2-Hydroxycurcuminoid induces apoptosis of human tumor cells through the reactive oxygen species-mitochondria pathway

Article abstract of DOI:10.1016/j.bmcl.2010.11.114

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI ₅₀ value of 7 μM, while the parent compounds, HCA and curcumin, displayed GI₅₀ values of 12 and 30 μM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.

Full text of DOI:10.1016/j.bmcl.2010.11.114

Bioorganic & Medicinal Chemistry Letters 21 (2011) 747–751
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Hydroxycurcuminoid induces apoptosis of human tumor cells through
the reactive oxygen species–mitochondria pathway
Young-Min Han a,b, Dae-Seop Shin a,b, Yu-Jin Lee a,b, Ismail Ahmed Ismail c,d, Su-Hyung Hong d,
a,b,
Dong Cho Han a,b, , Byoung-Mog Kwon
⇑
⇑
a Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 52 Uendong, Yoosung, Daejeon 305-806, Republic of Korea
b University of Science and Technology, 52 Uendong, Yoosung, Daejeon 305-806, Republic of Korea
c Laboratory of Molecular Cell Biology, Department of Zoology, Faculty of Science, Assiut University, Assiut 71516, Egypt
d Department of Dental Microbiology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against
various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were
synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The
hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7)
Received 8 September 2010
Revised 23 November 2010
Accepted 24 November 2010
Available online 28 November 2010
strongly inhibited the growth of SW620 colon tumor cells with a GI50 value of 7
lM, while the parent
compounds, HCA and curcumin, displayed GI50 values of 12 and 30 M, respectively. HCC-7 was found
to induce apoptosis through the reactive oxygen species–mitochondria pathway and cell cycle arrest
at G2/M phase.
l
Keywords:
Hydroxycinnamaldehyde
Curcuminoid
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis
Reactive oxygen species
Many natural products or synthetic compounds with antitumor
activity have been studied in the pursuit of novel therapeutic agent.
However, drug resistance and the genomic instability of cancer cells
remain significant problems in the advancement of new cancer
chemotherapies. Compared with normal cells, tumor cells seem to
have higher levels of endogenous oxidative stress in culture and in
vivo.1,2 For example, lymphocytes isolated from the blood of pa-
tients with chronic lymphocytic leukemia produced more reactive
oxygen species (ROS) than normal lymphocytes.3 In solid tumors,
products of oxidative damage, such as the oxidized DNA base 8-hy-
droxy-20-deoxyguanosine (8-OHdG), and lipid peroxidation prod-
ucts, were detected in patient tumor specimens and cancer cells.4
It has also been reported that levels of antioxidant enzymes, such
as SOD and catalase, were decreased in some primary cancer cells.5
Cancer cells that produce excessive levels of ROS are vulnerable to
oxidative stress by treatment with an exogenous agent. Addition-
ally, inhibition of ROS scavenging systems could be a promising
strategy to selectively target cancer cells without toxicity to normal
cells. Therefore, several redox-modulating agents, ROS-generating
agents and ROS elimination inhibitors, are currently in clinical trials
as single agents or in combination therapy.6
2-Hydroxycinnamaldehyde (HCA), isolated from the stem bark
of Cinnamomum cassia, has been shown to inhibit on farnesyl pro-
tein transferase in vitro, as well as angiogenesis, and tumor cell
growth.7–9 Although HCA has strong anti-proliferative activity,9
cinnamaldehyde (CA) has relatively weak cytotoxicity against
human tumor cells lines grown in vitro and model murine xeno-
grafts.10,11 A previous study showed that HCA and its synthetic
analog BCA (2-benzoyloxycinnamaldehyde) induced apoptosis in
SW620 human colorectal cancer cells via ROS generation.12 There-
fore, we hypothesized that the 2-hydroxy functional group of HCA
critically enhances the antitumor effects of HCA derivatives.
Pharmacokinetic experiment showed that BCA was rapidly
hydrolyzed to HCA, followed by rapid oxidation of the aldehyde
functional group by aldehyde dehydrogenase to form coumaric
acid.13 We also reported that the oxidized HCA derivative 2-hydroxy
cinnamicacid, as well as the reducedHCA derivative 2-hydroxycinn-
amyl alcohol, did not show any cytotoxicity against various human
tumor cell lines.14 To overcome the metabolic instability of HCA in
vivo, we first thought to replace the aldehyde functional group of
HCA with an enol or enone.
The rhizome of Curcuma longa, commonly known as turmeric, is
used worldwide as a spice (e.g., curry), food preservative and color-
ing agent.15 Curcumin, the main medicinal component of Curcuma
species, has a variety of biological activities including suppression
of inflammation, angiogenesis, tumorigenesis, diabetes, and neuro-
logical systems.16 Perhaps the most important aspect of curcumin
⇑
Corresponding authors. Tel.: + 82 42 860 4568; fax: +82 42 861 2675 (D.C.H);
tel.: + 82 42 860 4557; fax: +82 42 861 2675 (B.-M.K.).
E-mail addresses: dchan@kribb.re.kr (D.C. Han), kwonbm@kribb.re.kr (B.-M.
Kwon).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.114

748
Y.-M. Han et al. / Bioorg. Med. Chem. Lett. 21 (2011) 747–751
O
O
R
O
CH3
CH3
H
CH3
R
R
2,9
3,10
OH
O
OH
O
CH3
4,11
5,12
R
R
R
R=OH, H
O
O
O
O
CH3
R
R
R
6,13
7,14
OH
O
OH
O
O
O
O
O
O
O
H3C
CH3
H3C
CH3
15
HO
OH
O
TBDMS
TMDMS
O
O
O
O
O
O
O
H3C
CH3
H3C
CH3
O
O
HO
OH
16
TBDMS
TMDMS
Scheme 1. Synthesis of curcuminoid derivatives. Reagents: (i) Ba(OH)2ꢀ8H2O, 3,3-dimethylpentane-2,4-dione, MeOH; (ii) KOH, acetone, EtOH; (iii) B2O3, acetylacetone,
tributyl borate, HCl, DMF; (iv) iodomethane, K2CO3, acetone; (v) tert-butyldimethylchlorosilane, imidazole, THF; (vi) tetrabutylammonium fluoride, THF.
O
O
O
O
O
O
OH
O
O
O
O
H
H
H3C
CH3
OH
1
2
3
4
5
6
7
8
15
16
17
OH
OH
OH
OH
OH
OH
HO
O
O
O
CH3
CH3
H3C
CH3
OH
9
HO
O
O
CH3
CH3
CH3
CH3
O
10
H3C
CH3
OH
OH
O
O
O
O
OH
OH
O
O
O
O
HO
OH
CH3
CH3
11
12
13
HO
CH3
CH3
O
O
O
O
14
OH
HO
Figure 1. Structures of HCA and curcuminoids.

Y.-M. Han et al. / Bioorg. Med. Chem. Lett. 21 (2011) 747–751
749
is its effectiveness against various types of cancer, having both che-
A
2.2
2.0
1.8
1.6
1.4
1.2
1.0
mopreventive and chemotherapeutic properties. In this context,
curcumin is currently in human clinical trials against a number
of cancers, including multiple myeloma, pancreatic cancer, myelo-
dysplastic syndromes, and colon cancer.17 Several in vitro studies
suggest that curcuminoid-induced apoptosis is associated with
ROS production and/or oxidative stress in transformed cells.18,19
Curcumin predominantly exists in its enol-tautomer form, and it
exhibits poor solubility in water and only modest solubility in
MeOH. These properties may be responsible for its low bioavail-
ability.20 To improve upon its pharmacological properties, numer-
ous efforts have been made to study curcumin’s chemistry and
biology.19,21–23 To date, however, direct chemical connections
between curcumin’s structure and its mode of action remain
incompletely understood.
0.8
0.0
5
12
15
13 14
16
1
2
3
4
6
7
8
9
10 11
R = OH
R = H
Compound Numbers
In this present study, we designed a series of curcuminoids
based on the structures of HCA and curcumin, and synthesized a
panel of analogs with or without a 2-hydroxy group and cucumi-
noids eliminated the unstable b-diketone moiety.
To improve the metabolic stability and antitumor activity of
these compounds, HCA and curcuminoid analogs (compound
1–14, 16) were synthesized as shown in Scheme 1.
B
2.0
1.5
1.0
As shown in Scheme 1, compounds 1–14 were synthesized by
using benzaldehyde or 2-hydroxybenzaldehyde as starting materi-
als and compound 16 was prepared from curcumin 15. In order to
replace the reactive aldehyde group with a more stable enone, HCA
derivatives 2, 3, 9, and 10 were prepared by acylation in the pres-
ence of potassium or barium hydroxide.24 The enolic compounds 4,
5, 11, and 12 were prepared through a one-pot reaction se-
quence.25 The central b-dicarbonyl moiety of curcumin is subject
to keto–enol tautomerization, which is hypothesized to influence
the stability and bioavailability of curcumin.16,17 To overcome this
limitation, compounds 16 and 17 were prepared, in which the two
hydrogen atoms on the central carbon of curcumin were replaced
with geminal dimethyl substituents or a cyclohexyl ring com-
pound, respectively (Fig. 1).16 We synthesized the 6, 7, 13, and
14 by the reaction of enol-containing derivatives with iodometh-
ane in presence of acetone and potassium carbonate and cucumi-
noid 16, eliminated the unstable b-diketone moiety, also
prepared. The structures of these curcuminoids were confirmed
by the presence of methyl protons and the absence of enolic pro-
tons in the 1H NMR spectra.
0hr
2hr
1hr
Figure 2. ROS production by treatment with compounds 1–16. (A) SW620 cells
were treated with compounds 1–16 (20 M) and ROS were measured fluorimet-
l
rically with DCF-DA, using an excitation wavelength of 485 nm and an emission
wavelength of 535 nm. (B) Time-dependence of ROS generation by SW620 cells
following treatment with HCC-7 (20
fluorescence microscope.
lM). Images were captured by a Nikon
ROS are required by both malignant and non-malignant cells for
proliferation. However, the irregular elevation of ROS can selec-
tively reduce proliferation and induce apoptosis of cancer cells.
Therefore, we determined the degree to which ROS production in-
creased following treatment with HCA and curcumin derivatives.
ROS was measured by using DCF-DA, a specific oxidation-sensitive
fluorescent probe of total intracellular ROS. As shown in Figure 2A,
those compounds with a 2-hydroxy group (1–7) enhanced ROS
generation by 1.5- to 2-fold in SW620 cells treated for 2 h. How-
ever, compounds lacking the hydroxyl group (8–14) weakly en-
hanced ROS generation in comparison with DMSO-treated cells.
Especially HCC-7 (compound 7) induced ROS on time dependent
manner (Fig. 2B). To evaluate the effect of the b-diketone moiety
of curcuminoids on ROS generation, we measured the ROS level
after treatment of curcumin 15 or cucuminoid 16 in SW620 cells.
As expected, it was found that cucuminoid 16 strongly increased
the ROS level in comparison with that of curcumin 15 (Fig. 2A).
These results suggest that hydroxyl group works as an enhancer
and b-diketone moiety inhibits ROS generation. Therefore hydroxyl
group and b-diketone moiety in these compounds play critical
roles in ROS production.
DLD-1
Colon
HCT116
SW620
Melanoma SK-MEL-28
Pancreatic
Lung
MiaPaCa2
A549
MCF-7
Breast
MDA-MB-468
MDA-MB-231
0
10
20
GI50 (µM)
30
Figure 3. HCC-7 strongly inhibited human tumor cell growth. Proliferation of
human tumor cells was measured by WST-1 assay kit at 24 h after the treatment of
different concentrations of in HCC-7.
line). Cells were counted after treatment with different concentra-
tions of each compound for 48 h. The GI50 value of compound 1, 5,
6, 7, 15, and 16 is 15, 35, 40, 7, 32, and 7 lM, respectively. HCC-7
(compound 7) and cucuminoid 16 showed stronger cytotoxicity
against SW620 cells than HCA and curcumin. To further under-
stand its biological activity in cancer cells, we investigated whether
the growth of other human tumor cells could be inhibited by
To determine the biological activity of compounds 1–16 in
human cancer cells, we investigated whether the compounds could
inhibit the growth of SW620 cells (a human colorectal tumor cell

750
Y.-M. Han et al. / Bioorg. Med. Chem. Lett. 21 (2011) 747–751
incubation with HCC-7 for 48 h (Fig. 3). In particular, colon
(HCT116, DLD-1, and SW620), lung (A549), breast (MDA-MB-231,
-468, MCF-7), melanoma (SK-MEL-28), and pancreatic (MiaPaCa2)
cell lines were examined. As shown in Figure 3B, colon cell lines
including HCT116, SW620, and DLD-1 were more sensitive than
other cell lines to HCC-7. Even though the actual mechanism of
curcuminoid antitumor activity is still far from understood, a hy-
droxyl group greatly enhances the anti-proliferative effects of curc-
uminoid and the ability to generate ROS.
We have reported that the production of ROS was elevated in
tumor cells after treatment of HCA, and that this effect was
abrogated by pretreatment of cells with N-acetyl-cysteine
(NAC) or GSH.13 We observed by morphology recovery experi-
ments with MDA-MB-231 cells that ROS were quenched when
HCC-7 was co-administered with GSH or NAC, antioxidant mole-
cules containing a thiol group (Fig. 4A). This is consistent with
previous reports that ROS production was elevated in tumor cells
following treatment of HCA, but not if the cells were pretreated
with glutathione.13
PARP participates in DNA repair in response to environmental
stress, and its cleavage by caspase-3 is an established marker of
apoptosis. We found that HCC-7 induced PARP degradation and
caspase-3 activation in SW620 cells. Apoptotic cell death induced
by HCC-7 was also confirmed by FACS analysis (Fig. 4B). Cell mor-
phology (Fig. 4A), PARP degradation, and activation of caspase-3
induced by HCC-7 were recovered by co-treatment with NAC or
GSH.13 It means that oxidative stress may contribute to HCC-7-in-
duced apoptosis.
Figure 4. Apoptosis was induced by HCC-7 through morphology changes, cleavage of PARP, and activation of caspase-3. (A) MDA-MB-231 cells were treated with HCA
(20 M) or HCC-7 (10 M) in the presence or absence of 1 mM GSH. Cell morphology was imaged by Nikon fluorescence microscope. (B) SW620 cells were treated with HCC-7
l
l
in the presence or absence of 1 mM NAC for 48 h and stained with annexin V and propidium iodide. Stained cells then were subjected to FACScalibur analysis to determine the
distribution of cells.

Y.-M. Han et al. / Bioorg. Med. Chem. Lett. 21 (2011) 747–751
751
mitochondria perturbation that require ROS accumulation. This
study thus provides a rationale for the development of curcumi-
noid as chemotherapeutic agent against human tumors.
0
0
3
6
12 24 48 time (hrs)
Cytosolic
Bax
Membrane
Acknowledgments
3
6
12 24 48 time (hrs)
This work was supported by the Korea Research Institute of Bio-
science and Biotechnology Research Initiative Program, the Na-
tional Chemical Genomics Research Program, and the Center for
Biological Modulators of the 21st Century Frontier Research
Program.
Cytosolic
Cyto-C
Membrane
Figure 5. The effect of HCC-7 on the localization of apoptosis regulatory proteins
Bax and cytochrome c (Cyto-c). SW620 cells treated with 10 M of HCC-7 for the
indicated time intervals and then the Bax and Cyto-c protein expression level were
investigated in the cytosolic and membrane fractions by Subcellular fractionation
followed by Western blot analyses.
l
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ROS accumulation is critical for apoptosis induction through medi-
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decrease of the mitochondrial membrane potential by the reduc-
tion in the ratio of A590 (red):A520 (green). We observed that
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fractionation and Western blot analysis. When 10 lM of HCC-7
was treated in SW620 cells, the level of cytochrome c was mark-
edly increased in the cytosolic fraction. Bax, on the contrary to
cytochrome c, is remarkably increased at the membrane fraction
with the concurrent decrease at cytosolic fraction (Fig. 5).
Previous studies have shown that HCA and its analogs induced
cell cycle arrest at the G2/M phase.13,27 Therefore, we chose to
examine the mechanism of action of HCC-7 by cytofluorimetric
analysis, using propidium iodide to label DNA. After treatment of
15. Itokawa, H.; Shi, Q.; Akiyama, T.; Morris-Natschke, S. L.; Lee, K. H. Chin. Med.
2008, 3, 11.
16. Anand, P.; Thomas, S. G.; Kunnumakkara, A. B.; Sundaram, C.; Harikumar, K. B.;
Sung, B.; Tharakan, S. T.; Misra, K.; Priyadarsini, I. K.; Rajasekharan, K. N.;
Aggarwal, B. B. Biochem. Pharmacol. 2008, 76, 1590.
17. Hatchera, H.; Planalpb, R.; Chob, J.; Tortia, F. M.; Torti, S. V. Cell. Mol. Life Sci.
2008, 65, 1631.
18. Hail, N., Jr. Free Radical Biol. Med. 2008, 44, 1382.
19. Ravindran, J.; Subbaraju, G. V.; Raman, M. V.; Sung, B.; Aggarwal, B. B. Biochem.
Pharmacol. 2010, 79, 1658.
20. Payton, F.; Sandusky, P.; Alworth, W. L. J. Nat. Prod. 2007, 70, 143.
21. Amolins, M. W.; Peterson, L. B.; Blagg, B. S. Bioorg. Med. Chem. 2009, 17, 360.
22. Liang, G.; Shao, L.; Wang, Y.; Zhao, C.; Chu, Y.; Xiao, J.; Zhao, Y.; Li, X.; Yang, S.
Bioorg. Med. Chem. 2009, 17, 2623.
SW620 and MCF-10A cells with HCC-7 (10 lM), the cells were har-
vested and analyzed with a FACScalibur flow cytometer. Treatment
of SW620 cells with HCC-7 for 15 h increases DNA content and
chromosome count to a tetraploid state, indicating cell cycle arrest
at the G2/M phase. However, when MCF-10A (an immortalized
23. Lin, L.; Shi, Q.; Nyarko, A. K.; Bastow, K. F.; Wu, C.-C.; Su, C.-Y.; Shih, C. C.-Y.;
Lee, K.-H. J. Med. Chem. 2006, 49, 3963.
24. Liu, X. H.; Cui, P.; Song, B. A.; Bhadury, P. S.; Zhu, H. L.; Wang, S. F. Bioorg. Med.
Chem. 2008, 16, 4075. The spectroscopic characterization of compound 6 as a
sticky yellow oil; ESI MS m/z: 231.0985 (MꢁH)+ Calcd for C14H16O3: 232.1099;
1H NMR (CDCl3): d 8.05 (d, 1H, J = 16.2 Hz), 7.48 (m, 1H), 7.26 (m, 1H), 6.94 (m,
3H), 2.15 (s, 3H), 1.41 (s, 6H); 13C NMR (CDCl3): 208.99, 199.18, 156.35, 140.89,
132.13, 129.96, 121.44, 121.18, 120.66, 116.50, 61.88, 26.63, 21.13. The
spectroscopic characterization of compound 7 as a sticky yellow oil; ESI MS m/
z: 336.0732 (MꢁH)+ Calcd for C21H20O4: 336.1362; 1H NMR (CDCl3): d 8.00 (d,
2H, J = 16 Hz), 7.44 (d, 2H, J = 8.1 Hz), 7.20 (t, 2H, J = 7.2 Hz), 6.87 (m, 6H), 1.48
(s, 6H); 13C NMR (CDCl3): 198.27, 157.30, 138.68, 132.00, 129.27, 121.27,
120.76, 119.37, 116.16, 59.62, 20.89.
25. Weber, W. M.; Hunsaker, L. A.; Roybal, C. N.; Bobrovnikova-Marjon, E. V.;
Abcouwer, S. F.; Royer, R. E.; Deck, L. M.; Vander Jagt, D. L. Bioorg. Med. Chem.
2006, 14, 2450. The spectroscopic characterization of compound 13 as a sticky
yellow oil; ESI MS m/z: 216.0951 (M+H)+ Calcd for C14H16O2: 216.1150; 1H
NMR (CDCl3): d 7.71 (d, 1H, J = 15.3 Hz), 7.54 (m, 3H), 7.39 (m, 2H), 7.39 (m,
3H), 6.79 (d, 1H, J = 15.3 Hz), 2.13 (s, 3H), 1.40 (s, 6H); 13C NMR (CDCl3):
207.92, 197.77, 144.49, 134.15, 130.84, 128.93, 128.58, 120.61, 61.85, 26.51,
21.00. The spectroscopic characterization of compound 16 as a sticky red oil;
ESI MS m/z: 395.5201 (MꢁH)+ Calcd for C23H24O6: 396.4311; 1H NMR (CDCl3):
d 7.65 (d, 2H, J = 9.3), 7.09 (m, 2H), 6.98 (d, 2H, J = 1.14), 6.88 (d, 2H, J = 4.92),
6.62 (d, 2H, J = 9.3), 5.89 (s, 2H), 3.91 (s, 6H), 1.46 (s, 6H).
non-tumorigenic cell line) was treated with HCC-7 (10 lM), DNA
content at the G0/G1 phase was increased. It means that the com-
pound exhibits a different cell cycle effect in a non-tumorigenic
human cell line. It has been reported that immortalized human
umbilical vein endothelial (ECV304) cells undergo arrest at the
G0 phase by curcumin (a structural relative of HCC-7), and that cur-
cumin induced G2/M phase arrest in tumor cells.28 These results
suggest that HCC-7 selectively arrests the cell cycle at the G2/M
phase in tumor-derived cells and retains mother compound’s origi-
nal mechanism of action in tumor cells.
In summary, HCC-7 and compound 16 is a relatively strong gen-
erator of ROS generator in comparison to the other compounds and
has the strongest antitumor activity. These results suggest that ROS
production is an optional source of curcuminoid bioactivity, and
that the presence of a specific hydroxyl group critically enhances
the anti-proliferative effects of curcuminoids and the ability to
generate ROS. It is also found that the b-diketone moiety of
curcuminoids affected the stability and antitumor effects of the
compounds. And our results demonstrate that HCC-7-induced
apoptosis in human colorectal tumor cell is mediated by the
26. Hong, S. H.; Kim, J.; Kim, J. M.; Lee, S. Y.; Shin, D. S.; Son, K. H.; Han, D. C.; Sung,
Y. K.; Kwon, B. M. Biochem. Pharmacol. 2007, 74, 557.
27. Jeong, H. W.; Han, D. C.; Son, K. H.; Han, M. Y.; Lim, J. S.; Ha, J. H.; Lee, C. W.;
Kim, W. M.; Kwon, B. M. Biochem. Pharmacol. 2003, 65, 1343.
28. Sa, G.; Das, T. Cell Div. 2008, 3, 14.