12885 2015 Article 1929

12885 2015 Article 1929
Koehler et al. BMC Cancer (2015) 15:919
DOI 10.1186/s12885-015-1929-y
Open Access
Pan-Bcl-2 inhibitor Obatoclax is a potent
late stage autophagy inhibitor in colorectal
cancer cells independent of canonical
autophagy signaling
Bruno Christian Koehler1*†, Adam Jassowicz1†, Anna-Lena Scherr1, Stephan Lorenz1, Praveen Radhakrishnan2,
Nicole Kautz1, Christin Elssner1, Johanna Weiss3, Dirk Jaeger1, Martin Schneider2
and Henning Schulze-Bergkamen1,4
Background: Colorectal cancer is the third most common malignancy in humans and novel therapeutic
approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells
collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in
order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to
contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central
regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic
members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small
molecules inhibiting those proteins are in clinical trials.
Methods: Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D
cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied
to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling.
Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and
autophagy flux.
Results: This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer
(CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax
causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor,
failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in
cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions
such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is
independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12.
(Continued on next page)
* Correspondence: bruno.koehler@nct-heidelberg.de
Equal contributors
National Center for Tumor Diseases, Department of Medical Oncology,
Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany
Full list of author information is available at the end of the article
© 2015 Koehler et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Koehler et al. BMC Cancer (2015) 15:919
Page 2 of 11
(Continued from previous page)
Conclusions: The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells
and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1
independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy
inhibition as therapeutic strategy for CRC.
Keywords: Autophagy, Colorectal cancer, Apoptosis, Autophagy related gene, LC3, p62 (SQSTM1), Obatoclax,
Colorectal tumors are one of the major causes for cancer
related death in humans [1]. Even if novel and targeted
therapeutic approaches are rapidly emerging, the prognosis in the metastatic stage (UICC IV) is restricted.
Autophagy is an evolutionarily conserved process of cellular self-digestion, which is indispensable in situations of
cellular stress such as hypoxia, energy deprivation or an
acidic environment [2]. In gut development and pathophysiology, autophagy plays a decisive role [3]. An imbalance within the tightly regulated autophagy network has
implications for various human diseases including cancer
[4, 5]. It has been proven in various cancer models that
autophagy represents an important mechanism by which
cancer cells maintain their highly active metabolism [6, 7].
Furthermore, autophagy may provide resistance towards
antitumor therapy [8]. The exact mechanism by which autophagy modulates malignant transformation has been a
matter of controversy in current research [7, 9].
By now, there is a huge and growing body of preclinical and clinical data investigating genetic or chemical
approaches to block autophagy as a therapeutic strategy
in cancer [6, 10, 11]. Potent autophagy inhibitors such as
the malaria drug chloroquine have entered late stage
clinical trials [12]. Little is known regarding autophagy
inhibition in colorectal cancer. Studies present heterogeneous data regarding autophagy in CRC [13–15].
The Bcl-2 family of proteins is mainly known for its
pivotal role in the regulation of mitochondrial apoptosis
[16]. A variety of small molecules targeting antiapoptotic
Bcl-2 proteins have been developed with the aim to
overcome cell death resistance in cancer [17]. Several
Bcl-2 inhibitors have entered clinical trials [17, 18]. In
addition, it has been shown that antiapoptotic Bcl-2 proteins interfere with autophagy signaling. For instance,
Bcl-2 binds, among others, to proautophagic Beclin-1,
thereby blocking autophagy induction [19, 20].
In order to prove the feasibility and potency of autophagy inhibition as concept for colorectal cancer treatment, this study first aimed at investigating chloroquine
in CRC cells as a model agent for autophagy inhibition.
There are several Bcl-2 mimicking small molecules
available. In this study we investigated pan Bcl-2 inhibition by obatoclax compared to the Mcl-1 sparing
inhibitor ABT-737. We have recently shown that the
pan-Bcl-2 inhibitor obatoclax induces cell cycle arrest
and blocks migration in CRC cells rather than causing
direct cell death, arguing for pleiotropic antitumor effects of this agent [21]. Since others have reported a
contribution of obatoclax to autophagy signaling in cancer, we focused on autophagy in CRC cells treated with
obatoclax [22, 23]. Therefore, the aim of this study was
to further dissect the anti-cancer properties of obatoclax
in colorectal cancer cells and to further elucidate the
contribution of antiapoptotic Bcl-2 proteins in the crosstalk between apoptosis and autophagy.
Reagents and cell lines
The colorectal cancer cells HT29 and SW480 were obtained from ATCC and cultured under standard conditions as described previously, supplemented with 10 %
fetal bovine serum (PAA laboratories, Cölbe, Germany)
in a humidified atmosphere [21]. Starvation was induced
using OptiMEM (Invitrogen, Karlsruhe, Germany) as a
reduced medium without supplements.
Chloroquine was purchased from Sigma-Aldrich
(Hamburg, Germany). Obatoclax and ABT-737 were
obtained from Selleckchem (Munich, Germany), staurosporine was from Enzo Life Science (Farmingdale, NY,
RNA-interference and transfection
Transfections were carried out in OptiMEM without supplements using Lipofectamine RNAi-Max (Invitrogen,
Karlsruhe, Germany) as described [24]. Sequences for
siRNA targeting Mcl-1, Beclin-1, Atg7 and Atg12 are available upon request; a non-targeting siRNA (siScramble) was
used as a control (MWG Biotech, Ebersberg, Germany).
24 h post transfection cells were treated for 48 h
Cell death and growth assays
Flow cytometry analyses were performed using a FACS
CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA)
as described previously [21]. Cells in the sub-G1 fraction
were depicted as apoptotic.
Cell growth was assessed using a colorimetric 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Koehler et al. BMC Cancer (2015) 15:919
(MTT) based assay as described previously [25]. Absorbance was measured at 550 nm using a plate reader
(Infinite 200 pro; Tecan, Männedorf, Switzerland).
Values were normalized to untreated controls.
Immunohistochemistry and 3D cell culture
Sections (8 μm thickness) were fixed in 4 % PFA and
stained with haematoxylin and eosin according to standard
procedures. Immunohistochemistry was performed using
NovoLink Polymer detection System (Leica Microsystems,
Wetzlar, Germany) according to the manufacturer’s instructions. Images were captured using an inverted microscope
(Keyence, Neu-Isenburg, Germany). Images were analyzed
using CellSense® and ImageJ software.
Long term cell culture in 3-dimensional ALVETEX
scaffolds (Reinnervate, Sedgefield, UK) fosters cell-cellinteractions in a tissue mimicking environment and has
been described previously [24]. Fresh medium was added
every 48 h. After 7 days of treatment, scaffolds were collected, shock frozen and subsequently sectioned and
stained as described [24]. The following antibodies were
used for detection: anti-LC3b (Cell Signaling, Boston,
MA, USA) anti-p62 (BD, Franklin Lakes, NJ, USA).
SDS-Page, western blotting and q-RT PCR
Cell lysis, SDS-page and western blotting were performed according to standard procedures as described
previously [21]. The following antibodies were used:
anti-Mcl-1 (Santa Cruz Biotechnology, Heidelberg,
Germany), anti-LC3b (Cell Signaling), anti-p62 (BD),
anti-Atg12 (Cell Signaling), anti-Atg7 (Cell Signaling),
anti-Tubulin (Sigma, St. Louis, MO, USA.
RNA isolation, cDNA synthesis and quantitative real
time polymerase chain reaction (q-RT PCR) were carried
out as described using primer assay kits [21]. Each reaction was run in duplicates, values were normalized to
GAPDH as housekeeping gene.
Supravital cell-staining with acridine orange
Cells grown on cover slips were treated as described.
24 h following initial treatment, cells were incubated
with medium containing 2 μg/mL acridine orange
(Sigma-Aldrich) for 20 min. Subsequently, medium containing acridine orange was removed and cells were
washed twice with PBS and fixed using 4 % PFA (Merck)
for 15 min at room temperature. Pictures were obtained
using a Zeiss LSM 780 confocal laser scanning microscope
with ZEN microscopy software at 63x magnification.
Cells grown on cover slips were treated as described
above. 24 h following initial treatment, cells were washed
twice with PBS and fixed using ice-cold methanol at −20 °
C for 20 min. Fixed cells were washed twice with PBS and
Page 3 of 11
blocked in PBS containing 5 % BSA and 0.3 % Triton™ X100 (Merck) for 1 h. After blocking, cells were incubated
with primary antibodies in PBS with 1 % BSA and 0.3 %
Triton™ X-100 at 4 °C overnight. The next day, cells were
incubated with Alexa Fluor 488-conjugated goat antirabbit secondary antibodies (Invitrogen, Carlsbad, California, USA) for 1 h at room temperature and subsequently
counterstained with PBS containing 2 μg/mL Hoechst
33342 trihydrochloride trihydrate (Invitrogen) for 15 min
at room temperature. Pictures were obtained using a Zeiss
LSM 780 confocal laser scanning microscope with ZEN
microscopy software at 63 × magnification and 10 x magnification for counting of LC3II puncta. The average of 5
visual fields at 10 x magnification was used.
In vitro lysosomal staining
Cells grown in 12-well cell-culture plates were treated as
described above. 24 h following initial treatment, cells
were washed twice with PBS and incubated with reaction
buffer containing 2 μL Cyto-ID® Green Detection Reagent (Enzo), 1 μL Hoechst 33342, Trihydrochloride,
Trihydrate (Enzo) and 5 % FCS per mL reaction buffer
for 25 min at 37 °C in a cell incubator [26]. Subsequently, the reaction buffer was removed, cells were
washed and fresh reaction buffer was added. Cell-culture
plates were analyzed using a Biorevo BZ-9000 Inverted
fluorescence phase-contrast microscope with BZ-observer
microscopy software (Keyence) at 40x magnification.
Statistical analysis
Statistical analysis was done using student’s t-test (paired,
two sided) based on normal data distribution. All statistics were calculated using SPSS 20 (IBM, NY, USA), pvalues < 0.05 were considered to be significant.
Late stage autophagy inhibition induces apoptosis in
starved CRC cells
Chloroquine (CQ) has been widely used as a model agent
for autophagy inhibition [27, 28]. Here, we demonstrate
that CQ has limited impact on HT29 and SW480 CRC
cells in fully supplemented growth medium. In contrast,
CQ is able to induce cell death in CRC cells under conditions of starvation (Additional file 1: Figure S1 A left and
right graph, p < 0.001).
In order to investigate autophagy signaling upon CQ
treatment, we assessed protein levels of Light Chain Enhancer 3 (LC3) and the conversion from soluble LC3I to
membrane-bound LC3II, which is indicative for autophagy
flux activation (Additional file 1: Figure S1 B) [29, 30]. In
addition, we observed accumulation of p62, also termed
sequestosome 1 (SQSTM1) in cells under CQ treatment.
P62 gets degraded in autophagic vesicles and is therefore
an indicator of autophagic flux [31, 32]. Lysotracker and
Koehler et al. BMC Cancer (2015) 15:919
acridine orange (AO) staining revealed marked cytosolic
accumulation of acidic vesicles in HT29 and SW480 cells
(Additional file 1: Figure S1 C and Additional file 2: Figure
S2) [33–35]. Of note, hypoxia and cell death induced by
staurosporine (STS) failed to modulate autophagy as
shown in Additional file 1: Figure S1 B.
Obatoclax inhibits cell growth and induces cell death in
autophagy promoting conditions
In starving cells, catabolic autophagy becomes crucial to
keep cell metabolism running. Our data show that
obatoclax-induced growth inhibition in SW480 and
HT29 colorectal cancer cells is profoundly augmented
under starvation (Fig. 1 a, left and right upper graphs).
Of note, starvation alone did not reduce growth of
HT29 and SW480 cells (data not shown).
HT29 cells are resistant to obatoclax-induced cell
death even under starving conditions. By contrast,
SW480 cells show a high sensitivity towards obatoclax,
which is strikingly enhanced upon starvation. The
amount of propidium iodide (PI) positive SW480 cells
increased from 21 % to 46 % when exposed to starvation
instead of fully supplemented medium after 48 h obatoclax treatment (Fig. 1a, p < 0.001).
In order to investigate the role of obatoclax in autophagy of CRC cells, we performed western blot analyses.
We show that obatoclax, but not ABT-737, induces profound inhibition of autophagy in CRC cells pointing to a
unique feature of obatoclax regarding autophagy regulation. As shown in Fig. 1 b, immunoblotting reveals a
massive obatoclax induced accumulation of LC3 in both
cell lines, as well as an increased conversion from LC3I
to LC3II. This effect is already induced by sublethal
doses. In addition, we observed p62 accumulation in
cells treated with obatoclax. Collectively, the coinciding
accumulation of LC3II and p62 indicates a late stage autophagy inhibition by obatoclax.
Next, we sought to explore whether the impact of obatoclax on autophagy relevant proteins is limited to the
protein level. Q-RT PCR revealed an increase of LC3
mRNA transcription in obatoclax treated cells (Fig. 1 c).
This effect holds true under starvation.
Obatoclax is a late stage autophagy inhibitor in CRC cells
In order to validate our observations made by immunoblotting and to investigate the subcellular distribution of
autophagy relevant proteins, we performed immunohistochemistry of cells treated with obatoclax. Two different cell growth conditions were applied to study LC3
and p62 in obatoclax treated CRC cells. First, we seeded
and cultured HT29 on glass slides and applied obatoclax
treatment for 24 h. In mock treated control cells, there
was only a marginal amount of LC3 detectable. In line
with our findings from western blots, we observed a
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striking accumulation of LC3 in cells treated with obatoclax (Fig. 2 a, right). P62 staining revealed ubiquitous expression in control cells. The staining was cytosolic and
homogeneous in a granular fashion. In comparison, obatoclax treatment caused a much more intense staining
with emphasized large granules (Fig. 2 a, left).
3D polystyrene scaffolds foster cell-cell-interaction
mimicking tissue growth. Furthermore long-time studies
are feasible within this in vitro system. In line with our
earlier reports, obatoclax reduced cell growth and decelerated migration (data not shown). LC3 was not detectable in untreated control cells but showed massive
accumulation after long term obatoclax treatment. In
sections of polystyrene scaffolds p62 showed a weak expression in mock treated cells. By contrast, strong cytosolic signal for p62 was detected in cells after 7 days of
obatoclax treatment (Fig. 2 b). In summary, these observations point towards a swift but sustained late stage autophagy blockade.
In order to visualize the autophagic flux in cells
treated with obatoclax, we applied acridine orange (AO)
staining. In untreated cells the unprotonated form of
AO shows a homogeneous cytosolic distribution (Additional file 2: Figure S2). Under obatoclax treatment, the
protonated form becomes visible in a granular stain with
a predominantly perinuclear localization. This observation augments our hypothesis that autophagosomes are
built and formed but not degraded anymore in cells
treated with obatoclax (Additional file 2: Figure S2).
CQ has been introduced as a model agent for autophagy inhibition (Additional file 1: Figure S1). We stained
obatoclax and CQ treated HT29 cells with a fluorophore
labeled antibody against LC3. Mock treated cells showed
virtually no positivity for LC3. In comparison to CQ,
obatoclax caused a much more intense fluorescence signal by means of cytosolic LC3 puncta (Fig. 3 a and b, p
< 0.001).
Obatoclax blocks autophagy independently of Beclin-1,
Atg7 and Atg12
Autophagy related gene (Atg) 7 and Atg12 mediate important early steps during the formation of the autophagosome downstream of Beclin-1. In untreated cells,
siRNA mediated downregulation of Atg7 or Atg12 prevents LC3 conversion, leading to an accumulation of the
soluble LC3I form. Nevertheless, the obatoclax-induced
increase of LC3II was unaltered after Atg7 or Atg12
knockdown. Of note, p62 expression is weaker in obatoclax treated cells after Atg7 knockdown (Fig. 4 a). The
independence of obatoclax effects from Atg7 and Atg12
held true for cells under fully supplemented and starving
Beclin-1 is a central pro-autophagic protein involved
in early autophagosome biogenesis. Moreover, it has
Koehler et al. BMC Cancer (2015) 15:919
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Fig. 1 Obatoclax blocks cell growth and induces apoptosis in starving CRC cells. a and b HT29 and SW480 cells were treated with obatoclax
in full supplemented medium or starvation as indicated. a Cell growth was assessed by MTT-Assay after 48 h. Cell death was assessed by Flow
Cytometry after 48 h. Assays were done in triplicates. Values are expressed as mean ± SD. b Representative Western blots for p62 and LC3 I/II
in HT29 and SW480 treated with obatoclax or ABT-737 in full supplemented medium as indicated. c Rel. mRNA levels of LC3 in HT29 cells after
24 h treatment with obatoclax. mRNA levels were quantified by qRT-PCR and normalized to GAPDH as housekeeping gene. Assays are
representative of at least three independent experiments . ** = p < 0.01; *** = p < 0.001
been demonstrated that Beclin-1 interacts with antiapoptotic Bcl-2 and Bcl-xL, representing a key switch between apoptosis and autophagy signaling. We therefore
decided to investigate the role of Beclin-1 for the autophagy inhibition caused by obatoclax. siRNA mediated
knockdown of Beclin-1 led to high expression of LC3I.
The conversion from LC3I to LC3II was insufficient
after knockdown of Beclin-1 (Fig. 4 b). Furthermore, we
did not observe an accumulation of p62. The
described pattern of autophagy relevant proteins is
Koehler et al. BMC Cancer (2015) 15:919
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Fig. 2 Autophagy regulation by Obatoclax in 3D long term cell culture. a HT29 cells were seeded on glass slides, grown for 7 days and treated
with 0.25 μM obatoclax. Immunhistochemical staining was done for p62 and LC3b. b HT29 cells in scaffolds after 7 days treatment with
obatoclax. Immunhistochemical staining for p62 and LC3b. Representative pictures for at least 3 experiments are shown. Scale bar indicates
200 μm for longitudinal sections and 50 μM for corresponding insets. DMSO served as vehicle
indicative for an early block of autophagy caused by
downregulation of Beclin-1 (Fig. 4 b). Impressively,
obatoclax treatment led to a massive accumulation
of both, p62 and LC3II, even after knockdown of
We showed a starvation-dependent cell death induction caused by obatoclax in SW480 cells (Fig. 1).
However, we further investigated cell death under
obatoclax treatment after knockdown of Atg7 and
Beclin-1. We observed an unchanged cell death induction pattern in cells treated with obatoclax after
RNAi mediated knockdown of either Atg7 or Beclin-1
(Fig. 4 c).
Mcl-1 is a regulator of autophagy in colorectal cancer
Since we could show that ABT-737 failed to interfere
with autophagy (Fig. 1), we further focused on Mcl-1
and investigated its role in autophagy and its importance for the effects of obatoclax. A siRNA mediated
knockdown of Mcl-1 did not influence autophagy signaling as shown by an unchanged expression of LC3I
and LC3II as well as p62 in untreated cells (Fig. 4 b).
Intriguingly, knockdown of Mcl-1 led to an impaired
autophagy inhibition in HT29 cells, treated with
obatoclax. The impaired effectiveness of obatoclax on
autophagy becomes obvious in a decreased expression
Koehler et al. BMC Cancer (2015) 15:919
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Fig. 3 LC3 accumulates in autophagosomes in cells treated with Obatoclax. a Immunofluorescence staining for LC3 in untreated (upper panel),
Chloroquine treated (30 μM, middle panel) and obatoclax treated (0.25 μM, lower panel) cells. Representative pictures are shown. Assays were
done at least three times. b Analysis of LC3 aggregates per cell. Values are expressed as mean ± SD. *** = p < 0.001
of LC3I and a reduced conversion from LC3I to
LC3II (Fig. 4 b).
Autophagy has a decent role in colorectal organogenesis,
mucosal homeostasis and disease development. In colorectal cancer onset, autophagy plays a protective role for
cancer cells by means of energy delivery. However, targeting autophagy has been recently discovered as promising and novel treatment approach [9, 36]. Autophagy is
regulated by a tightly balanced signaling network of
enormous complexity. Within the autophagy signaling
network, crosstalks with classical apoptosis pathways
have been identified [37, 38]. For instance, Bcl-2, an
important antiapoptotic protein, binds proautophagic
Beclin-1 thereby inhibiting autophagy [20, 39].
First, we explored the potential impact of autophagy
inhibition in CRC cells. The malaria drug chloroquine
inhibits the execution of autophagy at a late stage via an
elusive mechanism [40]. Our data indicates that CQ is
an effective autophagy inhibitor in CRC cells. A moderate apoptosis induction by CQ is massively augmented
in cells under starvation. Hypoxia and direct autophagy
induction via Staurosporine failed to regulate autophagy.
This observation argues for a pivotal role of autophagy
and substantiates our hypothesis that inhibition of autophagy can trigger apoptosis induction in CRC.
In our present study, we sought to investigate the role
of Bcl-2 inhibition for autophagy. There are various
Koehler et al. BMC Cancer (2015) 15:919
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Fig. 4 Obatoclax blocks autophagy independent of canonical autophagy signaling. a and b Representative western blotting for HT29 cells
treated with obatoclax. siRNA mediated knockdown of Atg12 and Atg7 is shown in (a), siRNA mediated knockdown of Mcl-1 or Beclin-1 is shown
in (b). c Flow cytometric analysis for apoptosis induction in full supplemented (left) or starving (right) HT29 cells after knockdown of Atg7 or
Beclin-1 and treatment with obatoclax. Values are expressed as mean ± SD. Staurosporine 2 μM for 2 h served as a positive control for cell
death induction
small molecules available, mimicking proapoptotic proteins, which may lead to apoptosis induction. We have
recently shown that the pan-Bcl-2 inhibitor obatoclax
blocks invasiveness of colorectal cancer cells and causes
cell cycle arrest in G1-Phase [21]. Furthermore our earlier studies demonstrated that Mcl-1 sparing ABT737 but
not obatoclax induced apoptotic cell death in CRC cells.
Here, we show that cell growth inhibition as well as cell
death induction caused by obatoclax is profoundly
augmented when cells are challenged by starvation. The
degree of cell death induction and the subtype of death
in this context depend on the cell type. HT29 compared
to SW480 cells are likely to die through necroptosis rather than apoptosis [41].
This observation suggests a major role of autophagy,
since it becomes crucial for cell survival and growth in
stressful situations. Immunoblotting showed a massive
upregulation of LC3 on the protein level and an increased shift from LC3I to the activated and membrane
bound form LC3II. The increase of LC3 was accompanied by an accumulation of p62, a scaffold protein of
autophagic vesicles, in cells treated with obatoclax. This
protein pattern is indicative for a late stage inhibition of
autophagy, because high levels of LC3 indicate an
Koehler et al. BMC Cancer (2015) 15:919
increased autophagy flux whereas high levels of p62
occur in case of a disrupted enzymatic degradation of
autophagosome cargo [30]. The similarity of the expression pattern of p62 and LC3 under obatoclax and CQ
treatment further argues in favor of a late stage autophagy inhibition induced by obatoclax.
Interestingly, LC3 is not only upregulated on the protein level. On the transcriptional level we observed an
upregulation of LC3 mRNA. Little is known regarding
transcriptional regulation of autophagy. LC3 mRNA is
upregulated upon obatoclax treatment and, to an even
greater extent, in starved cells. Even not fully understood
yet, transcriptional regulation has recently moved into
research focus [42, 43] The transcription factor E2F has
been shown to be a mediator of LC3 upregulation [44].
In nasopharyngeal carcinoma, there is some evidence for
an involvement of the JNK-pathway in LC3 transcription
[45]. Nevertheless, the presented experimental set up is
unable to distinguish between a compensatory upregulation as a consequence of an autophagy blockade or a direct impact of obatoclax on transcription.
Subcellular localization of LC3 and the cytosolic LC3I/
II ratio has been widely used to visualize and quantify
autophagy [46, 47]. Nevertheless, choosing the right
assay to monitor and asses autophagy has been a matter
of debate [30, 48, 49]. We applied immunohistochemistry and fluorescence microscopy to analyze subcellular
distribution of autophagy proteins p62 and LC3. Staining
for LC3 revealed a weak expression in CRC cells in full
supplemented conditions. Upon obatoclax treatment, a
fundamental increase of LC3 can be observed in a
homogenous cytosolic and nucleic fashion. The translocation of LC3 to the nucleus as well as its interactions
with nucleic proteins has been recently described. Solely
the cytosolic fraction of LC3 after lipidization mediates
autophagy initiation, whereas the biological relevance of
nucleic LC3 remains elusive [49]. Immunofluorescence
for LC3 showed a strong expression but rather perinucleic distribution after obatoclax treatment.
P62 is a scaffold protein involved in a variety of cellular
processes including NF-kB-signaling [31, 50, 51]. Recently,
a correlation of p62 expression with clinicopathologic parameters has been shown for prostate cancer and gastrointestinal carcinoma [52, 53]. Under obatoclax treatment
we observe an accumulation of p62, which is indicative for
a late stage autophagy inhibition, since the scaffold protein
is trapped in the autophagic vesicle.
This hypothesis is underpinned by our immunohistochemical findings. Indeed, control cells showed a
homogenous cytosolic staining, turning into a granular
stain upon obatoclax treatment. Since the specificity of
p62 for autophagy is limited, an interaction of obatoclax
with NF-kB signaling or oxygen signaling should be addressed in future studies [31, 54].
Page 9 of 11
The decent mechanism by which obatoclax regulates
autophagy remains elusive. Here, we show that obatoclax
is capable of modulating autophagy even in the absence of
Beclin-1. Autophagy initiation is crucially regulated by
Beclin-1 [39, 55]. We observed that obatoclax treatment
caused LC3 accumulation in CRC cells after Beclin-1
knockdown to the same extent as in control cells. Beclin1’s interactions with Bcl-2 proteins have been reported to
play a role in autophagy regulation [56, 57].
Obatoclax seems to interfere with autophagy independently of the Beclin-1 complex. Therefore we further
investigated autophagy relevant proteins with regard to
implications for obatoclax. Atg7 belongs to the ubiquitin
like conjugation system (E1-like enzyme) mediating early
steps of phagophore formation. Atg12 is conjugated to
Atg5 and plays an important role in the conjugation of
LC3 to phosphatidylethanolamine (E3-like enzyme) [58,
59]. Strikingly, RNA interference leading to a knockdown of the respective protein (Atg7 and Atg12) left
LC3 activation unaltered. Taken together, our data indicate a regulative impact of obatoclax on autophagy independently of the cascaded and canonical autophagy
pathway. By contrast, others reported Beclin-1 and Atg5
dependent effects of obatoclax [59]. Liang and coworkers
propose an autophagy inducing effect of obatoclax,
which cannot be confirmed by our data. In line with our
presented findings in colorectal cancer, a Cathepsin
dependent inhibition of autophagosomal lysis by obatoclax has been reported in breast cancer [60]. Interestingly, knockout of Atg7 abolished LC3 processing but
failed to prevent obatoclax induced death in lung cancer
cells [61]. Furthermore, obatoclax has been recently
linked to the emerging concept of necroptosis [62]. In
addition, endoplasmatic reticulum stress and reactive oxygen species may contribute to obatoclax effects [63, 64].
Next, we decided to investigate Mcl-1 in the context
of obatoclax and autophagy, since Mcl-1 sparing
ABT737 failed to inhibit autophagy. The interaction of
Mcl-1 and Beclin-1 is crucial for chemotherapy resistance in leukemic B cells [65]. Mcl-1 has been described
as key factor for a governed resistance towards obatoclax
[66]. Interestingly, knockdown of Mcl-1 markedly suppressed LC3I level and decreased the LC3I/II ratio in
cells treated with obatoclax. A regulatory role of Mcl-1
in autophagy signaling has not yet been described mechanistically and deserves further attention.
In summary, our data prove that autophagy inhibition
represents a potent approach to inhibit CRC cell growth
in starving conditions reflecting a common phenomenon
in growing tumor tissues. Furthermore, we demonstrate
that pan-Bcl-2 inhibition by obatoclax utilizes autophagy
inhibition as a main effector. This late stage autophagy
Koehler et al. BMC Cancer (2015) 15:919
inhibition is independent of canonical autophagy signaling pathways. Finally, our data indicate a decisive role of
Mcl-1 in autophagy regulation, which needs further attention in future studies.
Additional files
Additional file 1: Figure S1. Chloroquine induces apoptosis in starving
CRC cells via autophagy inhibition. A) HT29 and SW480 cells were grown
in full supplemented or reduced medium to induce starvation. Cells were
treated with 20 μM Chloroquine for 48 h and apoptosis induction was
subsequently assessed by flow cytometry. Values are expressed as mean
± SD. B) HT29 and SW480 cells were treated with escalating Chloroquine
doses in full supplemented medium for 24 h. Representative Western
blots for p62 and LC3 I/II was performed. Hypoxia (1% O2) and
Staurosporine (2 μM, 6 h) served as control. Tubulin served as loading
control. Assays were done in triplicates. C) Fluorescence microscopy in HT29
with vital Lysotracker dye after 48 h Chloroquine (20 μM) treatment versus
mock treated cells. DMSO as a vesicle. *** = p < 0.001. (PDF 5657 kb)
Additional file 2: Figure S2. Obatoclax is a very late stage autophagy
inhibitor in CRC cells and allows acidification of autophagosomes. Cells
were treated with either Chloroquine (30 μM, middle) or obatoclax (0.25
μM, lower) for 48 h. Acridine orange was applied. Green dots indicate
unprotonated (left panel) and red dots (middle panel) protonated
Acridine Orange. The right panel shows a merged overlay. Pictures are
representative for three independent experiments. AO = Acridine Orange.
(PDF 6121 kb)
Page 10 of 11
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
BCK, AJ, ALS and HSB conceived and designed the experiments. BCK, AJ, SL,
ALS, NK and CE performed the experiments. BCK, AJ, ALS, HSB, PR, JW, and
MS analyzed the data. BCK, AJ, ALS, JW, PR, DJ, MS and HSB drafted the
manuscript. All authors read and approved the final version of the
Author details
National Center for Tumor Diseases, Department of Medical Oncology,
Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany.
Department of General, Visceral and Transplantation Surgery, University of
Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany.
Department of Clinical Pharmacology and Pharmacoepidemiology,
University Hospital Heidelberg, University of Heidelberg, Im Neuenheimer
Feld 410, 69120 Heidelberg, Germany. 4Department of Internal Medicine II,
Marien-Hospital, Wesel, Germany.
Received: 5 August 2015 Accepted: 12 November 2015
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