The antibacterial substance taurolidine exhibits anti-neoplastic action based on a mixed type of ...

17 Pages·2009·1.63 MB·English

[Autophagy 5:2, 194-210; 16 February 2009]; ©2009 Landes Bioscience
Research Paper
The antibacterial substance taurolidine exhibits anti-neoplastic action
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based on a mixed type of programmed cell death
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Ruediger Stendel,1,* Hector Rodriguez Cetina Biefer,2 Gabriela Marta Dékány,2 Hisashi Kubota,2 Christian Münz,3 Sheng
Wang,2 Hanns Mohler,4 Yasuhiro Yonekawa2 and Karl Frei2 i
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1Department of Neurosurgery; Charité—University Medicine Berlin; Berlin, Germany; 2Department of Neurosurgery; University Hospital Zurich; Zürich, Switzerland; 3Laboratory
s
of Viral Immunobiology; Institute of Experimental Immunology; University Hospital Zurich; Zurich, Switzerland; 4Institute of Pharmacology and Toxicology; University of Zurich and
Department of Applied Biosciences; Swiss Federal Institute of Technology; Zurich, Switzerland i
d
Abbreviations: 3-MA, 3-methyladenine; Ab, antibody; AIF, apoptosis inducing factor; AO, acridine orange; Atg, autophagy gene; ATP,
adenosine triphosphate; AVO, acidic vesicular organelles; BfA1, bafilomycine A1; BSO, DL-buthionin-(S,R )-sulfoximine; CP, cytoplasm;
t
CQ, chloroquine; DNA, deoxyribo-nucleic acid; Cyt-C, cytochrome-C; EC , effective concentration ; FasL, fas-ligand; GBM, glioblas-
50 o50
toma; GFM, glucose free medium; GFP, green fluorescent protein; LC3, microtubule-associated protein light chain 3; MV, microvilli; NAC,
n
N-acetylcysteine; OD, optic density; OM, oligomycin; PBS, phosphate-buffered saline; PCD, programmed cell death; PS, phosphatidyl-
serine; Rapa, rapamycin; ROS, reactive oxygen substrates; RFU, relative fluorescence units; SA- β-gal, senescence-associated-β-galactosidase;
o
siRNA, small interference ribo-nucleic acid; SMAC, second mitochondria derived activator of caspase; SOD, superoxid dismutase; TAU,
taurolidine; TEM, transmission electron microscopy; TMZ, temozolomide; V, vacuoles; WD B, western blot
Key words: autophagy, apoptosis, glioma, brain tumor, glioblastoma, programmed c ell death
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The antibacterial amino-acid derivative taurolidine (TAU) has bneen shown that this resistance could be related to alterations of
been recently shown to exhibit anti-neoplastic activity based on genes participating in the apoptotic pathway.2 This is the rationale
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a mechanism, which is still unknown in detail. Cytotoxicity and for extending the search for anti-neoplastic drugs acting on alterna-
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clonogenic assays were performed and the impact of apoptosis tive mechanisms.
c
modulators, a radical scavenger, autophagy inhibitors, silencing TAU has been recently shown to exert direct and selective
of apoptosis inducing actor (AIF) and cytochrome-c s(Cyt-C) by anti-neoplastic activity in vitro3-7 and in vivo in animals8-10 and
siRNA, and knockdown of autophagy related genoes were evalu- in humans.11 Moreover, TAU has been demonstrated to inhibit
ated in vitro. The intracellular ATP-content, rielease of AIF and endothelial cell adhesion and angiogenesis in vitro.12
Cyt-C, and DNA-laddering were investigateBd. This study could Induction of PCD was identified as the underlying mechanism
demonstrate cell killing, inhibition of proliferation, and inhibition of the anti-neoplastic effect, which has shown to be based on the

or prevention of colony formation in hsuman glioma cell lines and generation of ROS.3-5,13 Although a defined mechanistic model
ex vivo glioblastoma cells after incubation with TAU. This effect is for the role of ROS in the apoptotic pathway is still lacking, it has
e
based on the induction of a mixed type of programmed cell death been reported that the increase of the intracellular ROS level leads
with the main preference of audtophagy, and involvement of senes- to induction of apoptosis.14 In this case, special ultrastructural and
cence, necroptosis and necnrosis. This mechanism of action may biochemical changes should occur following initiation of cell death
open a new approach for therapeutic intervention. by TAU. We therefore investigated the mechanisms of TAU-induced
a
cell death in glioma cells.
Introduction
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Results
Despite inte nsive research, currently available treatment modali-
9
ties have only marginally improved survival of patients with
Experimental design. Morphological alterations following TAU
malignan0t gliomas, especially with the most malignant form, the
treatment have been investigated by light and electron microscopy.
glioblastoma.1 A known feature of glioblastoma is its intrinsic relative
0 The effect on tumor cell viability and proliferation has been deter-
resistance to cytotoxic chemotherapy and ionizing radiation. It has
mined using cytotoxicity and colony assays.
2
To test the hypothesis that apoptosis is the underlying mechanism
© of TAU-induced cell death, flow cytometric analysis, pan caspase
*Correspondence to: Ruediger Stendel; Department of Neurosurgery; Charité— inhibition and highly sensitive caspase 3 and 7 assays have been
University Medicine Berlin; Charitéplatz 1; Berlin 10117 Germany; Tel.: [phone];
performed. Furthermore, it has been investigated whether Cyt-C
Fax: [phone]; Email: [email protected]
is released in response to TAU treatment, and if knockdown of the
Submitted: 11/14/07; Revised: 10/24/08; Accepted: 11/13/08 Cyt-C gene protects cells from TAU-induced cell death. In addition,
Previously published online as an Autophagy E-publication: it has been tested if DNA laddering occurs following TAU treatment.
http://www.landesbioscience.com/journals/autophagy/article/7404
194 Autophagy 2009; Vol. 5 Issue 2
TAU exhibits anti-neoplastic action
The effect of co-incubation with SMAC peptide and a PARP inhibi- 7.9 ± 0.6%, GBM-2: 5.0 ± 0.4%, GBM-3: 2.0 ± 0.1%, GBM-4: 3.0
tion has been investigated. ± 1.4%, GBM-5: no colony formation. Incubation with TAU caused
Because the results of the delineated experiments had shown that dose dependent inhibition or prevention of colony formation of all
the apoptotic pathway is not really effective in TAU-induced cell tumor cell lines and ex vivo GBM cells investigated. The mean EC50 .
death, other forms of PCD came into focus. The results of electron of TAU regarding inhibition of colony formation differed signifi- e
microscopy had suggested autophagy as a potential mechanism cantly and was significantly lower as compared to the concentratiton
involved. During induction of autophagy, phosphatidylethanolamine needed for acute cytotoxicity (t-test, p < 0.05, Table 1). u
is covalently linked to the cytosolic protein LC3-I to yield LC3-II, Cell membrane PS externalization and flow cytometric anal-
b
which then associates with the autophagosome.21-23 In this way, the ysis. Detection of cell membrane PS externalization demonstrated
tumor cells were transfected with GFP-LC3 expression vector and an increase of SYTOX-/AnnexinV+ (apoptotic) anid SYTOX+/
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conversion of LC3-I into LC3-II had been observed. Furthermore, AnnexinV- (necrotic) cells after TAU treatment (Fig. 2).
t
raised AVO formation is a typical feature in autophagy17 and can Pan-caspase inhibition and caspase 3/7 activity. While z-VAD.
s
therefore be inhibited by the autophagy inhibitor BfA1. BfA1 is fmk had no significant influence on the cell killing (Fig. 3A), there
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an H+-ATPase inhibitor and blocks the fusion of lysosomes and was a dose- and time-dependent increased of caspase 3 and 7 activity
autophagosomes.24 AVO-formation could be observed following following incubation with TAU (Fig. 3B left). However, no signifi-

AO staining. cant correlation could be found bettween cytotoxicity and caspase 3
Since knockdown of Atg-7 and Atg-12 led only to partial protec- and 7 activation upon TAU treoatment (Fig. 3B left). In addition,
tion from the cell death induced by 50 μg/ml TAU, and autophagy z-VAD.fmk completely prohibited caspase 3 and 7 activation (Fig.
n
inhibitors could only provide partial protection, the involvement of 3B right) without having significant impact on the cell killing (Fig.
other forms of PCD—necroptosis and senescence—has been inves- 3A). In contrast, NAC prevented caspase 3 and 7 activation (Fig.
o
tigated. 3B right) and was highly protective against TAU-induced cell killing
Finally, the influence of the radical scavenger NAC and gluta- (Fig. 5G). D
thione depletion has been investigated, considering the role of ROS Cyt-C and AIF release after incubation with TAU. Incubation

in the TAU-induced cell death. of U-373, .LN-229 and ex vivo GBM 5 cells with 100 μg/ml TAU
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Morphological features. Light microscopy after TAU exposure was associated with a translocation of AIF into the cytosol while no
revealed a concentration-dependent appearance of cell pyknosis releacse was seen in untreated cells. Cyt-C translocation could only be
while the outer nuclear structures were preserved. The cells lost their observed in LN-229 and U-373 cells to a very small extent, but not
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processes, eventually resulting in complete destruction of cell struc- in ex vivo GBM 5 cells (Fig. 3C).
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tures. Pre-incubation with the radical scavenger NAC protected the Silencing of AIF and Cyt-C genes by siRNA. Silencing of the
cells against TAU-mediated cell killing (Fig. 1A). i AIF gene by siRNA provided partial but significant protection by
c
A series of distinct chronological morphological changes could 50%–60% in U-373 cells at 50, 100 and 200 μg/ml TAU and in
be observed in electron microscopy: formation of intracsytoplasmic LN-229 cells by 15% only at 100 μg/ml TAU. There was no protec-
vacuoles, which appeared to increase in number and size with tive effect by silencing of the Cyt-C gene (Fig. 3D).
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increasing duration of incubation, loss of cytoplasmic microvilli, DNA laddering. U-373 and LN-229 cells did not show DNA
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onset of streaky condensation and partial marginalization of chro- ladder formation after incubation with 100 μg/ml TAU for 6 hours.
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matin, disintegration of cytoplasm, ongoing patchy condensation of There was likewise clear DNA ladder formation after 6-hour incuba-
chromatin with progression to stripe-shap ed condensation and full tion of the U-373 cells with 1 μg/ml anti-Fas antibody (Fig. 3F).
s
condensation of chromatin. The vacuoles seemed to contain partially Transfection with GFP-LC3 expression vector. The U-373
digested material (see magnificatione) (Fig. 1B). The mean count of cells were transiently transfected as described earlier, which resulted
vacuoles per U-373 cell increasesd from 1 ± 1 in untreated cells to 42.3 in >90% expression level after 6 weeks of transfection and G-418
± 4.2 cells after incubation with 100 μg/ml TAU (Chi-square-test; p selection. GFP-LC3 transfected U-373 cells were left untreated as a
n
< 0.05) for 3 hours and to 46.3 ± 10.2 following incubation with 100 control or treated by TAU at 20, 40 and 100 μg/ml. In addition, a
μg/ml TAU for 24 houras (Chi-square-test; p < 0.05). positive control with rapamycin was included. The vast majority of
Effect on tumorL cell viability and proliferation. Cell prolifera- the GFP-LC3-transfected U-373 cells showed diffuse distribution of
tion of U-373 cells was inhibited at about 60% at a concentration of GFP-LC3 without TAU, whereas treatment with TAU (20–100 μg/

2 μg/ml with9 no significant cytotoxicity. In the proliferation assay, ml) for 6 and 24 hours resulted in a punctate pattern of GFP-LC3
EC was <2 μg/ml (Fig. 1C), which is much lower as in the cyto- fluorescence (Fig. 4A). This pattern represents the autophagic vacu-
50 0
toxicity assay (Fig. 1D, Table 1). The higher sensitivity regarding cell oles in U-373 cells upon TAU treatment. Rapamycin induced the
proli0feration is also reflected by the differences in the EC values. same fluorescence pattern in U-373 cells (Fig. 4A). A histogram
50
I2ncubation with TAU caused cell killing in all human glioblastoma/ model from Leica LT software was used to measure the green fluo-
astrocytoma tumor cell lines and ex vivo GBM cells investigated in rescence in pixels and calculate mean values for quantification of the
©
a dose-dependent manner (Table 1, Fig. 1D for U-373, LN-229, amount of GFP-LC3 dots. Values are shown in Figure 4A on the
LN-18 and ex vivo GBM 1 cells). lower right corner of each figure.
Effect on colony formation. The colony formation rate R Conversion of LC3-I into LC3-II. Exposure of U-373 glioma
C
varied in the different cell lines and ex vivo GBM cells: LN-18: cells to TAU (100 μg/ml, 6 h) led to significant increase of the
17.0 ± 2.2%, LN-229: 19.3 ± 1.2%, U87-MG: 9.5 ± 2.5%, U-138: LC3-II and a decrease of the LC3-I level (Fig. 4B). The intensities
22.7 ± 1.6%, U-251: 16.2 ± 1.2%, U-373: 13.6 ± 0.9%, GBM-1: of each band of western blotting were measured in comparison to
www.landesbioscience.com Autophagy 195
TAU exhibits anti-neoplastic action
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196 Autophagy 2009; Vol. 5 Issue 2
TAU exhibits anti-neoplastic action
Figure 1 (See previous page). TAU induces cell killing and inhibition of proliferation in human astrocytoma/glioblastoma cell lines with ultrastructural features
of autophagy. (A) U-373 cells were left untreated or incubated with 100 μg/ml TAU (TAU), 100 μg/ml TAU + 5 mM NAC (TAU + NAC), and Fas-Ligand
5% (FasL) for 24 hours and morphological alterations observed under phase contrast microscopy at a magnification of 200x. (B) U-373 human glioblastoma
cells were left untreated or incubated with 100 μg/ml TAU for 3 (TAU 3 h) and 24 (TAU 24 h) hours, and ultrastructural alterations observed using TEM.
Following incubation with TAU, formation of numerous intracytoplasmatic vacuoles containing material, loss of microvilli, streaky condensation of the chroma- .
e
tin and degradation of the cytoplasm could be observed. Higher magnification is shown in the insert (CP = cytoplasm; MV = microvilli; C = chromatin; V =
vacuoles). (C) For proliferation assay 1 μCi/ml of 3H-thymidine were added for the final 7 hours of culture to U-373 cells before harvesting and counting ton
a liquid scintillation counter. Data are presented as means ± SEM of 10 individual experiments (t-test). (D) U-373, LN-229, LN-18 and ex vivo GBM 1u cells
were treated with TAU at various concentrations for 2, 4, 8 and 24 hours to assess cytotoxicity. After incubation, the cells were stained with crystal violet
and the OD red on a microtiter plate reader at an absorption wavelength of 540 nm. Data are given as means ± SEM of 5 individual experimebnts.
i
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the loading control to quantify the conversion of LC3 by TAU. t
Table 1 Cytotoxicity of TAU on human glioma cell lines
Rapamycin (10 μM, 6 h) was used as a positive control (Fig. 4B). s
and low passages of ex vivo glioblastoma cells
AVO-detection and AO staining. U-373, LN-229 and ex vivo i
GBM 1 cells were treated with TAU 5 and 50 μg/ml and BfA1 5, 25 Cell type Cytotoxicity assaya d Colony assayb
and 100 nM. Using the fluorochrome AO as an indicator of H+-pump EC (μg/ml) maximum cell EC (μg/ml)
50 50
activity, AVO formation was measured by FACS analysis and the tkiling (%)c
effects of the administered agents visualized per confocal microscopy. LN-18 48.6 ± 3.2 o 66.3 ± 1.2 1.9 ± 0.1
Results showed a dose-dependent increase of AVO-formation for LN-229 38.9 ± 6.9n 79.5 ± 1.1 1.1 ± 0.2
both tested TAU concentrations (basal level: U-373: 6.2, LN-229: U87-MG 79.2 ± 8.2 69.3 ± 0.7 1.2 ± 0.8
4.1, ex vivo GBM 8.5; TAU 5 μg/ml: U-373: 26.2, LN-229 20.1, U-138 92.4 o± 10.2 52.3 ± 5.3 2.4 ± 1.9
ex vivo GBM 5 31.9; TAU 50 μg/ml: U-373: 38.2, LN-229: 36.4, U-251 D49.8 ± 4.5 73.8 ± 10.5 0.5 ± 0.2
ex vivo GBM 5: 56.4), which could be completely prevented by
U-373 72.3 ± 5.2 69.4 ± 4.9 1.1 ± 0.2
BfA1. Irradiation at 10 Gy (positive control) resulted only in a minor
GBM-1 . 79.2 ± 2.8 82.5 ± 0.8 1.4 ± 0.2
increase in AVO formation due to radioresistance of the cells (Fig.
GBM-2 e 102.3 ± 6.3 60.5 ± 5.1 0.5 ± 0.1
4C and D).
GBM-c3 62.5 ± 2.9 73.5 ± 3.5 0.9 ± 0.1
Effect of Atg-7 and Atg-12 knockdown on TAU induced cyto-
toxicity. Silencing of Atg-7 and Atg-12 had a partial protective GnBM-4 72.3 ± 6.8 89.3 ± 4.4 0.9 ± 0.1
effect (30–40%) in U-373 and LN-229 cells only at 50 μg/ml TAU, GBM-5 85.4 ± 12.1 68.4 ± 4.3 no colony
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formation
which has been statistically significant only for the Atg-7 knockdown
i Mean ± SD 71.2 ± 19.6 71.3 ± 10.2 1.2 ± 0.6
(t-test; Fig. 5A). Western-Blot analysis served as a control for the
c
effectiveness of the Atg knockdown, which could be demonstrated Data are presented as mean values ± SD of 5 individual experiments. aCytotoxicity after 24 hours of
s
only for the Atg-7 knockdown (Fig. 5B). treatment was calculated from OD measured following crystal violet staining (for details see “Materials
Autophagy inhibitor treatment. 3-MA is an inhibitoor of autophagy and methods”). EC50 = mean effective concentration at which the cell number is reduced to 50% of that in
the untreated control cultures. bCytotoxicity after 2 weeks of treatment was calculated from OD measured
and acts as a PI3K inhibitor, interfering with the formation of pre-
i following crystal violet staining (for details see “Materials and methods”). EC = mean effective concen-
autophagosomal structures. Using U-373 and BLN-229 cells, 3-MA tration at which the number of cell colonies is reduced to 50% of that in th5e0 untreated control cultures.
applied in concentrations of 1, 2.5 and 5 mM revealed a slight but cMaximum cell killing after 24 hours of treatment was calculated from OD measured following crystal violet

significant protection (max. 21%), bust demonstrated an intrinsic staining (for details see “Materials and methods”). maximum cell killing = percentage of maximum cell
killing achieved with 100 μg/ml TAU.
cytotoxic activity (7.4 ± 2.1% at a 3-MA concentration of 1 mM; 8.0
e
± 9.6% at 2.5 mM; 9.0 ± 1.9% at 5 mM, Fig. 5C).
Intracellular ATP content. Indtracellular ATP content was partially Involvement of senescence. From the observation that TAU
preserved following a 6 hnour incubation with 100 μg/ml TAU, induces growth arrest in glioma cell lines at low concentrations with
whereas it was significantly lower after incubation with FasL 25%. no obvious cell killing (Fig. 1C), an induction of senescence as a
a
Incubation of U-373 and LN-229 cells for 24 hours with TAU possible cause was investigated in U-373 and LN-229 cells. SA-β-gal
reduced the intraceLllular ATP content to nearly zero (Fig. 5D). Cell was used as a marker for senescence. It reflects the increased lyso-
cultures in whic h all sources of ATP production were eliminated by somal mass of senescent cells and therefore can be used for their
3-h incubatio9n with OM and GFM served as positive controls. visualization. At a low TAU concentration of 20 μg/ml a 4–5 fold
Involv0ement of necroptosis. Based on the finding that necrosis increase in senescent cells was seen (Fig. 5F), which was identical to
plays a part in TAU-induced killing, the role of necroptosis, a the positive control of 10 Gy irradiated cells. Irradiation is known
0
recently discovered cell death mechanism25 that morphologically to induce senescence.26 At TAU concentrations revealing >75%
r2esembles necrosis, was questionable. Therefore, the effect of necro- cytotoxicity, the number of senescent cells was similar to untreated
© statin-1—a specific inhibitor of H2O2-induced necroptosis25—on cells (Fig. 5F).
the TAU-induced cell killing in LN-229 cells and ex vivo GBM cells Influence of radical scavenger and glutathione depletion.
has been investigated (Fig. 5E). Necrostatin-1 at a concentration Pre-exposure to 5 mM NAC significantly inhibited cell death of
of 5 μM was able to reduce cell killing induced by 50 μg/ml TAU U-373 and LN-229 cells. The average decrease in cell killing was
by 46.2% in U-373 and by 58.9% in LN229 cells, suggesting an 41.4 ± 13.0% (Fig. 5G). The susceptibility of the tumor cells to TAU
involvement of necroptosis at a variable degree in TAU-induced cell after depletion of glutathion by BSO pre-treatment for 20 hours led
killing (Fig. 5E). to significant increased cell killing by a mean of 20.3% (Fig. 5H).
www.landesbioscience.com Autophagy 197
TAU exhibits anti-neoplastic action
Figure 2. Flow cytometric analysis of LN-18, LN-229 and
U-373 human astrocytoma/glioblastoma cells following
TAU treatment. The cells were cultured in the presence of
TAU or left untreated and then stained with annexin V-PE
and the vital dye SYTOX Green (Vybrant apoptosis assay .
e
kit) according to the instructions of the manufacturer
(Invitrogen AG, Basel, Switzerland). (A) Flow cytomettric
analysis of LN-229 cells prior to and following inucuba-
tion with TAU at a concentration of 25 and 100 μg/ml
for 24 hours. Incubation of cells with 200 μM bH O and
2 2
250 μM cisplatin served as positive controls. Data were
i
calculated directly from the plots using WinMDI soft-
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ware. (B) Flow cytometry of U-373, LN-229 and LN-18
t
cells prior to and following incubation with 100 μg/ml
s
TAU for 6 and 24 hours. Data were calculated directly
from the plots using WinMDiI software. (C) Percentage
of apoptotic (SYTOX- AnndV+), necrotic (SYTOX+ AnnV-)
and late apoptotic/necrotic (SYTOX+ AnnV+) cell death

after treatment witht TAU for 24 hours. Control cells were
treated with cisoplatin and H2O2. Data are given as
means ± SEM of 3 individual experiments. An increase
of apoptoticn (U-373, LN-229), necrotic (U-373, LN-229
and LN-18) and late apoptotic/necrotic cells (LN-229)

couldo be observed upon treatment with 100 μg/ml TAU
for 24 hours.
D
Influence of PARP inhibition and of SMAC
.
peptide. Co-incubation with 3 aminobenzamide
e
(3-AB) had no significant effect on the cytotoxic
c
activity of TAU (t-test). 3-AB alone had no intrinsic
n cytotoxic activity (Suppl. Fig. 6A). Co-incubation
with the SMAC peptide was not associated with a
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significant increase in TAU-mediated cytotoxicity.
i In contrast, SMAC peptide significantly ampli-
c
fied the cytotoxicity induced by cisplatin (20 μM
s SMAC: 7.3 ± 2.8%; 50 μM SMAC: 13.0 ± 3.8%;
o t-test; p < 0.05; Suppl. Fig. 6B).
i Discussion
B
TAU induced dose-dependent killing and inhi-

s bition of proliferation and colony formation of
all investigated tumor cell lines and ex vivo GBM
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cells. Inhibition of tumor cell proliferation and
d
colony formation required significantly lower TAU
n concentrations as compared to the acute cell killing
(Fig. 1, Table 1).
a
However, the results of the present study do not
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support the hypothesis that “classical” apoptosis
plays a significant role in the mechanism of action
9
of TAU. An increasing number of late-apoptotic/
0 necrotic cells could be observed using flow cyto-
metric analysis (Fig. 2), which is inconsistent with
0
apoptosis, but not with other forms of PCD such
2
as autophagy. Cell membrane PS-externalization
© may occur independently of caspase activation
outside the “classical” apoptosis pathway27 and
thus enables efficient phagocytosis for cells that
do not undergo apoptosis.28 Apoptosis is strongly
related to caspase activation, eventually leading to
the demolition of cellular structures and culmi-
nating in cellular shrinkage with nuclear chromatin
198 Autophagy 2009; Vol. 5 Issue 2
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F2igure 3. For figure legend, see page 200.
©
condensation and nuclear fragmentation.22,29,30 Pan-caspase inhibi- In contrast, co-treatment with the radical scavenger NAC could
tion resulted in an entire abolition of caspase activation while there prevent both, cytotoxicity and caspase activation (Fig. 3B). Caspase-
was no effect on cytotoxicity (Fig. 3A). Although activation of the independent cell death mechanisms, mainly those that were executed
most downstream effector caspases 3 and 7 could be demonstrated by mitochondrial pathways, can cause structural and functional
using a highly sensitive assay, there was no significant time- and damage to trigger the release of other death factors independently
concentration-dependent correlation with cytotoxicity (Fig. 3B). of upstream signals.31 This may explain the activation of caspases
www.landesbioscience.com Autophagy 199
TAU exhibits anti-neoplastic action
Figure 3A–C. Caspase 3/7 activation upon TAU treatment can be explained as an epi-phenomenon, since cytotoxicity of TAU was not significantly influ-
enced by pre-incubation with zVAD.fmk. TAU treatment causes release of AIF in human U-373 and LN-229 astrocytoma/glioblastoma cell lines and human
ex vivo GBM cells. Silencing of AIF—but not of the Cyt-C gene leads to significant partial protection from TAU induced killing in U-373 (50, 100 and 200
μg/ml TAU) and LN-229 (100 μg/ml) cells. TAU does not cause DNA-laddering in U-373 and LN-229 human astrocytoma/glioblastoma cells. (A) U-373
and LN-229 cells were incubated with TAU at various concentrations for a period of 24 hours with or without pre-treatment with 50 μM z-Vad.fmk 30 min in .
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advance. Then, crystal violet staining and measurement of OD using a microtiter plate reader has been performed. Incubation with Fas-ligand 25% served
as a positive control. Results are presented as means ± SEM of 5 independent experiments (t-test). (B) Activation of caspases 3 and 7 upon TAU treatmentt in
U-373 and LN-229 glioma cells: time- and dose-dependent increase of caspase 3 and 7 activity (left y-axis) and concomitant demonstration of cytotouxicity
(right y-axis). Cells incubated with cisplatin served as a positive control. There was no time- and dose dependent correlation between TAU-induced cytotoxicity
and caspase 3 and 7 activation. The results are presented as means ± SEM of 5 independent experiments (left, t-test). The activation of caspasbe-3 and -7
by TAU treatment could be completely prevented by pre-treatment with z-VAD-fmk and NAC, respectively (right, t-test). (C) Western blot analysis of AIF and
i
Cyt-C in the cytosolic and mitochondrial fraction (200 μg/lane) of U-373, LN-229 and ex vivo GBM 5 cells 6 hours following incubation with 100 μg/ml
r
TAU. The p65 subunit of NFκB served as a loading control for the cytosolic fraction and the mitochondrial SOD-2 for the mitochondrial fraction.
t
s
3 and 7 upon TAU treatment as an epi-phenomenon, which has concentration-dependent manner. This suggests a partial effect of
i
no relevance for TAU-induced cell death. There are more findings this pathway on the TAU-induced cell deadth. Probably, other mecha-
arguing against a significant role of apoptosis: An internucleosomal nisms of cell death are more active at TAU concentrations <50 μg/

chromatin degradation involving fragmentation of DNA into sizes ml (e.g., senescence) and >100 μgt/ml (e.g., necroptosis, necrosis),
of about 200 base pairs or multiples thereof, which can be visual- whereas autophagy is the main moechanism within the concentration
ized as “DNA ladders” in an electrical field,32 could not be observed range of 50–100 μg/ml TAU.
n
following incubation with TAU (Fig. 3F). Neither co-incubation Furthermore, all processes involved in the first step of autophagy,

with the PARP inhibitor 3-AB, which prevents DNA repair,33,34 the sequestration, are ATP-dependent. Therefore, the intracellular
o
nor with SMAC-peptide35,36 led to significant amplification of ATP content would have to be preserved for some time.21 In the
D
TAU-induced cell death (Suppl. Fig. 6A and B). present study, the intracellular ATP content was only partially
Morphological alterations of the tumor cells observed in light and preserved aft er 6 hours of incubation with TAU, but significantly
electron microscopy after TAU treatment indicated a major role of higher as c.ompared to the incubation with FasL 25% (Fig. 5D).
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autophagy on its mechanism of action.37-39 During autophagy, cyto- It has been reported that caspase inhibition does not prevent
plasm and intracellular organelles are sequestered into characteristic deathc-domain receptor (DR)-induced cell death.25 Cell death
multimembraned vacuoles (autophagosomes) and finally delivered to innduced under such conditions lacks the typical features of apoptosis
lysosomes for further degradation.23 Using TEM, which is consid- and instead resembles necrosis, thus indicating a caspase-indepen-
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ered to be the “gold standard” in autophagy detection,40 important dent, nonapoptotic, necrotic-like cell death mechanism.25 This newly
features of autophagy could be demonstrated: cytoplasmic blebibing, discovered death pathway had been named ‘necroptosis’ and its exis-
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the disintegration of cytoplasmic components with a progressive loss tence proved with a specific and potent inhibitor of DR-stimulated
of electrodensity that precedes the degradation of the nucsleus,41 and cell death (necrostatin-1) without caspase signaling.25,46 Consistently,
the appearance of autophagosomes containing digesteod material (Fig. the appearance of necrotic morphology was completely inhibited.
1B).37,38 The localization of GFP-LC3 was used to detect autopha- Moreover, it has been reported that necroptosis could additionally
i
gosomes by confocal microscopy.37,38,42,43 A dBiffuse distribution of induce autophagy as a downstream consequence, however, oneself
GFP-LC3 could be observed in the absence of TAU, and a massive not being dependent on autophagy.25 Necroptosis was shown to be
punctated pattern in the presence, which is known to be caused by involved in the TAU-induced cell killing as the specific necroptosis-
s
the localization of LC3-II on the autophagosome membrane (Fig. inhibitor necrostatin-1 reduced the TAU-induced cytotoxicity at a
4A).42,44 Furthermore, conversion eof LC3-I into LC3-II could be variable degree (Fig. 5E).
demonstrated by immunoblottindg (Fig. 4B). Cellular senescence is the phenomenon where cells enter a state
Raised AVO formation is a typical feature in autophagy and can of terminal growth arrest and express SA-β-gal, which is its most
n
therefore be inhibited by the autophagy inhibitor BfA1.17 BfA1 widely used marker.26,47,48 Senescence has lately attracted interest
is a proton pump inhibaitor of ATPase,45 which prevents fusion of in cancer research, because limitless replicative potential is a feature
autophagosome anLd lysosome.24 Interestingly, AVO formation, that is essential for the development of malignancy.47 Using SA-β-gal
which was obviously increased upon treatment with TAU, could for visualizing senescent cells, a 4-fold increase of SA-β-gal positive

be totally pre9vented by pre-treatment with BfA1 (Fig. 4C and D), cells at a TAU concentration of 20 μg/ml could be demonstrated.
whereas there was no significant protective effect of BfA1 in the cell However, at a TAU concentration of 50 μg/ml there were less
0
killing assays (data not shown). This may indicate that other cell senescent cells as compared to a TAU concentration of 20 μg/ml
0
death mechanisms are involved. (Fig. 5F). A possible explanation is, that at a TAU concentration of
2Knockdown of the autophagy gene Atg-7 led to significant, but 50 ug/ml other mechanisms of cell death (autophagy, necroptosis,
only partial protection at a TAU concentration of 50 μg/ml (Fig. necrosis) are more apparent, so that the cells are killed rather then
©
5A). However, there were more arguments against autophagy as the go into senescence. As stated in the precedent studies that TAU leads
only mechanism underlying the anti-neoplastic effect of TAU. The to oxidative stress in glioma cells and as senescence can be induced
class III PI3K/BECLIN-1 induces autophagy when upregulated, through oxidative stress,26 the induction of senescence contributes to
which can be inhibited by the autophagy inhibitor 3-MA.18,19 the cytotoxic effect of TAU.
In the present study, co-exposure to 3-MA was found to signifi- It has been shown by demonstration of AIF translocation into the
cantly reduce cell killing of 50 and 100 μg/ml TAU (Fig. 5C) in a cytosol that the mitochondrial pathway is involved in TAU-induced
200 Autophagy 2009; Vol. 5 Issue 2
TAU exhibits anti-neoplastic action
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Figure 3D–F. For figure legend, see page 202.
cell killing. Furthermore, the preceding results revealed that TAU autophagosomal membrane, and the conversion of LC3-I into
initiates cell death also at the lysosomal level leading to autophagy, LC3-II. It has been shown previously that TAU induces the genera-
which was confirmed by the TEM findings, the demonstra- tion of ROS in human glioma/glioblastoma cell lines and ex vivo
tion of AVO formation, the redistribution of GFP-LC3 on the cells from patients with a glioblastoma.3 ROS are a group of highly
www.landesbioscience.com Autophagy 201
TAU exhibits anti-neoplastic action
Figure 3D–F. Caspase 3/7 activation upon TAU treatment can be explained as an epi-phenomenon, since cytotoxicity of TAU was not significantly influenced
by pre-incubation with zVAD.fmk. TAU treatment causes release of AIF in human U-373 and LN-229 astrocytoma/glioblastoma cell lines and human ex vivo
GBM cells. Silencing of AIF—but not of the Cyt-C gene leads to significant partial protection from TAU induced killing in U-373 (50, 100 and 200 μg/
ml TAU) and LN-229 (100 μg/ml) cells. TAU does not cause DNA-laddering in U-373 and LN-229 human astrocytoma/glioblastoma cells. (D) Silencing
of AIF and Cyt-C genes by siRNA at different TAU concentrations in U-373 and LN-229 cells. Data are presented as mean values ± SEM of two individual .
e
experiments. A significant decrease in cytotoxicity could be observed at a TAU concentration of 50, 100 and 200 μg/ml in U-373 cells and of 100 μg/ml
in LN-229 cells and silencing of the AIF gene (t-test). The silencing of the Cyt-C gene had no influence on the TAU-induced cell killing. (E) Reductions in tthe
corresponding proteins by siRNA compared with nonspecific (NS) oligoribonucleotides as shown by WB analysis in total lysates (200 mg/lane) of Uu-373
and LN-229 glioma cells. As a loading control, α-tubulin was used. Nonspecific siRNA oligoribonucleotides (NS), AIF and Cyt-C siRNA were purchased
from Santa Cruz Biotechnology (Santa Cruz, California) and used according to the instructions of the manufacturer. (F) U-373 and LN-229 cellbs were left
untreated or incubated with TAU 100 μg/ml or anti-Fas antibody 1 μg/ml for 6 hours, and then prepared using the apoptosis DNA ladder detection kit
i
(Medical & Biological Laboratories) as described by the manufacturer. Finally, the DNA probes were loaded onto 1% agarose gel with SYBR Safe staining
r
solution (Molecular Probes). The stained DNA was visualized by transillumination with UV light and documented on photographs.
t
s
reactive molecules with unpaired electrons that are generated in higher concentrations has no significant siide effects.11,54 TAU has
d
normal physiologic processes or by external stress such as UV been shown to represent a very promising new anti-neoplastic agent,
light, ionizing irradiation, and DNA-damaging drugs.49 Known which leads to growth inhibition, inh ibition or prevention of colony
cellular defences to ROS include but are not limited to antioxidant formation and cell killing of tumort cells by the induction of a mixed
o
scavengers, like GSH (glutathione) and ascorbate, and antioxidant type of programmed cell death.
enzymes, such as superoxide dismutase, glutathione peroxidase and n
Material and Methods
thioredoxin reductase. The regulation of the oxidation-reduction

reactions is critical in cell signalling and programmed cell death.49 Stock solutions weore used containing TAU at 10 mg/ml prepared
Pre-treatment with the radical scavenger NAC could protect the
from ultrapure pDowder (kindly provided by Geistlich Pharma
cells from TAU-induced cell death (Fig. 5G), whereas pre-treatment
AG, Wolhusen, Switzerland), sterile filtered and diluted with PBS
with BSO led to significant increase of cell killing (Fig. 5H). BSO
(Sigma Aldrich) to achieve the respective concentrations. LN-18,
.
is a selective inhibitor of gamma-glutamylcysteine synthase and in
LN-229,e U-373 human glioblastoma/astrocytoma cell lines were
this way reduces biosynthesis of glutathione,50 which has protec-
kindly provided by N. de Tribolet, Geneva, Switzerland. Different ex
tive activity against hydrogen peroxide and ROS.51 An increase in c
vivo GBM cells (GBM 1–5, passage 3–7) from the Department of
ROS generation, or a reduction in their detoxification, is known n
Neurosurgery, University Hospital Zurich, were used.
to lead to apoptosis by depolarization and permeabilization of the
e Morphological characterization. For light microscopy, LN-229
mitochondrial membrane.14 In opposite, it could be shown here
cells were seeded into 75-cm2 plastic cell culture flasks (Falcon, BS
that the apoptotic cascade is not significantly activated by iTAU
c Biosciences) with cell culture medium (DMEM; Gibco, Invitrogen
treatment. The results indicate, rather, that through generation of
AG, Basel, Switzerland) containing 10% FCS, gentamicin (10 μg/
ROS, upon TAU treatment more than one cell death smechanism
ml, Gibco), 50 ml of fetal calf serum (FCS; PAA Laboratories) heated
is involved, having seen that caspase-independenot programmed
to 37°C, 5 ml of 100 mM sodium pyruvate (ICN Biomedicals,
cell death pathways such as autophagy, senescence, necroptosis and
i Aurora, Ohio), and 5 ml N-acetyl L-alanyl L-glutamine (Biochrom
necrosis occur. An unanswered question is the Bmechanism that leads
AG, Berlin, Germany). The cells were cultured at 37°C in an atmo-
to the switch between or the parallel action of the different death
pathways. Recently, pathways that link di fferent types of PCD have sphere of 5% CO2 until about 80% confluence. Following counting,
s a suspension containing 5 x 104 cells/ml was prepared and a 200-μl
been deciphered at the molecular level.23 After TAU treatment, a
e aliquot transferred into each well of a 96-well cell culture plate. After
mixed type of PCD has been detected. Taking into account that
12 hours, cell adhesion was evaluated microscopically, medium was
different TAU concentrations adppear to involve different forms of
exchanged, and the cells were incubated with 100 μg/ml TAU with
PCD, the conclusion seems justifiable that the choice among them
n or without 5 mM NAC for 24 hours. Cells prepared equally and
depends on the level of stress, namely the concentration and applica-
tion period of TAU. Thais meets the earlier observation that different incubated with 25% Fas-ligand were used for positive control. Cells
were then evaluated under the phase contrast microscope (Axiovert,
forms of PCD can Lbe activated as a result of variable thresholds for
Zeiss).
the different processes—as observed in the present study—or from
a “cellular de9cis ion” between the responses that may be linked to a For electron microscopy, U-373 cells were seeded and incubated
mutual inhibition of the forms of PCD.23 with 100 μg/ml of TAU. Identical amounts of medium were added
In this0 way, TAU represents a new strategy, especially for, but not to control cells. After 6 or 24 hours, nonadherent cells were harvested
restri0cted to the therapy of GBM, because these tumors are highly by centrifugation (Hermle; 5000 rpm, 5 min) and fixed for 30 min
resistant to the current chemotherapeutic strategies, which mainly at 4°C in 0.05 M cacodylate buffer containing 2% glutaraldehyde
2
target apoptosis.52 The pharmacokinetic profile of TAU permits and 0.8% paraformaldehyde. Postfixation was done in 0.1 M caco-
© therapeutic use, leading to mean plasma concentrations of TAU of 83 dylate buffer with 1% OsO and 1.5% K (FeCN) . Subsequently the
4 4 6
± 18.1 μg/ml during intravenous application in humans.53 This value cells were embedded in epon. Ultrathin sections stained with uranyl
compares favorably to the facts that TAU at very low concentrations acetate and lead citrate were examined in a TEM (TEM 420, Philips).
leads to a dramatic reduction or even prevention of proliferation Qualitative and quantitative evaluation has been performed. For
(from ca. 2 μg/ml) and inhibition or prevention of colony forma- quantitative evaluation the number of vacuoles has been counted for
tion (EC = 1.2 ± 0.6 μg/ml), and at higher concentrations to acute at least five cells and averaged to the mean number per cell. The count
50
cell killing (EC = 71.2 ± 19.6 μg/ml). In addition, TAU at even in untreated cells has been compared with that of treated cells.
50
202 Autophagy 2009; Vol. 5 Issue 2
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