoltenburg-Didinger G, Brock M, Mohler H:
Enhancement of Fas-ligand-mediated programmed cell death by
taurolidine. Anticancer Res 2003, 23:2309-2314.
Daigeler A, Chromik AM, Geisler A, Bulut D, Hilgert C, Krieg A, KleinHitpass L, Lehnhardt M, Uhl W, Mittelkotter U: Synergistic apoptotic effects
of taurolidine and TRAIL on squamous carcinoma cells of the
esophagus. Int J Oncol 2008, 32(6):1205-20.
McCourt M, Wang JH, Sookhai S, Redmond HP: Taurolidine inhibits tumor
cell growth in vitro and in vivo. Ann Surg Oncol 2000, 7:685-691.
Nici L, Monfils B, Calabresi P: The effects of taurolidine, a novel
antineoplastic agent, on human malignant mesothelioma. Clin Cancer Res
2004, 10:7655-7661.
Opitz I, Veen Van der H, Witte N, Braumann C, Mueller JM, Jacobi CA:
Instillation of taurolidine/heparin after laparotomy reduces
intraperitoneal tumour growth in a colon cancer rat model. Eur Surg Res
2007, 39:129-135.
Griffith OW, Meister A: Potent and specific inhibition of glutathione
synthesis by buthionine sulfoximine (S-n-butyl homocysteine
sulfoximine). J Biol Chem 1979, 254:7558-7560.
Estrela JM, Ortega A, Obrador E: Glutathione in cancer biology and
therapy. Crit Rev Clin Lab Sci 2006, 43:143-181.
Braumann C, Henke W, Jacobi CA, Dubiel W: The tumor-suppressive
reagent taurolidine is an inhibitor of protein biosynthesis. Int J Cancer
2004, 112:225-230.
Chromik AM, Daigeler A, Hilgert C, Bulut D, Geisler A, Liu V, Otte JM, Uhl W,
Mittelkötter U: Synergistic effects in apoptosis induction by taurolidine
and TRAIL in HCT-15 colon carcinoma cells. J of Investigative Surgery 2007,
20:339-348.
Daigeler A, Brenzel C, Bulut D, Geisler A, Hilgert C, Lehnhardt M,
Steinau HU, Flier A, Steinstraesser L, Klein-Hitpass L, et al: TRAIL and
Taurolidine induce apoptosis and decrease proliferation in human
fibrosarcoma. J Exp Clin Cancer Res 2008, 27:82.
Abramjuk C, Bueschges M, Schnorr J, Jung K, Staack A, Lein M: Divergent
effects of taurolidine as potential anti-neoplastic agent: Inhibition of
36.
37.
38.
39.
40.
41.
42.
43.
Page 16 of 16
bladder carcinoma cells in vitro and promotion of bladder tumor in
vivo. Oncol Rep 2009, 22:409-414.
Rodak R, Kubota H, Ishihara H, Eugster H-P, Könü D, Möhler H, Yonekawa Y,
Frei K: Induction of reactive oxygen intermediates-dependent
programmed cell death in human malignant ex vivo glioma cells and
inhibition of the vascular endothelial growth factor production by
taurolidine. J Neurosurg 2005, 102:1055-1068.
Wang J, Yi J: Cancer cell killing via ROS: to increase or decrease, that is
the question. Cancer Biol Ther 2008, 7:1875-1884.
Conklin KA: Chemotherapy-associated oxidative stress: impact on
chemotherapeutic effectiveness. Integr Cancer Ther 2004, 3:294-300.
Engel RH, Evens AM: Oxidative stress and apoptosis: a new treatment
paradigm in cancer. Front Biosci 2006, 11:300-312.
Ozben T: Oxidative stress and apoptosis: impact on cancer therapy.
J Pharm Sci 2007, 96:2181-2196.
Chan DW, Liu VW, Tsao GS, Yao KM, Furukawa T, Chan KK, Ngan HY: Loss
of MKP3 mediated by oxidative stress enhances tumorigenicity and
chemoresistance of ovarian cancer cells. Carcinogenesis 2008,
29:1742-1750.
Constantinou C, Papas KA, Constantinou AI: Caspase-independent
pathways of programmed cell death: the unraveling of new targets of
cancer therapy? Curr Cancer Drug Targets 2009, 9:717-728.
de Bruin EC, Medema JP: Apoptosis and non-apoptotic deaths in cancer
development and treatment response. Cancer Treat Rev 2008, 34:737-749.
doi:10.1186/1756-9966-29-21
Cite this article as: Chromik et al.: Comparative analysis of cell death
induction by Taurolidine in different malignant human cancer cell lines.
Journal of Experimental & Clinical Cancer Research 2010 29:21.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit