Upregulation of both heme oxygenase-1 and ATPase inhibitory factor 1 renders tumoricidal activity by synthetic flavonoids via depleting cellular ATP

Article abstract of DOI:10.1016/j.bmcl.2014.08.055

Heme oxygenase-1 (HO-1) and ATPase inhibitory factor (ATPIF) 1 is often overexpressed in different types of cancer cells. Chrysin is a naturally-occurring flavonoid with antioxidant potentials, but also known to promote apoptosis. We have synthesized four chrysin derivatives and found compounds 1 and 4 remarkably upregulated the expression of HO-1, a cytoprotective enzyme. A robust expression of ATPIF1 was only seen in compound 4. Upregulation of both proteins triggers cell death in hydrogen peroxide-primed cells. Ten derivatives of compound 4 were synthesized and measured the expression of HO-1 and ATPIF1. Again, upregulation of both proteins by compound 8 killed the cells via apoptosis. To gain a physiological significance, we treated the synthetic flavonoids in colon cancer cells, HT29 and HCT116 cells and confirmed that overexpression of both HO-1 and ATPIF1 was critical for tumor cell death with an impaired mitochondrial energetics. It would provide a strategy for developing selective anti-tumor candidates.

Full text of DOI:10.1016/j.bmcl.2014.08.055

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4845–4849
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Upregulation of both heme oxygenase-1 and ATPase inhibitory factor
1 renders tumoricidal activity by synthetic flavonoids via depleting
cellular ATP
Phil Jun Lee a, , Iljin Shin a, , Seung-Yong Seo b, Hyoungsu Kim a, Hong Pyo Kim a,
⇑
a College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea
b College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Heme oxygenase-1 (HO-1) and ATPase inhibitory factor (ATPIF) 1 is often overexpressed in different
types of cancer cells. Chrysin is a naturally-occurring flavonoid with antioxidant potentials, but also
known to promote apoptosis. We have synthesized four chrysin derivatives and found compounds 1
and 4 remarkably upregulated the expression of HO-1, a cytoprotective enzyme. A robust expression
of ATPIF1 was only seen in compound 4. Upregulation of both proteins triggers cell death in hydrogen
peroxide-primed cells. Ten derivatives of compound 4 were synthesized and measured the expression
of HO-1 and ATPIF1. Again, upregulation of both proteins by compound 8 killed the cells via apoptosis.
To gain a physiological significance, we treated the synthetic flavonoids in colon cancer cells, HT29
and HCT116 cells and confirmed that overexpression of both HO-1 and ATPIF1 was critical for tumor cell
death with an impaired mitochondrial energetics. It would provide a strategy for developing selective
anti-tumor candidates.
Received 13 June 2014
Revised 22 August 2014
Accepted 26 August 2014
Available online 3 September 2014
Keywords:
Chrysin derivatives
Heme oxygenase
ATPIF1
Apoptosis
Energetics
Ó 2014 Elsevier Ltd. All rights reserved.
Chrysin is one of the natural flavonoids present in plants, and
large amounts are present especially in honey and propolis. In
addition to its antioxidative, anti-inflammatory activities, and
aromatase inhibitor, chrysin has also been reported to confer an
anti-tumor activity.1–3
Heme oxygenase-1 (HO-1) is a well-recognized, stress-induc-
ible enzyme that degrades heme into biliverdin, carbon
monoxide (CO), and ferrous ion.4 The activity of HO-1 serves to
maintain cellular integrity upon several stressors. This cytoprotec-
tive potential of HO-1 activity may be attributed to the catabolic
removal of toxic heme, and to the anti-apoptotic activity of by-
products produced during heme catabolism.5,6 For example, both
HO-1 and its enzymatic products confer cytoprotection from
hyperoxic insults, cisplatin nephrotoxicity and endotoxic shock
by attenuating apoptotic signals. Despite its cytoprotective activi-
ties, cumulating evidences suggest a paradoxical role for HO-1 in
tumorigenesis.5–10 Indeed, upregulation of HO-1 expression is fre-
quently observed in different types of cancer cells. Likewise, basal
overexpression of HO-1 provides survival benefit for several types
of cancer, including liver and lung carcinoma, whereas silencing
HO-1 expression reduces viability of hormone refractory prostate
cancer.11 Hence the protective activity of HO-1 seems to be a dou-
ble-edged sword, since cancer cells adopt the cytoprotective activ-
ity of HO-1 for their survival strategies against chemotherapeutic
agents.4,5 Revelation of its effects on tumor growth and metastasis
has undoubtedly increased the significance of HO-1 in cancer biol-
ogy. Many studies have targeted inhibition of this enzyme as an
approach to sensitize cancer cells to therapies.8–11
Mitochondria are both the target and the source of reactive oxy-
gen species. The mitochondrial membrane potential,12 which is the
driving force for mitochondrial ATP synthesis, declines during
apoptosis, and maintenance of membrane potential can prevent
apoptosis. Since apoptosis involves the vigorous activity of
hydrolytic enzymes, chromatin condensation and vesicle forma-
tion, apoptosis is likely to require high energy.13 It is well con-
ceived that the cellular ATP level is an important determinant for
cell death.
Abbreviations: DOR1, delta opioid receptor 1; PARP-1, poly(ADP-ribose) poly-
merase-1.
⇑
Corresponding author. Tel.: +82 31 219 3452.
E-mail address: ghim@ajou.ac.kr (H.P. Kim).
These authors contributed equally. The manuscript was written through contri-
butions of all authors. All authors have given approval to the final version of the
manuscript.
In addition, altered metabolism in cancer cells is likely to con-
tribute for the chemo-resistance and intracellular ATP levels are
http://dx.doi.org/10.1016/j.bmcl.2014.08.055
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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P. J. Lee et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4845–4849
a major determinant in acquisition of the drug resistance in human
colon cancer cells. Drug-resistant cells were characterized by ele-
vated aerobic glycolysis and higher absolute levels of intracellular
ATP.13 Metabolic reprogramming by tumor cells aimed to inhibit
the H+-ATP synthase activity can be mediated by an overexpression
of the ATPase inhibitory factor 1 (ATPIF1),14 which is a physiolog-
ical inhibitor of the mitochondrial synthase. A number of investiga-
tion demonstrated that the mitochondrial content of ATPIF1
increases significantly in carcinomas, suggesting the participation
of ATPIF1 in oncogenesis.10,11
The expression of ATPIF1 varies significantly in cancer cell lines.
Overexpressed ATPIF1 seemed to activate NF-kB via reactive oxy-
gen species generated from mitochondria in a retrograde fashion.13
The present study evaluated the tumoricidal activities of chry-
sin derivatives which we have chemically synthesized in the con-
text of relative expression of both HO-1 and ATPIF1, which are
known to frequently overexpress in various tumors.5,14 We have
found that the chemical(s) which could upregulate both protein
expressions in cells paradoxically trigger cell death and further
confirmed in colon cancer cell lines, HT29 and HCT116 cells.
First, we have examined the induction of HO-1 in MRC5 cells
with four synthetic chrysin derivatives15 (Fig. 1A). The MRC5 cells
were chosen because the cells are very sensitive to several stimuli,
including heme, lipopolysaccharide and hypoxia treatment, result-
ing in robust induction of HO-1 (Supporting information SI Fig. 1
and not shown). Among four synthetic flavonoids inspired by nat-
ural product from honey, chrysin,1 a substantial induction of HO-1
was demonstrated in MRC5 cells treated with compounds 1 and 4
(Fig. 1B).
concentration-dependent manner (SI Fig. 2). Unexpectedly, how-
ever, compound 4 induced cell death at a 30 M dose (Fig. 1C),
l
with which the induction of HO-1 was nicely achieved with con-
comitant expression of ATPIF1. Despite stronger induction of HO-
1 by compound 1 than compound 4, this flavonoid did not affect
cell viability upon oxidative stress.16 This initial result led us to
hypothesize that upregulation of both HO-1 and ATPIF1 would
affect the cytotoxicity synergistically.17 To test the cytotoxic effect
of C(5), C(7), and C(40)-phenolic hydroxyls in compound 4 and to
confirm the relationship between induction of HO-1/ATPIF1 and
cytotoxic effect of analogues of compound 4, we synthesized ten
derivatives of the flavonoid 4 by methylation of three phenolic
hydroxyl (7–13) or B or D ring modification (5, 6 and 14) in 4
(Fig. 2A). These analogues were easily prepared through the proce-
dures previously developed by Kim and co-workers.15 With those
ten analogues, we have repeated the experiments described above,
and found that compounds 8, 11, and 12 exhibited remarkable
upregulation both HO-1 and ATPIF1 (Fig. 2B and SI Fig. 3). These
structure–activity relationship analysis suggested that protection
of C(7) hydroxyl is crucial for the upregulation both HO-1 and
ATPIF1 and also indicated that C(5) and (or) C(40) hydroxyl are
required for the upregulation both proteins. Modification of B or
D ring in compound 4 (5, 6, and 14) did not affect the co-induction
of HO-1 and ATPIF1 expression. Compound 8 showed most cyto-
toxicity among four synthetic chrysin derivatives (Fig. 2C). How-
ever, compound 14 with robust expression of ATPIF1 but not HO-
1 did not affect cell viability in MRC5 cells (Fig. 2B and C). Cellular
damage upon treatment with compound 8 was accompanied by
the downregulation of anti-apoptotic proteins, Bcl2 and Bcl-xL
(Fig. 2D).
Collectively, these data suggest that some of synthetic chrysin
derivatives selectively induced cell death via inducing apoptosis
in MRC5 cells. The balance between the synthesis and turnover
of ATP might be differentially influenced by these flavonoids.
Indeed the expression of ATPIF-1 was remarkably augmented by
compound 8, but not by compound 1 (Figs. 1B and 2B). Since the
overexpression of ATPIF-1 turns out to diminish ATP production
and impairs mitochondrial membrane potential, reduced ATP pro-
duction by compound 8 may exacerbate the cellular integrity if not
supported by alternative essential components such as heme.
Heme is quintessential factor for many enzymes and electron
transport chains in the mitochondria.18 Heme degradation by
HO-1 activity results in the consumption of the important mole-
cules for diverse cellular processes.18 In this regard, we propose
that the overexpression of both HO-1 and ATPIF1 may trigger cel-
lular damage by synthetic, chrysin derivatives and alteration of cel-
lular energetics by cytotoxic chrysin derivatives can mediate cell
death.
However, the expression of ATPIF1 by compound 1 was negligi-
ble while the expression of the protein by compound 4 was mod-
erate (Fig. 1B). Since it is generally accepted that induction of HO-1
confers cytoprotection against noxious stimuli,4 we tested the
activity of those flavonoids in MRC5 cells primed with low dose
of H2O2. Cell death upon H2O2 treatment was increased in a
1RO
(A)
OMe
D
H
OBn
MeO
B
2RO
O
A
C
O
O
3RO
O
OBn O
2: R1 = Bn, R2 = H, R3 = Bn
1
: R = Bn, R2 = Bn, R3 = Bn
1
3
4
1
: R = H, R2 = H, R3 = H
To gain insights for the activity of chrysin-derived synthetic
flavonoids, we examined the expression of antioxidant enzyme,
HO-1 and cellular energetics in colon cancer cells. The basal
expression of HO-1 was markedly high in HCT116, moderate in
HT29 cells but little in SW480 cells (Fig. 3A and not shown).
HO-1 expression seemed cell-specific since the expression of
DOR-1 was almost same among the colon cancer cells (Fig. 3A).
We sought that administration of chrysin derivatives which aug-
mented ATPIF1 expression could effectively induce cell death in
the context of basal HO-1 expression. Treatment of compound 8
in both HT29 and HCT116 cells exhibited contrasting responses
(B)
(C)
20 μM
30 μM
p<0.05
C
1
2
3
4
p<0.01
HO-1
1.0 3.0 1.1 0.6 1.7
1.0 1.0 1.5 2.0 2.3
β-actin
IF-1
in cell death. Compound 8 at 20 lM evoked cell death only in
−
veh
1
4
μM)
HCT116 cells which basally expressed high levels of HO-1
(Fig. 3B) and exhibited cell death in a time- and dose-dependent
fashion (SI Fig. 4A and B). In addition, compound 8 increased in
ATPIF1 expression as expected (Fig. 3C). To validate the role of
HO-1 in our experimental settings, we did downregulate the activ-
ity or expression of the protein by a chemical inhibitor, zinc
β-actin
H2O2 (100
Figure 1. Upregulation of both HO-1 and ATPIF1 confers sensitivity to MRC5 cells
against H2O2-primed cell death by a chrysin derivative, compound 4. Four synthetic
chrysin derivatives (A). Expression of HO-1 and ATPIF-1 (B). Cytotoxicity of MRC5
cells were treated with chrysin derivatives (C).

P. J. Lee et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4845–4849
4847
(A)
(B) cpds
HO-1
1.0
1.7
2.4
2.3
1.6
3.4
1.8
1.1
2.2
2.2
1.5
1.1
ATPIF1
(C)
1.0
100 μM H2O2
p<0.01
C
4
2.3
1.5
1.4
1.3
1.8
ns
5
6
7
8
9
1.6
1.9
2.0
2.3
1.6
1.8
Ctl
−
1
8
11
12
14
10
11
12
13
14
Bcl2
(D)
Bcl-xL
β-actin
C
−
1
8
H2O2 (100 μM)
Figure 2. Ten synthetic derivatives of compound 4 (A) and their cytotoxic activity in MRC5 cells via apoptosis. Quantitation of HO-1 and ATPIF1 expression (B). Cytotoxicity of
MRC5 cells by selected chrysin derivatives (C). Expression of antiapoptotic proteins (D).
contrary, overexpression of HO-1 by hemin (2.5 lM) induced cell
(C)
veh
8
(A)
death in HT29 cells treated with compound 8 (SI Fig. 5A and B).
Proteolytic cleavage of PARP supported the apoptotic cell death
in HCT116 cells (Fig. 3C). Time and dose-dependent experiments
have also shown that compound 1 with low ATPIF1 expression
did not affect cellular integrity at all in both HT29 and HCT116 cells
(SI Fig. 4C and D). But compound 8 triggered cell death in both
HT29 and HCT116 cells at higher doses and longer duration, which
is more pronounced in HCT116 cells accompanied with high HO-1
expression (Fig. 3B).
HO-1
IF-1
PARP
cl. PARP
DOR-1
β-actin
HO-1
The endogenous levels of ATP are likely consistent with cell
injury, in which compound 8 significantly diminished ATP levels
regardless of cell types. Approximately 60% of cellular ATP was
depleted when treated with compound 8 in both HT29 and
HCT116 cells (Fig. 4A, SI Fig. 6A). Intriguingly, compound 1 could
diminish the intracellular ATP levels of HCT116 by 20%, which fur-
ther support the cellular heme metabolism is critical to maintain
the cellular energetics. It is of note that the basal levels of ATP were
much less in HCT116 cells compared with those in HT29 cells
(Fig. 4A).
GADPH
140
(B)
p <0.01
120
100
80
60
40
20
0
HT29
HCT116
Morphological studies of basal mitochondrial architecture also
accounted for the different amounts of ATP in both cells (Fig. 4B).
There was no notable difference in the number of mitochondria
of both HT29 and HCT116 cells (SI Fig. 6B). Perinuclearly distrib-
uted mitochondria exhibited electron dense phenotype and well
preserved cristae in HT29 cells. However, mitochondria in
HCT116 cells showed swelling and damaged mitochondria (black
arrowhead) membrane. The alteration of mitochondria architec-
ture reflected the basal contents of ATP levels in both HT29 and
HCT116 cells. Interestingly lipid bodies (Fig. 4B white arrowhead)
near mitochondria were observed frequently in the HT29 cells
which provided further support that the cells are well-equipped
with energy reserviour17 which may benefit the cell survival of
HT29 cells against cytotoxic stimuli (Fig. 4B, SI Fig. 4B).
veh
1
8 (20 μM)
Figure 3. Cytotoxicity of compound 8 in HCT 116 cells with higher levels of both
HO-1 and ATPIF1. Basal expression of HO-1 in tumor cell lines (A). Expression of
ATPIF1 (IF1) and apoptotic cleavage of PARP1 (B). Cytotoxicity of cells treated with
compound 1 and 8 (C).
protoporphyrin or small interference RNA. Inhibition of HO-1
activity masked the cytotoxic effect of compound 8, even aug-
mented the viability of HCT116 cells. Similar result was obtained
in cells transfected with siRNA for HO-1 (not shown). On the

4848
P. J. Lee et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4845–4849
to heme deficiency, for example, heme degradation by increased
(A)
HO-1 activity. Therefore it is plausible to speculate that upregula-
tion of both HO-1 and ATPIF1 can trigger tumor cell death as
shown in current study.
cells
HT29
HCT116
treat /w
veh
1
8
veh
1
8
Interestingly, recent observation suggests that there is a causal
link between ATPIF1 activity and expression of an enzyme for
heme biosynthesis, ferrochelatase in erythroblasts and confirmed
in a zebra fish model.21 However, their connection with cancer
biology is not examined yet.
ATP 772,031 766,046 291,743 16,099 12,763 5,993
SD
%
64,568 38,094 28,064
100 99.2 37.8
625
100
196
460
79.3 37.3
Fatty acids are the building blocks for the synthesis of triacylglyc-
erol, which are the main storage form of surplus energy.22,23 Phos-
pholipids, together with cholesterols and sphingolipids, comprise
of biological membranes. There is increasing evidence that cancer
cells show specific alterations in different aspects of lipid metabo-
lism.24 These alterations can affect the availability of lipids for the
construction of membranes, the synthesis and degradation of lipids
that contribute for maintaining energy homeostasis. These changes
are thought to contribute the resistance of tumor cells against vari-
ous stressors including oxidative stress.17 Thus, peri-mitochondrial
distribution of lipid droplets in HT29 cells is consistent with the
resistance of the cells against H2O2 induced cellular damage.
Lastly, upon pharmacological aspects, chrysin bioavailability is
likely limited due to their poor absorption in the gastrointestinal
tracks in vivo and human.25,26 Therefore, to use the synthetic flavo-
noids as therapeutic candidates, it would require an optimal deliv-
ery system.
(B)
HT29
HCT116
Overall, the study provides evidences that some synthetic flavo-
noids modified from chrysin upregulated the expression of HO-1
and ATPIF1 simultaneously, which induced an effective cytotoxic-
ity in cells including colon cancer cells. This proposal would also
provide
a novel strategy to design or establish anti-tumor
interventions.
Funding sources
Figure 4. Impaired cellular energetics (A) in tumor cells by compound 8 and
mitochondrial morphology (B). Mitochondria (black arrowhead), Lipid droplets
(white arrowhead).
This research was supported by Basic Science Research Program
through the National Research Foundation (NRF) of Korea funded
by the Ministry of Science, ICT
2012M3A9C3050632 and NRF-2012R1A1A1009271).
& Future Planning (NRF-
Metabolic switch from oxidative phosphorylation to glycolysis
is a universal feature of highly proliferating cells including immune
cells and tumors.19 The ATP synthase is specialized in synthesizing
ATP by using a proton gradient generated across a mitochondrial
inner membrane.12 One of the survival strategies of tumor cells
to induce metabolic switch can be mediated by upregulation of
ATPIF1 which downregulates ATP synthase.12,13 Recently, Formen-
tini et al. demonstrated that ATPIF1 overexpression in colon cancer
cells inhibited the activity of the ATP synthase and promoted the
upregulation of aerobic glycolysis.13 Besides its role in cellular
energetics, the ATP synthase is also critical for the maintenance
of the mitochondrial membrane potential and cristae structure.
In addition, it is fundamental for an optimal supramolecular orga-
nization of the respiratory chain, and for regulating the mobiliza-
tion of cytochrome c during apoptosis.12 Derangement of cristae
architecture detected in HCT116 cells (Fig. 4B) might be due to
the suppression of ATP synthase activity by synthetic flavonoid o
through ATPIF1 overexpression.
Although many cancer cells exhibit increased aerobic glycolysis
for energy production,20 evidences show that mitochondrial respi-
ration is still important in the bioenergetics and supply for building
blocks of cells. Accordingly, cancer cells exhibit elevated heme syn-
thesis and uptake.18 Heme stimulates the synthesis of many hemo-
proteins. Likewise, increased hemoproteins in turn lead to
intensified oxygen consumption and cellular energy production,
thereby fueling cancer cell progression.18 Epidemiological studies
support the idea that heme positively impacts cancer progres-
sion.18 These data imply that HCT116 cells may be more vulnerable
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.0 8.0 55.
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