Oncotarget, Vol. 7, No. 3
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Inhibition of hypoxia inducible factor 1 and topoisomerase
with acriflavine sensitizes perihilar cholangiocarcinomas to
photodynamic therapy
Ruud Weijer1,2,*, Mans Broekgaarden1,*, Massis Krekorian1, Lindy K. Alles1, Albert
C. van Wijk1, Claire Mackaaij3, Joanne Verheij3, Allard C. van der Wal3, Thomas M.
van Gulik1, Gert Storm2,4, Michal Heger1,4
1
Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The
Netherlands
2
Department of Controlled Drug Delivery, MIRA Institute for Biomedical Technology and Technical Medicine, University of
Twente, 7500 AE Enschede, The Netherlands
3
Department of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
4
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The
Netherlands
*
These authors have contributed equally to this work
Correspondence to: Michal Heger, e-mail: m.heger@amc.uva.nl
Keywords: cancer therapy, drug delivery system, extrahepatic cholangiocarcinoma, hypoxia, tumor targeting
Received: August 03, 2015
Accepted: November 16, 2015
Published: November 27, 2015
ABSTRACT
Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative
stress and hypoxia but also survival signaling through activation of hypoxiainducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively
recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the
role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3)
determine whether HIF-1 inhibition reduces survival signaling and enhances PDT
efficacy.
Results: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was
confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered
zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased
transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells
and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment
of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and
topoisomerases I and II.
Methods: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was
determined by immunohistochemistry in human perihilar cholangiocarcinomas. In
addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT
with liposome-delivered zinc phthalocyanine was investigated under both normoxic
and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1β dimerization inhibitor and a
potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on
PDT efficacy.
Conclusions: HIF-1, which is activated in human hilar cholangiocarcinomas,
contributes to tumor cell survival following PDT in vitro. Combining PDT with
acriflavine pretreatment improves PDT efficacy in cultured cells and therefore
warrants further preclinical validation for therapy-recalcitrant perihilar
cholangiocarcinomas.
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cells [28] and human bladder cancer (UROtsa, RT112, and
J84 but not RT4) cells [29] as well as in murine Kaposi’s
sarcoma- [30], BA mouse mammary carcinoma- [31, 32],
and CNE2 nasopharyngeal carcinoma xenografts [33].
Inhibition of HIF-1 activity and corollary survival signaling
may consequently improve the therapeutic efficacy of PDT.
This study therefore investigated the therapeutic
potential of the HIF-1 dimerization inhibitor acriflavine
(ACF) in an in vitro PDT setting for the treatment of
human perihilar cholangiocarcinoma (SK-ChA-1)
cells [34], i.e., a cell line derived from a type of cancer
that is recalcitrant to different types of treatment. The
photosensitizer used in this study was zinc phthalocyanine
(ZnPC), a second-generation photosensitizer that was
encapsulated in cationic liposomes designed to target
tumor cells and tumor endothelium [35, 36]. ACF was
selected due to its selective inhibition of HIF-1 activation
[37] and due to its clinical safety [38]. In a recent study, it
was shown that ACF downregulates the HIF-1 target gene
vascular endothelial growth factor (VEGF) and reduces the
amount of tumor microvessels in murine breast carcinoma
(4T1)-bearing mice [39]. Moreover, Wong et al. revealed
that treatment of human breast carcinoma (MDA-MB-231
and MDA-MB-435)-xenografted mice with ACF inhibited
HIF-1-mediated invasion and metastasis [40]. Besides
HIF-1 inhibition, ACF was also investigated in the context
of its dual topoisomerase I and II inhibitor activity, as
discovered by Hassan et al. [41]. Topoisomerases are
involved in the cleavage and resealing of DNA breaks
during transcription and cell replication, and inhibition
of these topoisomerases may lead to cell cycle arrest and
apoptosis in dividing cells (reviewed in [42]).
The most important findings were that HIF-1
is activated by sublethal PDT in SK-ChA-1 cells.
Immunostaining
of
patient-derived
perihilar
cholangiocarcinoma biopsies demonstrated extensive
neovascularization in desmoplastic tissue and
heterogeneous glucose transporter 1 (GLUT-1)
overexpression, hinting towards the possible involvement
of hypoxia- and HIF-1-mediated angiogenesis. In vitro,
pretreatment of tumor cells with ACF improved PDT
outcome and reduced the PDT-induced expression of
VEGF and PTGS2. Lastly, incubation of SK-ChA-1 cells
with ACF resulted in induction of S-phase cell cycle arrest,
DNA damage, and apoptosis, altogether underscoring
ACF’s dual topoisomerase I/II inhibition potential and
utility to act as a neoadjuvant chemotherapeutic in PDT.
INTRODUCTION
Photodynamic therapy (PDT) is a non-to-minimally
invasive treatment modality for a variety of solid cancers.
This therapy is based on the accumulation of a lightsensitive drug (photosensitizer) in the tumor following
systemic administration. Next, the photosensitizer-replete
tumor is locally irradiated with (laser) light, resulting in the
activation of the photosensitizer and subsequent production
of reactive oxygen species (ROS) via type I (superoxide)
and/or type II (singlet oxygen) photochemical reactions.
Consequently, PDT locally induces a state of hyperoxidative
stress, culminating in tumor cell death, destruction of
the microvasculature that causes tumor hypoxia and
hyponutrition, and an anti-tumor immune response [1, 2].
PDT is effective in the curative treatment of (pre-)
malignant skin lesions (actinic keratosis, basal/squamous
cell carcinoma) [3], but is also employed as (last-line)
treatment of head and neck cancer [4], early central stage
lung tumors [5], esophageal cancer [6], nasopharyngeal
carcinomas [7], bladder cancer [8], and non-resectable
perihilar cholangiocarcinomas [9]. Although PDT yields
complete response rates of 50–90% in the majority of the
abovementioned cancers, nasopharyngeal-, urothelial-,
and perihilar cholangiocarcinomas are relatively refractory
to PDT. This may be in part due to hypoxia-mediated
survival signaling that is triggered by the stabilization
of hypoxia inducible factor 1 (HIF-1) following PDT
[10–12]. In nasopharyngeal and superficial urothelial
carcinomas, the overexpression of HIF-1α has been
associated with poor overall survival [13, 14]. HIF-1
expression levels in perihilar cholangiocarcinomas are
currently elusive but may account for the recalcitrance of
these tumors to therapy [15].
HIF-1 is a transcription factor composed of HIF-1α
and HIF-1β subunits. During normoxia, prolyl-hydroxylases
(PHD) and factor inhibiting HIF (FIH) mediate the
hydroxylation of Pro402, Pro564, and/or Asn803 of HIF1α [16]. In turn, Von Hippel-Lindau tumor suppressor
protein (VHL) binds to hydroxylated HIF-1α, resulting
in complexation with E3 ubiquitin ligase and subsequent
proteasomal degradation of HIF-1α [17, 18]. In contrast,
hypoxia inhibits the activity of both PHDs and FIH, leading
to HIF-1α stabilization and nuclear translocation [19].
After translocation to the nucleus, HIF-1α dimerizes with
HIF-1β and mediates the transcription of various genes [20]
that are involved in glycolysis, angiogenesis, survival, and
apoptosis [21–23]. Alternatively, HIF-1 may be activated
through ROS, which also deter the activity of PHDs and
FIH, leading to the stabilization and nuclear translocation
of HIF-1α [24, 25].
HIF-1 is constitutively active in most tumors since
the tumor growth rate exceeds the rate of neoangiogenesis
[21, 23]. Moreover, HIF-1 is responsible for resistance to
chemotherapy and radiotherapy [26, 27]. PDT increases
HIF-1 activity in mouse mammary carcinoma (EMT-6)
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RESULTS
Expression of hypoxia-related proteins in human
perihilar cholangiocarcinoma
Although the incidence of tumor hypoxia and the
importance of HIF-1 expression in a large variety of tumors
have been widely established, literature on this phenomenon
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albeit several regions containing GLUT-1-expressing
cell clusters were observed in other sections of the tumor
(Figure 1F). Accordingly, these results provide compelling
evidence for the presence of hypoxia and HIF-1 activation
in perihilar cholangiocarcinomas, which likely drive
angiogenesis and regional upregulation of glycolysis.
Moreover, the perihilar cholangiocarcinomas are replete
with vasculature that may serve as a conduit for the
delivery of liposome-encapsulated photosensitizers.
in perihilar cholangiocarcinomas is scarce. Therefore, it
was investigated whether hypoxia-related proteins (VEGF
for angiogenic signaling, CD105 and CD31/Ki-67 for
neovascularization, and GLUT-1 for glycolysis) were present
in perihilar cholangiocarcinoma resection specimens. Of
note, immunostaining for HIF-1α directly was not performed
due to its high instability (protein half-life of 5–8 minutes)
[43]. Representative differently stained serial images are
presented in Figure 1.
The hematoxylin and eosin staining (Figure 1A)
revealed that perihilar cholangiocarcinomas were
characterized by clusters of tumor cells surrounded by
relatively large areas of desmoplastic tissue (i.e., stroma).
The tumor mass stained positively for VEGF (as did liver
tissue), whereas VEGF staining was less prominent in
the tumor stroma (Figure 1B). Nevertheless, the tumor
stroma was densely vascularized. The vasculature in the
desmoplastic tissue was not of pre-existent nature, as the
endothelium stained positively for CD105, a marker for
angiogenic endothelium (Figure 1C), and Ki-67, a marker
of proliferation (Figure 1D). Of note, the tumor mass was
largely devoid of Ki-67-positive cells, indicating that the
perihilar cholangiocarcinomas in our patient population
were slowly proliferating tumors. GLUT-1 was largely
absent in the tumor cell mass and stroma (Figure 1E),
HIF-1 is activated after PDT
To establish whether HIF-1 was activated by PDT,
the optimal PDT dose was first determined in perihilar
cholangiocarcinoma (SK-ChA-1) cells. SK-ChA-1
cells were incubated with increasing concentrations of
ZnPC-encapsulating cationic liposomes (ZnPC-ETLs)
and subsequently treated with PDT (500 mW, 15 J/
cm2). These liposomes have been shown to selectively
accumulate in tumor endothelium [44], which is expected
to translate to vascular shutdown and exacerbated tumor
hypoxia following PDT [15]. Moreover, the ZnPC-ETLs
are taken up by tumor cells, including SK-ChA-1 cells
(manuscript in preparation). After PDT, the cells were
either maintained under normoxic or hypoxic culture
Figure 1: Hypoxia-related protein expression in an extrahepatic perihilar cholangiocarcinoma (Klatskin tumor)
resection specimen. Serial histological sections were used for protein profiling in the same region. A. Hematoxylin and eosin staining
of a cholangiocarcinoma section containing tumor mass (intense purple staining, circular structure, bottom left, marked with an asterisk
in panels A-E), tumor stroma, and native tissue (e.g., pre-existent arterial structures). B. VEGF staining (brown), showing intense staining
of the tumor mass and vascular endothelium (insert) as well as pre-existent biliary structures (insert, arrow). The insert corresponds to the
demarcated region in the low-magnification image. C. CD105 staining (brown), showing no staining in the tumor mass and positive staining
of the vascular endothelial cells in the tumor stroma (insert, arrow, indicates neovessel formation). D. Angiogenesis was further confirmed
with CD31 (blue) and Ki-67 (red) double staining, showing that the blood vessels in the tumor stroma contain proliferating endothelial
cells (insert, arrows). E. GLUT-1 staining (brown) was largely absent in the tumor mass and stroma, indicating that these regions were not
affected by hypoxia. In some regions of the tumor, however, positive staining was observed (insert, arrow). F. Strong GLUT-1 staining was
found in another region of the histological specimen. Magnification: 4× (A-E, scale bar = 100 μm) and 10× (F, scale bar = 40 μm).
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conditions (Figure 2A) to mimic the PDT-induced
vascular shutdown [45, 46]. Cell viability was determined
24 hours after PDT using the WST-1 assay. Cells
exhibited a ZnPC concentration-dependent decrease in
cell viability following PDT, whereby the extent of cell
death was exacerbated by hypoxia (Figure 2A). Since
the IC50 concentration in normoxic and hypoxic cells
were approximately 10 and 5 μM ZnPC-ETLs (final lipid
concentration), respectively, these concentrations were
used in the rest of the experiments.
Next, the stabilization of HIF-1α and induction of
HIF-1α transcriptional targets were investigated following
PDT. As shown in Figure 2B, normoxic SK-ChA-1 cells
exhibited no notable HIF-1α expression. Stimulation of
cells with cobalt chloride is commonly used to induce
hypoxic signaling [47, 48] and was therefore used as
positive control. Indeed, cobalt chloride caused extensive
HIF-1α stabilization. Accordingly, SK-ChA-1 cells that
were placed in a hypoxic chamber stabilized HIF-1α in
a time-dependent manner, albeit less extensively than
after cobalt chloride stimulation. HIF-1α stabilization was
enhanced upon PDT.
The HIF-1α stabilization was associated with
upregulated transcription of several HIF-1 target genes,
including VEGF (angiogenesis), PTGS2 (survival), and
HMOX1 (survival) (Figure 2C). SK-ChA-1 cells also
upregulated SERPINE1 (angiogenesis) and baculoviral
inhibitor of apoptosis repeat-containing 5 (BIRC5, survival)
after PDT. It was therefore concluded that HIF-1α is
upregulated in SK-ChA-1 cells following PDT, albeit to a
minor extent in comparison to the cobalt chloride treatment.
ACF is translocated to the nucleus upon hypoxia
and/or PDT
Since PDT induced HIF-1 signaling in SK-ChA-1
cells, which may be responsible for the therapeutic
recalcitrance in vivo, we investigated whether the
Figure 2: Analysis of HIF-1α activation after PDT. A. SK-ChA-1 cells were incubated with increasing concentration of ZnPCETLs, treated with PDT, and maintained at normoxic (red bars) or hypoxic (blue bars) culture conditions. Cell viability was determined 24
hours post-PDT (n = 6 per group). B. SK-ChA-1 cells were treated with PDT (10 μM ZnPC-ETLs, final lipid concentration) or received a
control (CTRL) treatment, after which the cells were placed in a hypoxic chamber up to 240 minutes (min) post-PDT. HIF-1α protein levels
were determined using Western blotting. As a positive control, cells were incubated with 500 μM CoCl2 for 24 hours (top panel). Next, the
HIF-1α protein bands and their corresponding β-actin protein bands were quantified using ImageJ software [74] and each HIF-1α value
was divided by its corresponding β-actin value. All values were normalized to the positive control (CoCl2) (bottom panel). C. SK-ChA-1
cells were either left untreated (grey bars) or treated with PDT (white bars), and subsequently placed at hypoxic conditions for 4 hours.
Thereafter, downstream targets of HIF-1 were analyzed with qRT-PCR (n = 3 per group). Readers are referred to the experimental section
for the significance of the statistical symbols.
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HIF-1α/HIF-1β dimerization inhibitor ACF would
enhance PDT efficacy. First, the intracellular localization
of ACF was determined by confocal microscopy, whereby
the intrinsic fluorescence of ACF (λex = 453 nm and λem
= 507 nm) in combination with (intra)cellular membrane
staining (Figure 3). SK-ChA-1 cells were incubated with
ACF during normoxia, hypoxia, and/or after PDT to study
the cytosolic-to-nuclear translocation of ACF during these
processes.
As shown in Figure 3A, ACF was localized in both
the nucleus and cytosol under normoxic conditions. PDT
treatment was accompanied by a translocation of ACF
towards the nucleus under normoxic conditions, which
was further characterized by altered cell morphology that
entailed cell shrinkage and blebbing (Figure 3B). Hypoxia
(in the absence of PDT) triggered prominent translocation
of ACF from the cytosol to the nucleus (Figure 3C).
Interestingly, PDT-treated SK-ChA-1 cells that were
placed in a hypoxic environment revealed a similar ACF
distribution pattern as PDT-treated cells under normoxic
conditions. ACF was mainly found in the nucleus in PDTtreated hypoxic cells, albeit at relatively lower levels
compared to untreated hypoxic cells (Figure 3D).
To determine the most suitable concentration
of ACF for the improvement of PDT efficacy, the
concentration-dependent uptake and toxicity of ACF
were tested in SK-ChA-1 cells. ACF uptake followed a
concentration-dependent linear pattern up to 5 μM ACF
(Figure 4B). The toxicity of ACF was determined during
a 24-hour incubation period under either normoxic or
hypoxic conditions (Figure 4C). The IC50 concentration
during normoxia and hypoxia, determined with the WST-1
assay, was 29 and 73 μM, respectively. Inasmuch as SKChA-1 cells exhibited a relative viability of ~90% at 3 μM
ACF during normoxia, this concentration was used in the
rest of the experiments.
Next, SK-ChA-1 cells were incubated with ACF
for 24 hours under normoxic conditions and treated with
PDT (Figure 4D) to investigate ACF’s adjuvant efficacy.
As indicated, ACF was mildly toxic, which translated
to slightly increased cytotoxicity when combined with
PDT and normoxic incubation (Figure 4D). A similar
trend was observed in cells that were maintained under
hypoxic conditions after PDT (Figure 4E). In addition,
the levels of caspase 3 and 7 (i.e., apoptosis markers)
were assayed 4 hours post-treatment (Figure 4F, 4G).
Under normoxic conditions, neither ACF nor PDT
significantly affected caspase 3/7 levels, however, ACF
+ PDT resulted in a 8-fold higher caspase 3/7 activity
in SK-ChA-1 cells (Figure 4G). During hypoxia, PDT
resulted in a 4-fold increase in caspase 3/7 activity
and ACF + PDT resulted in a 10-fold higher caspase
3/7 activity, indicating that apoptosis constitutes an
important mode of cell death following combination
treatment of ACF + PDT. None of the conditions
induced the formation of DNA double-strand breaks,
as assessed by a phospho-H2AX staining 4 hours after
treatment (Figure 4I–4P), indicating that neither hypoxia
nor ACF or PDT induce direct damage to DNA in the
acute phase.
Lastly, inasmuch as HIF-1 signaling is a driving
force behind glycolysis and the consequent production
ACF potentiated PDT efficacy
For clinical application purposes, ACF should
remain stable during the application of PDT and during
conditions of oxidative stress in order to inhibit HIF-1
activation after PDT and the subsequent microvascular
shutdown. A model system was therefore used to study
the stability of ACF during PDT. ACF was dissolved in
buffer solution and exposed to increasing amounts of cell
phantoms (i.e., artificial cells) loaded with ZnPC, of which
it was demonstrated that ROS is produced upon irradiation
[36]. As shown in Figure 4A, the application of PDT only
marginally affected ACF fluorescence, confirming that
ACF remained stable during illumination and conditions
of hyperoxidative stress.
Figure 3: Intracellular ACF localization. A–D. SK-ChA-1 cells were either left untreated or treated with PDT and subsequently
incubated with ACF for 4 hours under normoxic (A, B) or hypoxic culture conditions (C, D). ACF localization was determined using
confocal microscopy (ACF in green; Nile Red (membrane staining) in red).
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of lactate [49], the production of lactate was quantified
in the cell culture medium 24 hours after ACF and
PDT treatment (Figure 4H). Lactate excretion levels
were substantially increased under hypoxic conditions
in all treatment groups compared to normoxic cells,
validating our hypoxic incubation model. However, no
further intergroup differences were observed in this cell
line with respect to lactate production. Neither ACF nor
PDT therefore induced notable metabolic catastrophe in
cells.
Figure 4: Combination treatment of ACF with PDT. A. Evaluation of ACF stability using increasing concentrations of ZnPCcontaining cell phantoms (ZnPC-CPs) with or without irradiation. ACF degradation was monitored using fluorescence spectroscopy (n =
4 per concentration). B. Cells were incubated with ACF for 24 hours, after which the uptake of ACF was determined using fluorescence
spectroscopy. Data were normalized to protein content (n = 4 per concentration). C. ACF toxicity was determined after 24-hour incubation
under either normoxic (red line) or hypoxic (blue line) conditions using the WST-1 method (n = 4 per group). Treatment efficacy of ACF
and ACF + PDT was tested in SK-ChA-1 cells after 4 hours at D. normoxic and E. hypoxic culture conditions (n = 6 per group). (F, G)
Relative caspase 3/7 activity was determined 4 hours after PDT at incubation at F. normoxic or G. hypoxic culture conditions (n = 6 per
group). H. Lactate production by SK-ChA-1 cells treated with ACF and ACF + PDT was evaluated after 24 hours at normoxic (red bars) or
hypoxic (blue bars) culture conditions (n = 6 per group). I–P. Analysis of DNA damage after control (CTRL), ACF, PDT, and ACF + PDT
treatment. Cells were kept for 4 hours under normoxic (I-L) or hypoxic conditions (M-P) post-treatment. Cells were stained with DAPI
(nuclei, blue) and phospho-H2AX (DNA double-strand breaks, red). The arrowhead in panel P indicates apoptosis. Readers are referred to
the experimental section for the significance of the statistical symbols.
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ACF interferes with the regulation of
HIF-1-induced target genes
However, the direction of the regulation is not always
consistent within one functional class.
To study whether pre-treatment with ACF
influences post-PDT HIF-1α signaling, SK-ChA-1 cells
were incubated with ACF and subsequently treated
with PDT and maintained under hypoxic conditions.
HIF-1 downstream targets were clustered in angiogenesis-,
glycolysis-, and survival-associated genes and analyzed
by quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR) at different time points after PDT
(Figure 5). Moreover, additional HIF-1 target genes were
included in the ACF-related transcriptomic analysis.
PDT induced the expression of VEGF, HMOX1, and
PTGS2, corroborating the data in Figure 2C. ACF reduced
the degree of PTGS2 upregulation (only in the 0-h and 2-h
group) and VEGF transcription post-PDT. Conversely, EDN1
was downregulated by hypoxia and PDT but upregulated
by ACF. In addition, SERPINE1 was highly induced upon
ACF treatment - an effect that was also observed after PDT
in the presence of ACF. Altogether, these findings indicate
that ACF by itself and in combination with PDT modulates
several important HIF-1-induced transcriptional targets.
Long-term exposure to ACF causes cell cycle
arrest and apoptosis
Although ACF is generally considered a specific
HIF-1α/HIF-1β dimerization inhibitor [37], Hassan
et al. have reported that ACF may also act as a dual
topoisomerase I/II inhibitor [41]. Topoisomerase I/II
inhibition is associated with cell cycle arrest and
consequent apoptosis as a result of DNA double-strand
breaks (reviewed in [42, 50]). In the acute phase after
PDT, DNA double-strand breaks were not observed
(Figure 4I–4P) but apoptotic signaling was pronounced,
particularly in the ACF + PDT and hypoxia groups (Figure
4F and 4G). To investigate the potential topoisomerase I/II
inhibitory effects, SK-ChA-1 cells were exposed to ACF
for longer time frames (24 and 48 hours) under normoxic
conditions, after which the cell cycle profile was analyzed
using propidium iodide staining (Figure 6A–6D).
As shown in Figure 6B and 6D, ACF led to an
increased fraction of cells in both the S- and G2/M-phase
Figure 5: Gene expression analysis after control (CTRL), ACF, PDT, and ACF + PDT treatment. Gene expression levels
were obtained by qRT-PCR from SK-ChA-1 cells as analyzed 0 hours, 2 hours, or 4 hours post-treatment under hypoxic conditions. The
plotted heat map data represents the log2-transformed fold change of each data point in relation to the 0-hour normoxic CTRL. Upregulated
genes are depicted in red, downregulated genes in green. Numeric values are provided in Table S2.
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after 24 and 48 hours of incubation. The most significant
effect of ACF was characterized by cell cycle arrest in
the S-phase after 48 hours of incubation. Furthermore,
ACF treatment was associated with increased apoptosis,
but not necrosis, after 24 and 48 hours (Figure 6E, 6F),
which concurred with elevated ROS production in cells
(Figure 6G, 6H). Finally, incubation of SK-ChA-1 cells
with ACF for 24 or 48 hours led to the formation of DNA
double-strand breaks (Figure 6I–6P), although not in a
concentration-dependent manner.
DISCUSSION
Perihilar cholangiocarcinoma is a relatively rare
cancer that is non-resectable in 70–80% of patients at
the time of diagnosis [51]. Although PDT is not curative
Figure 6: Response to ACF after prolonged exposure. A–D. SK-ChA-1 cells were incubated with ACF for either (A, B) 24 hours
or (C, D) 48 hours, after which the cell cycle profile was analyzed with flow cytrometry using propidium iodide staining (n = 3 per group).
E. Flow cytometric analysis of SK-ChA-1 cells that were incubated with ACF for either 24 hours (in grey) or 48 hours (in white), after
which the fraction of apoptotic (annexin V-positive) and F. necrotic (TO-PRO-3-positive) cells was determined (n = 3 per group). (G, H)
SK-ChA-1 cells were exposed to ACF for G. 24 hours or H. 48 hours and intracellular DCF fluorescence was determined as a measure of
ROS production. I–P. Analysis of DNA damage after control (CTRL) or ACF treatment. SK-ChA-1 cells received ACF or CTRL treatment
for (I-L) 24 hours or (M-P) 48 hours, and were subsequently stained with DAPI (nuclei, blue) and phospho-H2AX (DNA double-strand
breaks, red).
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in these patients, the treatment does prolong the median
survival of 6–9 months (stenting) to 21 months postdiagnosis (stenting + PDT) [9]. Driven by these promising
results, novel avenues are being explored to enhance PDT
efficacy in these refractory and rather lethal cancers.
PDT is associated with microvasculature shutdown
and consequent HIF-1 signaling that may contribute to
therapeutic recalcitrance [10]. Therefore, this study was
conducted to investigate the expression of HIF-1-induced
proteins in perihilar cholangiocarcinomas to gauge whether
inhibition of HIF-1 may be exploited as a therapeutic
target in the context of PDT. Histological analysis revealed
that human perihilar cholangiocarcinomas overexpress
VEGF homogeneously and GLUT-1 heterogeneously and
are replete with neoangiogenic vessels in the desmoplastic
tissue, suggesting that HIF-1 is constitutively active in
these tumors. Second, PDT of SK-ChA-1 cells with ZnPCencapsulating liposomes caused HIF-1α stabilization
and transcriptional upregulation of downstream targets
of HIF-1. Third, ACF was taken up by SK-ChA-1 cells,
especially during hypoxia, and translocated to the nucleus
upon hypoxia and PDT. Lastly, ACF pretreatment was
associated with S-phase cell cycle arrest and apoptosis
and enhanced PDT efficacy, likely via inhibition of HIF-1
inhibition and topoisomerase I/II.
HIF-1α stabilization after PDT has been observed
in various experimental settings. Ferrario et al.
revealed that porfimer sodium-PDT resulted in HIF1α stabilization in murine Kaposi’s sarcoma [30]. PDT
also upregulated the HIF-1-associated targets VEGF
and COX-2 [30]. In mouse mammary carcinoma (BA)
xenografts [31], porfimer-PDT led to an increase in
HIF-1α, BIRC5, and VEGF protein levels. Lastly,
murine mammary carcinoma (EMT-6) cells that were
treated with porfimer sodium-PDT exhibited HIF-1α
stabilization and its consequent translocation to the
nucleus [28]. In line with these findings, our study
demonstrated that HIF-1α was stabilized in SK-ChA-1
cells after incubation in a hypoxic chamber (to mimic
vascular shutdown) and after PDT. PDT also led to
the differential regulation of HIF-1-regulated genes,
including VEGF, PTGS2, SERPINE1, HMOX1, and
BIRC5. Consistent with these results, it was recently
demonstrated that SK-ChA-1 cells subjected to sublethal
PDT with neutral ZnPC-encapsulating liposomes
significantly upregulated HIF-1-associated genes 90
minutes post-PDT [52]. Altogether, these findings attest
that HIF-1α is activated following PDT and that this
transcription factor constitutes an important therapeutic
target, particularly in light of the fact that HIF-1
regulates biological processes that are important in PDT,
such as glycolysis, angiogenesis, and survival [10].
The combinatorial use of HIF-1 inhibitors with PDT
is a relatively new concept. For instance, Chen et al. used
HIF-1α siRNAs in combination with Photosan-PDT in
a head-and-neck cancer mouse model, which resulted in
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regression of tumor volume by ~40% within 10 days [53].
Besides HIF-1 inhibition, its downstream target VEGF
has been inhibited in various studies [32, 54, 55], which
generally led to improved therapeutic efficacy. Although
downstream targets of HIF-1 may be inhibited, from a
pharmacology point of view it would be more attractive
to inhibit HIF-1 itself, inasmuch as all the downstream
targets are blocked concomitantly. As such, the HIF-1α/
HIF-1β dimerization inhibitor ACF was evaluated for its
adjuvant potential in SK-ChA-1 cells. ACF specifically
binds the PER-ARNT-SIM (PAS) domain of HIF-1α and
HIF-2α, which prevents the dimerization of HIF-1, thereby
deterring its activation [37]. It was observed that ACF
was taken up by SK-ChA-1 cells and translocated to the
nucleus after hypoxia and/or PDT, presumably due to its
binding to HIF-1α and HIF-2α. Moreover, ACF remained
stable during the application of intense (laser) light
exposure as well as during conditions of oxidative stress,
suggesting that ACF will be able to inhibit HIF-1 after
PDT and consequent vascular shutdown. The IC50 value
of ACF in SK-ChA-1 cells was 29 μM during normoxia,
which is in the range that has been observed for other
cell lines [56]. Strese et al. found that human leukemic
monocyte lymphoma (U937) was most susceptible to
ACF, as demonstrated by an IC50 value of 4.6 μM, whereas
human breast cancer (MCF-7) cells exhibited an IC50 value
of 61 μM [56]. Pretreatment of SK-ChA-1 cells with ACF
significantly improved therapeutic efficacy, which was
partially mediated by the increase in caspase 3/7 levels
(apoptosis).
In addition to HIF-1 inhibition, ACF has also
been shown to act as a dual topoisomerase I/II inhibitor
[41]. Of note, topoisomerase inhibitors (e.g., topotecan
[57]) may also repress gene transcription, but to what
extent ACF is able to inhibit HIF-1-mediated signaling
via this mechanism is currently elusive. Topoisomerase
class I and II inhibitors cleave either one or both strands
of DNA, respectively. Both topoisomerase I and II
inhibitors may induce the formation of DNA doublestrand breaks, inasmuch as the single-strand break
that is induced by topoisomerase I inhibitors may turn
into a double-strand break when the topoisomerase I
cleavable complex collides with the replication fork
[58]. This type of DNA damage may culminate in
cell cycle arrest via tumor protein 53 (p53)-mediated
p21WAF1/CIP1 induction, cellular senescence, and both p53dependent and p53-independent apoptosis [42, 50, 58,
59]. To determine whether the observed cell death could
(in part) be explained by topoisomerase I/II inhibition,
SK-ChA-1 cells were incubated with ACF for 24 and
48 hours (i.e., a full cell cycle requires 48 hours [34]).
It should be noted that, although SK-ChA-1 cells have a
mutation (at codon 282) in the DNA binding domain of
p53 [60], their p53 is still functional. ACF incubation led
to cell cycle arrest in both the S-phase and the G2/Mphase and was associated with an increased percentage
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L-α-phosphatidylethanolamine,
distearoyl
methoxypolyethylene glycol conjugate (DSPE-PEG,
average PEG molecular mass of 2,000 amu), ZnPC,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), fibronectin, sodium chloride (NaCl),
β-mercaptoethanol, cholesterol, chloroform, Nile Red,
paraformaldehyde, sucrose, bovine serum albumin (BSA),
Tween 20, CoCl2, ACF, and pyridine were obtained from
Sigma-Aldrich (St. Louis, MO). Tris-HCl and dimethyl
sulfoxide (DMSO) were purchased from Merck KgaA
(Darmstadt, Germany). Ethanol was obtained from
Biosolve (Valkenswaard, the Netherlands). Protease
inhibitor cocktail and water-soluble tetrazolium-1 (WST1) were purchased from Roche Applied Science (Basel,
Switzerland). 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH2-DA) was obtained from Life Technologies
(Carlsbad, CA).
All lipids were dissolved in chloroform, purged
with nitrogen gas, and stored at −20°C. Phospholipid
stock concentrations were determined by the inorganic
phosphate assay modified from [66]. ZnPC was dissolved
in pyridine at a 178-μM concentration and stored at room
temperature (RT) in the dark, CoCl2 was dissolved in
MilliQ at a concentration of 50 mM, and ACF and DCFH2DA were dissolved in DMSO at a concentration of 50 mM.
of apoptotic cells. SK-ChA-1 cells also exhibited DNA
double-strand breaks as a result of ACF incubation.
Collectively, these findings support the notion that ACF
exhibits topoisomerase I/II inhibition activity that may
contribute to greater therapeutic efficacy.
An interesting finding of this study is the
upregulation of SERPINE1 after ACF treatment.
SERPINE1 is a downstream target of both HIF-1 and
p53 [61] and its protein product plasminogen activator
inhibitor 1 (PAI1) is known to exhibit pleiotropic effects.
PAI1 is involved in the inhibition of extracellular matrix
remodeling, but it also has anti-apoptotic and proproliferative capacities and is involved in angiogenesis
[62, 63]. This has been exemplified by Devy et al.,
who demonstrated that cultured mouse aortic rings
from PAI1-deficient mice, which were stimulated with
PAI1, exhibited a dose-dependent angiogenic response
[64]. Whereas low-dose levels of PAI1 were associated
with increased angiogenesis, high-dose levels of PAI1
inhibited microvessel formation [64]. To what extent
p53 is responsible for SERPINE1 induction after ACF
treatment is currently elusive, as are the consequences of
PAI1 induction in the context of PDT.
As stated earlier, the use of inhibitors of specific
survival pathways with PDT is a relatively novel
strategy. Several studies have indicated that inhibition of
survival pathways in conjunction with PDT may be an
attractive means to enhance PDT efficacy (reviewed in
[10]). Consistent with these results, the present findings
also encourage the use of small molecule inhibitors
(e.g., HIF-1 inhibitors) of survival pathways together
with PDT. These small molecule inhibitors can be coencapsulated with a photosensitizer into a single drug
delivery system, such as liposomes, in order to improve
treatment outcome.
In conclusion, HIF-1 is overexpressed in a variety
of solid cancers and is often associated with therapeutic
recalcitrance, inasmuch as it stimulates glycolysis,
angiogenesis, and survival. This study demonstrated that
HIF-1 inhibition via ACF may be an attractive method to
potentiate PDT efficacy in perihilar cholangiocarcinoma.
Interestingly, not only HIF-1 inhibition, but also
topoisomerase I/II inhibition by ACF may further
contribute to increased PDT efficacy. In vivo studies as
addressed in [65] are necessary to validate the potential of
ACF in combination with PDT.
Histology
Histology was performed on two patient-derived,
paraffin-embedded perihilar cholangiocarcinoma biopsies.
Tissue sections were dewaxed in xylene and rehydrated in
graded steps of ethanol. Endogenous peroxidase activity
was blocked with methanol containing 0.3% peroxide
(20 min, RT). Heat-induced epitope retrieval (HIER)
was performed in a pretreatment module (Thermo Fisher
Scientific, Fremont, CA) using Tris-EDTA (VEGF, Ki67, CD31, GLUT-1) or citrate buffer (CD105) for 20
minutes at 98°C. Throughout the staining procedure all
washing steps were performed with Tris-buffered saline.
Superblock (Immunologic, Duiven, the Netherlands) was
applied as a protein block prior to staining with primary
antibodies.
All antibodies were diluted with antibody diluent
(Scytek, Logan, UT). Single stains for CD105 (rabbit
anti-human, polyclonal, cat # RB-9291, Thermo Fischer
Scientific), VEGF (rabbit anti-human, polyclonal, cat.
# sc-152, Santa Cruz Biotechnology, Santa Cruz, CA),
and GLUT-1 (rabbit anti-human, polyclonal, cat. # RB9052, Thermo Fischer Scientific) were performed. These
primary antibodies were visualized with BrightVision
HRP-conjugated anti-rabbit polymer (Immunologic)
and BrightDAB. The sections were counterstained with
hematoxylin.
Sequential double staining [67] was performed for
CD31 (mouse anti-human, clone JC70A, cat. # M0823,
Dako, Glostrup, Denmark) and Ki-67 (rabbit anti-human,
MATERIALS AND METHODS
Chemicals
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine
(DPPS), and 3β-[N-(N’,N’-dimethylaminoethane)carbimoyl]cholesterol (DC-chol) were purchased
from Avanti Polar Lipids (Alabaster, AL).
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clone SP6, cat. # RM9106, Thermo Fischer Scientific).
Ki-67 was visualized with BrightVision AP-conjugated
anti-rabbit polymer (Immunologic) and Vector Red (Vector
Labs, Burlingame, CA). Next, an intermediate HIER step
using Tris-EDTA buffer (10 minutes at 98°C) was applied
to remove all antibodies but leaving the chromogen intact
[68]. Finally, CD31 was visualized with BrightVision
AP-conjugated anti-mouse polymer (Immunologic) and
PermaBlue plus/AP (Diagnostics Biosystems, Pleasanton,
CA). All slides were dried on a hotplate (50°C) and
permanently mounted with Vectamount (Vector Labs).
It should be noted that, as part of the clinical
diagnostics protocol at the Department of Pathology, all
antibodies had been validated for their cross-reactivity
and immunohistological staining efficacy using tissue that
overexpresses the respective marker. Immunostaining for
HIF-1α directly was not performed due to the instability
of the HIF-1α antigen (degrades within a few minutes after
biopsy).
RPMI 1640 medium) for 24 hours prior to PDT. Next,
cells were washed with PBS and incubated with ZnPCETLs (in serum-free supplemented RPMI 1640 medium)
for 1 hour at standard culture conditions. Cells were
washed twice with PBS and fresh serum- and phenol redfree supplemented RPMI 1640 was added to the cells.
Serum was deliberately withdrawn after PDT in order to
emulate the hyponutritional status of PDT-treated tumor
cells in vivo, which is caused by the vascular shutdown.
PDT was performed with a 671-nm solid state diode laser
(CNI Laser, Changchun, China) at a power of 500 mW
to achieve a cumulative radiant exposure of 15 J/cm2.
After PDT, cells were either placed at standard culture
conditions (normoxia) or placed in a hypoxic chamber
[69] (hypoxia) to mimic vascular shutdown.
Cell viability
Cell viability was assessed using the WST-1 assay
as described previously [36].
Liposome preparation
Western blotting
ZnPC-ETLs were composed of DPPC:DCchol:cholesterol:DSPE-PEG (66:25:5:4, molar ratio)
and ZnPC was incorporated at a ZnPC:lipid molar ratio
of 0.003. Liposomes were prepared using the lipid film
hydration technique as described previously [36]. ZnPCETLs were characterized for size and polydispersity by
photon correlation spectroscopy (Zetasizer 3000, Malvern
Instruments, Malvern, Worcestershire, UK). Liposomes
were purged with nitrogen and stored in the dark at 4°C
until use.
Cells were seeded in 6-wells plates and cultured
until confluence. Cells were incubated with 10 μM ZnPCETLs (final lipid concentration) and treated with PDT
(section “PDT protocol”). At 0, 30, 60, 120, and 240
minutes after PDT, cells were placed on ice and lysed in
ice-cold Laemmli buffer [70] supplemented with protease
inhibitor cocktail (1 tablet per 5 mL buffer). A 20-hour
incubation with 500 μM CoCl2 served as a positive
control for HIF-1α stabilization [71]. The lysates were
passed 10 × through a 25-gauge needle (BD Biosciences,
San Jose, CA) to shear DNA. Next, samples were placed
in a heat block for 10 minutes at 95°C, after which the
samples were centrifuged for 15 minutes at 13,000 × g
(4°C). Samples (30 μg) were loaded on a 10% SDS-PAGE
precast gel (50 μL slot volume, Bio-Rad Laboratories,
Hercules, CA) and the electrophoresis was performed for
90 minutes at 125 V. The gels were blotted onto methanolprimed PVDF membranes (Millipore, Billerica, MA) for
1 hour at 330 V at 4°C. Protein membranes were blocked
for 1 hour with 5% dried milk powder (Protifar, Nutricia,
Cuijk, the Netherlands) in 0.2% Tween 20 Tris-buffered
saline (TBST, 20 mM Tris-HCl, 150 mM NaCl, pH =
7.5). The membranes were incubated overnight at 4°C
on a rocker with anti-HIF-1α (1:500, clone 54/HIF-1α,
BD Transduction Laboratories (Franklin Lakes, NJ)) and
anti-β-actin (1:4,000, AC-74, Sigma-Aldrich). Next, the
membranes were washed 4 times in TBST and incubated
with HRP-conjugated goat-anti-mouse IgG1 (1:1,000,
Dako Cytomation (Glostrup, Denmark)) for 1 hour at RT.
Subsequently, membranes were washed 3 times with TBST
and 2 times with TBS. The enhanced chemiluminescence
(ECL) kit (Thermo Scientific) was used as substrate for
β-actin and ECL plus (Thermo Scientific) was used as
substrate for HIF-1α. Protein bands were visualized on
Cell culture
Human perihilar cholangiocarcinoma (SK-ChA-1)
cells were grown at standard culture conditions (37°C, 5%
CO2, and 95% air) and cultured in Roswell Park Memorial
Institute (RPMI) 1640 culture medium supplemented with
10% fetal bovine serum (FBS) (v/v) (both from Gibco,
Invitrogen, Carlsbad, CA), 1% penicillin/streptomycin
(v/v), 1% L-glutamine (v/v) (both from Lonza,
Walkersville, MD), and 1 × 10−5% β-mercaptoethanol
(v/v) (Sigma-Aldrich). Cells were passaged weekly at
a 1:10 ratio. For all experiments, SK-ChA-1 cells were
seeded in 24-wells (500 μL/well) or 6-wells plates (2 mL/
well) (Corning, Corning, NY) at a density of 2 × 105 cells/
mL. Confluent monolayers were achieved 48 hours after
cell seeding, whereas 70–80% confluency was reached 24
hours after cell seeding.
PDT protocol
Cells were seeded in either 24-wells or 6-wells
plates as indicated in the specific subsections and cultured
until confluence. In case of ACF pre-treatment, cells were
incubated with 3 μM ACF (in serum-free supplemented
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an ImageQuant LAS 3000 luminometer (GE Healthcare,
Little Chalfont, UK).
Cells were imaged on a Leica SP8 laser scanning
confocal microscopy system (Leica Microsystems,
Wetzlar, Germany). Fluorescence intensities were
measured at λex = 405 nm, λem = 415–480 nm for DAPI,
λex = 470 nm, λem = 480–550 nm for ACF, λex = 540 nm,
λem = 550–650 nm for Nile Red, and λex = 660 nm, λem
= 670–750 nm for phospho-H2AX. All experiments were
performed using the same laser and microscope hardware
settings.
qRT-PCR
Cells were seeded in 6-wells plates and treated
with PDT as described in the section “PDT protocol”.
RNA was extracted using TRIzol according to the
manufacturer’s protocol (Life Technologies). RNA was
quantified and analyzed with a Nanodrop 2000 UV-VIS
spectrophotometer (Thermo Scientific). cDNA synthesis
and RT-qPCR reactions were performed according to [52].
The primers that were used in this study are listed in Table
S1. The data was analyzed using the LinRegPCR software
in which relative starting concentrations of each cDNA
template (N0) were calculated [72], after which the N0
values of the target genes were corrected for the respective
N0 of the S18 rRNA. All S18 rRNA-corrected N0 values
of each gene were compared to the average N0 of the
untreated normoxic control samples. A log2 transformation
was performed in order to obtain absolute fold-differences
in expression levels of the genes of interest.
ACF degradation
To evaluate the stability of ACF during PDT, 450
μL of ACF (80 μM) in serum-free and phenol red-free
RPMI 1640 medium was added to 24-wells plates. Next,
50 μL of increasing concentrations of ZnPC-containing
cell phantoms (85% DPPC, 10% DPPS, 5% cholesterol,
molar ratio; ZnPC:lipid ratio of 0.003) in physiological
buffer (10 mM HEPES, 0.88% (w/v) NaCl, pH = 7.4,
0.292 osmol/kg) was added to the wells. The baseline
ACF fluorescence was read at λex = 460 ± 40 nm and
λem = 520 ± 520 nm using a BioTek Synergy HT multiwell plate reader (Winooski, VT). Subsequently, the
cells were subjected to PDT (500 mW, 15 J/cm2) and
ACF fluorescence was determined as a measure of ACF
degradation. The data was normalized to control wells (n
= 4 per group).
Confocal microscopy
Microscope cover slips (24 × 40 mm, VWR,
Lutterworth, UK) were first coated with 5 × 10−4% (w/v)
fibronectin in 0.9% NaCl (Fresenius Kabi, Bad Homburg,
Germany) for 2 hours at 37°C. Next, the fibronectincontaining solution was aspirated and cells were seeded
and allowed to grow overnight. To determine the ACF
subcellular localization, cells were either untreated
or subjected to PDT as described in the section “PDT
protocol”, and subsequently incubated with 3 μM ACF for
4 hours under normoxia and hypoxia as specified. Next,
cells were washed with 1 mL of PBS and fixed with a
mixture of 4% paraformaldehyde and 0.2% sucrose for
5 min. Cells were washed with 1 mL of PBS and stained
with 1 μM Nile Red (in PBS) for 1 min. Cells were washed
thrice with 1 mL PBS and mounted on microscope slides
using Vectashield mounting medium (Vector Laboratories,
Burlingame, CA). After 1 h, the slides were sealed with
nail polish.
For the assessment of DNA damage, cells were
fixed with a mixture of 4% paraformaldehyde and 0.2%
sucrose for 5 min and permeabilized in 0.1% TX-100
(in PBS) for 5 min. Next, cells were washed with 1 mL
of PBS and incubated for 16 hours with mouse antihuman phospho-H2AX-AlexaFluor647 (Cell Signaling
Technology, Danvers, MA) at a 1:100 dilution in 0.5%
BSA and 0.15% glycine (in PBS, staining buffer) at 4°C.
Next, cells were washed thrice with staining buffer and
mounted on microscope slides using Vectashield mounting
medium with 4′,6-diamidino-2-phenylindole (DAPI)
(Vector Laboratories). After 1 h, the slides were sealed
with nail polish.
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ACF uptake
Cells were cultured in 24-wells plates until
confluence. Cells were washed with PBS and incubated
with ACF in supplemented serum-free RPMI 1640
medium for 24 hours. After incubation, cells were washed
with PBS and fresh supplemented serum-free RPMI 1640
medium was added to the wells. Next, ACF fluorescence,
as a measure of uptake, was read at λex = 460 ± 40 nm and
λem = 520 ± 520 nm using a BioTek Synergy HT multiwell plate reader. Data were normalized to protein content
per well (n = 4 per group) as determined with the SRB
assay [73].
Caspase 3/7 activity
Cells were cultured in 24-wells plates and
subjected to treatment as described above. Cells were
incubated in 200 μL of serum- and phenol-red free
medium and maintained at either normoxic or hypoxic
conditions for 3.5 hours post-treatment. After treatment
and normoxic/hypoxic incubation, 25 μL of CaspaseGlo assay reagent (Promega, Madison, WI) was added
and cells were incubated for 30 minutes under the
aforementioned conditions. Luminescence was read on
a BioTek Synergy HT multiplate reader at 560 ± 20 nm
and a signal integration time of 1 s. Data were obtained
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from n = 5 measurements and corrected for background
luminescence.
Statistical analysis
Data were analyzed in GraphPad Prism software
(GraphPad Software, San Diego, CA). Data were analyzed
for normality using a Kolmogorov-Smirnov test. Normally
distributed data sets were analyzed with either a student’s
t-test or a one-way ANOVA and subsequent Bonferroni
post-hoc test. Non-Gaussian data were statistically
analyzed using a Mann-Whitney U or Kruskal-Wallis test
and a Dunn’s post-hoc test. All data are reported as mean
± standard deviation. In the figures, intergroup differences
are indicated with (*) and differences between treated
groups versus the untreated (CTRL) group are indicated
with (#). Differences between normoxic and hypoxic
data are, when relevant, indicated with ($). The level of
significance is reflected by a single (p < 0.05), double (p
< 0.01), triple (p < 0.005), or quadruple sign (p < 0.001).
Lactate production
Cells were cultured in 24-wells plates until
confluence and treated with ACF and PDT as indicated in
the section “PDT protocol”. After 24 hours, extracellular
lactate levels were determined using The Edge blood
lactate analyzer (Apex Biotechnology, Hsinchu, Taiwan).
Lactate concentrations were determined from a standard
curve and corrected for the average protein content per
group as determined with the bicinchoninic acid assay
(Thermo Scientific).
Flow cytometry
For cell cycle analysis, cells were seeded in 6-wells
plates and cultured until 70–80% confluence. Cells were
incubated with ACF (in supplemented serum-free RPMI
1640 medium) for 24 or 48 hours, after which cell cycle
analysis was performed using flow cytometry according
to ref. 69.
The mode of cell death following ACF incubation
was analyzed by flow cytometry using APC-conjugated
Annexin V (eBioscience, San Diego, CA) for apoptosis
and TO-PRO-3 (Life Technologies) for necrosis. Cells
were seeded in 6-wells plates and cultured until 70–80%
confluence. Next, cells were incubated with ACF (in
supplemented serum-free RPMI 1640 medium) for 24 or
48 hours. After incubation, the samples were prepared as
described previously [36] and assayed on a FACSCanto
II (Becton Dickinson, Franklin Lakes, NJ). Ten thousand
events were recorded in the gated region and data was
analyzed using FlowJo software (Treestar, Ashland, OR).
ACKNOWLEDGMENTS
The authors are grateful to Marcel Dirkes and Adrie
Maas for input regarding the hypoxic incubator, and Ron
Hoebe and Daisy Picavet for the technical assistance with
confocal microscopy.
CONFLICTS OF INTEREST
The authors declare that they have no competing
interests.
GRANT SUPPORT
This work was financed with grants from the
Phospholipid Research Center in Heidelberg (MH), the
Dutch Anticancer Foundation in Amsterdam (Stichting
Nationaal Fonds Tegen Kanker) (MH), and the Nijbakker
Morra Foundation (MH). The SK-ChA-1 cells were a kind
gift from Alexander Knuth and Claudia Matter from the
University Hospital Zurich, Switzerland.
Intracellular ROS assay
Cells were seeded in 24-wells plates and cultured
until 70–80% confluence. Thereafter, cells were washed
with PBS and incubated with ACF or vehicle (DMSO) in
supplemented serum-free RPMI 1640 medium for 24 or
48 hours. After the indicated time points, the medium was
removed, cells were washed with serum- and phenol redfree RPMI 1640 medium, and cells were incubated with
100 μM DCFH2-DA (in serum- and phenol red-free RPMI
1640 medium) for 1 hour at standard culture conditions.
Next, cells were washed with serum- and phenol red-free
RPMI 1640 medium, and fresh serum- and phenol red-free
RPMI 1640 medium was added to the wells. Intracellular
2′,7′-dichlorofluorescein (DCF) fluorescence, which
is a measure of ROS production, was read on a BioTek
Synergy HT multiplate reader at λex = 460 ± 40 nm and
λem = 520 ± 520 nm. Data were obtained from n = 6
measurements and corrected for ACF fluorescence, protein
content using the SRB assay, and DCF fluorescence (basal
metabolic rate) of control cells.
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Abbreviations
ACF,
acriflavine;
ANGPT,
angiopoietin;
BIRC5, baculoviral inhibitor of apoptosis repeatcontaining 5; COX-2, cyclooxygenase 2; DAPI,
4’,6-diamidino-2-phenylindole;
DC-chol,
3β-[N(N’,N’-dimethylaminoethane)-carbimoyl]cholesterol;
DCF,
2’,7’-dichlorofluorescein;
DCFH2-DA,
2’,7’-dichlorodihydrofluorescein diacetate; DMSO,
dimethyl
sulfoxide;
DPPC,
1,2-dipalmitoyl-snglycero-3-phosphocholine;
DPPS,
1,2-dipalmitoylsn-glycero-3-phospho-L-serine;
DSPE-PEG,
L-αphosphatidylethanolamine, distearoyl methoxypolyethylene
glycol conjugate; ECL, enhanced chemiluminescence;
EDN1, endothelin-1; ER, endoplasmic reticulum;
ETL, endothelium-targeted liposome; FIH, factor
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inhibiting HIF; GLUT-1, glucose transporter 1; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIER,
heat-induced epitope retrieval; HIF, hypoxia-inducible
factor; HK1, hexokinase 1; HMOX1, heme oxygenase
1 (gene); HO-1, heme oxygenase 1 (protein); LDHA,
lactate dehydrogenase A; NF-κB, nuclear factor κB; PAI1,
plasminogen activator inhibitor 1; PAS, PER-ARNT-SIM;
PHD, prolyl hydroxylase; PTGS2, prostaglandin synthase
2; qRT-PCR, quantitative reverse transcriptase polymerase
chain reaction; ROS, reactive oxygen species; RT, room
temperature; SERPINE1, serpin peptidase inhibitor, clade
E; TBS, tris-buffered saline; TBST, TBS supplemented with
0.2% Tween-20; VEGF, vascular endothelial growth factor;
VHL, Von Hippel-Lindau tumor suppressor protein; WST1, water-soluble tetrazolium-1; ZnPC, zinc phthalocyanine.
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