Dehydrozingerone, a structural analogue of curcumin, induces cell-cycle arrest at the G2/M phase and accumulates intracellular ROS in HT-29 human colon cancer cells

Article abstract of DOI:10.1021/np300465f

Dehydrozingerone (1) is a pungent constituent present in the rhizomes of ginger (Zingiber officinale) and belongs structurally to the vanillyl ketone class. It is a representative of half the chemical structure of curcumin (2), which is an antioxidative yellow pigment obtained from the rhizomes of turmeric (Curcuma longa). Numerous studies have suggested that 2 is a promising phytochemical for the inhibition of malignant tumors, including colon cancer. On the other hand, there have been few studies on the potential antineoplastic properties of 1, and its mode of action based on a molecular mechanism is little known. Therefore, the antiproliferative effects of 1 were evaluated against HT-29 human colon cancer cells, and it was found that 1 dose-dependently inhibited growth at the G2/M phase with up-regulation of p21. Dehydrozingerone additionally led to the accumulation of intracellular ROS, although most radical scavengers could not clearly repress the cell-cycle arrest at the G2/M phase. Furthermore, two synthetic isomers of 1 (iso-dehydrozingerone, 3, and ortho-dehydrozingerone, 4) were also examined. On comparing of their activities, accumulation of intracellular ROS was found to be interrelated with growth-inhibitory effects. These results suggest that analogues of 1 may be potential chemotherapeutic agents for colon cancer.

Full text of DOI:10.1021/np300465f

Article
pubs.acs.org/jnp
Dehydrozingerone, a Structural Analogue of Curcumin, Induces Cell-
Cycle Arrest at the G2/M Phase and Accumulates Intracellular ROS in
HT-29 Human Colon Cancer Cells
Shingo Yogosawa,† Yasumasa Yamada,‡ Shusuke Yasuda,† Qi Sun,† Kaori Takizawa,†
and Toshiyuki Sakai*,†
†Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of
Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
‡Department of Food Science and Nutrition, Faculty of Human Life and Science, Doshisha Women’s College of Liberal Arts,
Teramachi- Imadegawa, Kamigyo-ku, Kyoto 602-0893, Japan
S
* Supporting Information
ABSTRACT: Dehydrozingerone (1) is a pungent constituent
present in the rhizomes of ginger (Zingiber officinale) and
belongs structurally to the vanillyl ketone class. It is a
representative of half the chemical structure of curcumin (2),
which is an antioxidative yellow pigment obtained from the
rhizomes of turmeric (Curcuma longa). Numerous studies have
suggested that 2 is a promising phytochemical for the
inhibition of malignant tumors, including colon cancer. On the other hand, there have been few studies on the potential
antineoplastic properties of 1, and its mode of action based on a molecular mechanism is little known. Therefore, the
antiproliferative effects of 1 were evaluated against HT-29 human colon cancer cells, and it was found that 1 dose-dependently
inhibited growth at the G2/M phase with up-regulation of p21. Dehydrozingerone additionally led to the accumulation of
intracellular ROS, although most radical scavengers could not clearly repress the cell-cycle arrest at the G2/M phase.
Furthermore, two synthetic isomers of 1 (iso-dehydrozingerone, 3, and ortho-dehydrozingerone, 4) were also examined. On
comparing of their activities, accumulation of intracellular ROS was found to be interrelated with growth-inhibitory effects. These
results suggest that analogues of 1 may be potential chemotherapeutic agents for colon cancer.
On the other hand, dehydrozingerone (1), isolated from
Zingiber officinale Roscoe (Zingiberaceae), is a biosynthetic
intermediate of 2.17 Structurally, 1 is a half an analogue of 2 and
has also been found to possess antioxidant17−21 and antimuta-
genic effects.22,23 In addition, 1 was found to inhibit tumor
promotion in an in vitro short-term assay measuring TPA-
induced Epstein−Barr virus early antigen activation.23−25
Although there are a few preliminary reports about the
antiproliferative effect of 1 against cancer cells,26,27 no molecular
mechanisms of the action of 1 on cell growth have been clarified.
Therefore, the growth-inhibitory mechanisms of 1 were
investigated using HT-29 human colon cancer cells. While 2
caused cell death as previously reported,13,14 1 predominantly
induced cell-cycle arrest at the G2/M phase accompanied by
accumulation of intracellular ROS and up-regulation of p21.
Further analysis using compounds related in structure to 1
suggested that the growth-inhibitory effect was closely related to
intracellular ROS accumulation. These results suggest that 1
might be a promising candidate for development of novel
anticancer drugs.
olon cancer is a leading cause of death in many countries
Cdespite some clarification of its carcinogenic mechanisms.
Novel approaches are thus required for more effective prevention
or treatment. Many epidemiological studies have shown that
consumption of fruits and vegetables decreases the risk of
malignant tumors.1 Therefore, several dietary polyphenolic
phytochemicals that can prevent carcinogenesis and inhibit the
growth of colon cancer cells have been subjected to clinical
investigation.2
Curcumin (2), an active component of turmeric present in the
rhizomes of Curcuma longa L. (Zingiberaceae), is a well-known
dietary phytochemical and is currently being evaluated as a
potential agent for chemoprevention and/or chemotherapy.3−6
Many animal studies have shown that 2 prevents carcinogenesis
in various organs including the colon.7,8 Curcumin also exhibits
antiproliferative and/or apoptosis-inducing activities in many
human cancer cell lines, including colon cancer cell lines.9−14
Interestingly, there are many reports suggesting that the growth-
inhibitory effect of 2 is accompanied by an increase of reactive
oxygen species (ROS).9−13 Although 2 has been reported to have
free-radical-scavenging activity,15 this compound increases
intracellular ROS and induces cell death.16 These studies
strongly suggest that accumulation of intracellular ROS is
essential for the growth-inhibitory effect of 2.
Received: July 4, 2012
Published: December 17, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Dehydrozingerone (1) Causes Cell-Cycle Arrest at the
G2/M Phase. To investigate further the inhibitory effect of 1 on
cell growth, the cell-cycle distribution of HT-29 cells was
examined by flow cytometric analysis. As shown in Figure 2, the
RESULTS AND DISCUSSION
■
Dehydrozingerone (1) Inhibits the Growth of HT-29
Human Colon Cancer Cells. As previously reported,13,14,28−30
curcumin (2) induces apoptosis in HT-29 human colon cancer
cells. To confirm the cytotoxicicity of 2, its effects were examined
on the cell viability of HT-29 cells using a Guava VIACount
system. As shown in Figure 1A, the percentage of dead cells was
Figure 2. Dehydrozingerone (1) induces cell-cycle arrest at the G2/M
phase in HT-29 cells. Cells were treated with 1 at 125, 250, and 500 μM
for 24 h and stained with PI. Cells were then subjected to cell-cycle
analysis using FACSCalibur. (A) Representative histogram patterns of
the cell-cycle analysis of the HT-29 cells treated with 0.1% DMSO alone
and 1 at 125, 250, and 500 μM for 24 h. (B) Analytical data of the
percentages in the G1, S, and G2/M phases. The data are shown as
means SD (n = 3); *p < 0.05, **p < 0.01 compared with the control
(0.1% DMSO).
Figure 1. Induction of cell death by dehydrozingerone (1) and
curcumin (2) in HT-29 human colon cancer cells. Cells were treated
with 2 (A) or 1 (B) at the indicated concentrations or vehicle (0.1%
DMSO) for 72 h, and live and dead cells were counted using a VIAcount
kit with Guava EasyCyte Plus, as described in the Experimental Section.
Then, the percentage of dead cells was calculated. The data are shown as
means SD (n = 3).
percentage of cells at the G2/M phase was increased about 2- to
4-fold by treatment with 1 at 250 μM or more. Cyclin-dependent
kinase 1 (CDK1) and cyclin B complexes play important roles in
the G2/M transition, so the expression of these proteins was
examined by Western blotting. As shown in Figure 3A, the
expression of CDK1 and cyclin B1 did not appear to have been
changed. On the other hand, the expression of p21 protein
increased by treatment with 1 at 250 μM or more. p21 is known
to induce cell-cycle arrest at the G2/M phase31 accompanied by
dephosphorylation of Thr161 on CDK1.32 Consistent with this
report, phosphorylated CDK1 at Thr161 was decreased by
treatment with 1 at 500 μM.
increased markedly to 84−98% by treatment with 2 at 31.3 μM
or more. The cytotoxic effect of 1 was then evaluated on the same
cells. As shown in Figure 1B, the percentage of dead cells was
increased to only 22% or 37% by treatment with 500 or 1000 μM
1. Furthermore, at the concentration causing 70% inhibition of
cell growth, 2 induced the death of 45% of the cells, whereas 1
induced the death of only 11% of the cells. These results suggest
that the growth-inhibitory effect on HT-29 cells by 2 is mainly
caused by induction of cell death, whereas that by 1 is due to
repression of proliferation.
Next, the effects of knockdown of p21 by siRNA were
examined on the induction of cell-cycle arrest by 1. As expected,
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compared with 2, the natural fluorescence of 1 is weak, reducing
the possibility of false positives (Figure S1, Supporting
Information). Accordingly, CM-H2DCFDA was used as a ROS
indicator, and it was found that 1 at 250 μM or more increased
intracellular ROS (Figure 4).
Figure 4. Intracellular ROS accumulation induced by dehydrozingerone
(1). Cells were treated with 1 at the indicated concentrations for 24 h,
and intracellular ROS was measured using a ROS indicator, CMH2-
DCFDA, as described in the Experimental Section. The data are shown
as means SD (n = 3); *p < 0.05, **p < 0.01 compared with the control
(0.1% DMSO).
Effects on Growth Inhibition and Accumulation of
Intracellular ROS by Dehydrozingerone (1), Iso-Ddehy-
drozingerone (3), and ortho-Dehydrozingerone (4).
Dehydrozingerone (1)-related compounds (isomers) were
synthesized to examine the structure−activity relationship.
Their growth inhibitory effects against HT-29 cells were
examined by WST-8 assay as shown in Figure 5A. Among
them, 4 showed the most potent growth-inhibitory effect against
HT-29 cells when compared with 1 and 3. The effects of the three
isomers were examined on accumulation of intracellular ROS in
HT-29 cells. As shown in Figure 5B−D, 4 drastically induced
ROS with apoptosis, while 1 and 3 only slightly induced ROS
with G2/M arrest.
In this study, it was found that 1, 3, and 4 increased
intracellular ROS and repressed growth of HT-29 human colon
cancer cells. The contribution of ROS to the induction of cell-
cycle arrest at the G2/M phase by 1 was confirmed using N-
acetyl-L-cysteine, known widely as a ROS scavenger. As shown in
Figure S2A and B in the Supporting Information, N-acetyl-L-
cysteine repressed both the G2/M phase arrest and the increase
of p21 induced by 1. On the other hand, however, it has been
reported that 2 forms conjugates with GSH, which is a direct
metabolite of N-acetyl-L-cysteine, in Caco-2 colon cancer cells.40
Efflux of these conjugates depletes intracellular GSH and
eliminates 2 from the cell, thereby decreasing its potency.38 As
1 is structurally similar to 2, other antioxidants, such as L-ascorbic
acid, α-tocopherol, manganese(III) tetrakis (4-benzoic acid)
porphyrin chloride (MnTBAP), PEG-penetrated catalase, or
PEG-penetrated superoxide dismutase (SOD), were examined to
determine if they would repress the cell-cycle arrest at G2/M
phase by 1. The antioxidants other than N-acetyl-L-cysteine did
not clearly suppress this effect (Figure S2C, D, Supporting
Information), whereas MnTBAP showed only partial repression
(Figure S2E, Supporting Information). From the results above, it
could not be concluded that ROS is responsible for the G2/M
phase arrest induced by 1. On the other hand, L-ascorbic acid and
α-tocopherol have been reported not to attenuate but to enhance
the cytotoxic effects of 2,41 similar to the present results of 1.
These results might be explained by pro-oxidant property of
Figure 3. Dehydrozingerone (1) increases p21 levels in HT-29 cells. (A)
Cells were treated with 1 at the indicated concentrations or 0.1% DMSO
alone (−) for 24 h, and its effects on the expressions of each protein were
analyzed by Western blotting, with β-actin used as loading control. (B)
Effect of knockdown of p21 on protein expression by Western blotting.
HT-29 cells were transiently transfected with siRNA as indicated for 24
h after incubation with 0.1% DMSO alone or 1 at 500 μM for 24 h, with
β-actin used as loading control. (C) Effect of knockdown of p21 on cell-
cycle arrest by treatment with 1. Cells were transiently transfected with
siRNAs as indicated for 24 h after the treatment with 0.1% DMSO alone
or 1 at 500 μM for 24 h and then stained with PI. After staining, the
populations in different phases of the cell cycle were analyzed by flow
cytometry as described in the Experimental Section. The data are shown
as means SD (n = 3); *p < 0.01, compared with the DMSO-treated
control.
treatment with p21 siRNA resulted in a decrease of the protein
level of p21 (Figure 3B). As shown in Figure 3C, the induction of
cell-cycle arrest at the G2/M phase by 1 was repressed
significantly by p21 siRNA compared with that by control
siRNA. These results suggest that an increase of p21 at least
partially contributes to the G2/M phase arrest induced by 1 in
HT-29 cells.
Dehydrozingerone (1) Treatment Leads to Accumu-
lation of Intracellular ROS in HT-29 Cells. It has been
reported that 2 induces cell-cycle arrest9,33,34 or apopto-
sis9−11,13,15,16,34−37 via ROS generation. Therefore, it was
examined whether 1 similarly induces intracellular ROS.
However, it has been pointed out that fluorescence-based
approaches are not suitable for investigating ROS production in
cancer cells treated with curcumin (2), since this compound itself
is strongly fluorescent, leading to false positives.38,39 When
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Figure 5. Effects on growth inhibition and accumulation of intracellular ROS by treatment with compounds related in structure to dehydrozingerone
(1). (A) Growth-inhibitory effects of 1 (circles), 3 (squares), and 4 (triangles) on HT-29 cells. Cells were treated with each compound at the indicated
concentrations for 72 h, and the percentage of viable cells was evaluated by a WST-8 assay. The data are shown as means SD (n = 3). (B) Accumulation
of intracellular ROS by 1-related compounds. Cells were treated with or without each compound for 24 h, and intracellular ROS was measured by flow
cytometry using a ROS indicator, CMH2−DCFDA, as described in the Experimental Section. The data are shown as means SD (n = 3); **p < 0.01
compared with the control (0.1% DMSO). (C) Cells were treated with or without each 1-related compound for 24 h and then stained with PI. After
staining, the populations in different phases of the cell cycle were analyzed by flow cytometry as described in the Experimental Section. The data are
shown as means SD (n = 3); *p < 0.05, **p < 0.01 compared with the G2/M population of the cells treated with 0.1% DMSO. (D) Cells were treated
with or without each compound related in structure to dehydrozingerone (1) for 72 h and then stained with PI. After staining, populations of sub-G1
were analyzed by flow cytometry as described in the Experimental Section. The data are shown as means SD (n = 3); **p < 0.01 compared with the
control (0.1% DMSO).
antioxidants under certain circumstances. Taken together, the
combined effects of redox-active compounds are quite complex,
so further studies are required to confirm the contribution of
ROS to the cell-cycle arrest by 1.
stress.44−48 Treatment with compounds inducing oxidative stress
has increased p21 through a p53-independent but ERK-
dependent pathway.44,45 The induction of p21 by 1 may be
due to the same mechanism because HT-29 is known to have
mutated p53 and B-Raf. In addition, it has also been reported that
cell-cycle arrest is caused by p21 under moderate levels of
oxidative stress, whereas apoptosis can be induced under higher
oxidative stress conditions by destruction of the Nrf2-dependent
antioxidant system.46,47 Consistent with these reports, it was
found that 1 or 3 caused cell-cycle arrest with a slight increase of
intracellular ROS, whereas 4 induced apoptosis with an obvious
increase of intracellular ROS. In contrast, 1 at 250 μM did not
induce ROS or p21, while it clearly caused G2/M arrest.
Therefore, the significance of ROS and p21 has not been shown
clearly in the present study.
This is the first report on the interaction of 1 in relation to
ROS, cell-cycle arrest, and apoptosis. On one hand, ROS have
been shown to induce mutations of genes, possibly causing
carcinogenesis. In contrast, the antioxidant system has been
reported to promote carcinogenesis.49 If this is true, the
development of compounds related in structure to 1 that more
potently induce intracellular ROS may be a promising new
chemoprevention approach.
The effect of 1 on the growth of other colon cancer cell lines,
HCT-116 and HCT-15, was also examined (Figure S3,
Supporting Information). Similar to HT-29 cells, 1 inhibited
the growth of HCT-116 and HCT-15 cells with G2/M phase
arrest, which was inhibited by N-acetyl-L-cysteine, but not by L-
ascorbic acid or α-tocopherol (Figure S4, Supporting Informa-
tion). These results suggest that the G2/M phase arrest by 1 is
not specific for HT-29 cells. In human fibroblast WI-38 cells, 1
also weakly inhibited cell growth and induced G2/M arrest
(Figure S5, Supporting Information).
Although it was not elucidated if the accumulation of
intracellular ROS is essential for 1-induced cell-cycle arrest,
oxidative stress is known to play an important role in mediating
this phenomenon.42 In human nasopharyngeal carcinoma cells, 2
has been reported to induce G2/M arrest by ROS generation
accompanied by the reduction of CDK1 and cyclin B1.43 In the
present study, however, 1 did not clearly decrease CDK1 or
cyclin B1, but increased p21 with accumulation of ROS. There
are many reports stating that p21 is closely related to oxidative
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EXPERIMENTAL SECTION
ACKNOWLEDGMENTS
■
■
Reagents and Antibodies. The syntheses of compounds 1
(dehydrozingerone), 3 (iso-dehydrozingerone), and 4 (ortho-dehy-
drozingerone) and the spectroscopic data obtained are described in the
Supporting Information. The purities of 1, 3, and 4 were estimated to be
100%, 93.1%, and 100%, respectively, by HPLC analyses. Compound 2
was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and its purity
was 78.1%. Propidium iodide (PI) and anti-β-actin antibody were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies
against cyclin B1 and phospho-CDK1 were purchased from Cell
Signaling Technology (Beverly, MA, USA). An antibody against CDK1
was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,
USA). 5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diac-
etate, acetyl ester (CM-H2DCFDA) was purchased from Life
Technologies (Carlsbad, CA, USA). ECL anti-mouse IgG and ECL
anti-rabbit IgG were purchased from GE Healthcare (Piscataway, NJ,
USA).
Cell Culture. HT-29 human colon cancer cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v)
fetal bovine serum, 2 mM glutamine, 50 U/mL penicillin G, and 100 μg/
mL streptomycin at 37 °C in 5% CO2.
Cell Growth Assay. Cells were incubated with or without reagents
as indicated and then harvested. The cells were treated with a ViaCount
kit (Merck, Darmstadt, Germany), and viability was measured using a
Guava EasyCyte Plus flow cytometer (Merck), according to the
manufacturer’s instructions. After incubation with or without reagents as
indicated for 72 h, the WST-8 assay was performed using a Cell
Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the
manufacturer’s instructions.
We would like to thank Dr. Y. Sowa, Kyoto Prefectural University
of Medicine, for helpful discussions. This study was partially
supported by a grant-in-aid from the Japanese Ministry of
Education, Culture, Sports, Science and Technology, and a grant-
in-aid for the Encouragement of Young Scientists from the Japan
Society for the Promotion of Science.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The spectroscopic data of compounds 1, 3, and 4 and Figures
S1−S5 are available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-75-251-5339. Fax: +81-75-241-0792. E-mail: tsakai@
koto.kpu-m.ac.jp.
Notes
The authors declare no competing financial interest.
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