Anti-proliferative, apoptotic induction, and anti-migration effects of hemi-synthetic 1’S-1’-acetoxychavicol acetate analogs on MDA-MB-231 breast cancer cells

Article abstract of DOI:10.2147/DDDT.S130349

Nine analogs of 1′S-1′-acetoxychavicol acetate (ACA) were hemi-synthesized and evaluated for their anticancer activities against seven human cancer cell lines. The aim of this study was to investigate the anti-proliferative, apoptotic, and anti-migration effects of these compounds and to explore the plausible underlying mechanisms of action. We found that ACA and all nine analogs were non toxic to human mammary epithelial cells (HMECs) used as normal control cells, and only ACA, 1′-acetoxyeugenol acetate (AEA), and 1′-acetoxy-3,5-dimethoxychavicol acetate (AMCA) inhibited the growth of MDA-MB-231 breast cancer cells with a half-maximal inhibitory concentration (IC₅₀) value of <30.0 μM based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results, and were selected for further investigation. DNA fragmentation assays showed that these three compounds markedly induced apoptosis of MDA-MB-231 cells. Western blot analysis revealed increased expression levels of cleaved PARP, p53, and Bax, while decreased expression levels of Bcl-2 and Bcl-xL were seen after treatment, indicating that apoptosis was induced via the mitochondrial pathway. Moreover, ACA, AEA, and AMCA effectively inhibited the migration of MDA-MB-231 cells. They also downregulated the expression levels of pFAK/FAK and pAkt/Akt via the integrin β1-mediated signaling pathway. Collectively, ACA and its hemi-synthetic analogs, AEA and AMCA are seen as potential anticancer agents following their abilities to suppress growth, induce apoptosis, and inhibit migration of breast cancer cells.

Full text of DOI:10.2147/DDDT.S130349

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O r i g i n a l r e s e a r c h
anti-proliferative, apoptotic induction, and
anti-migration effects of hemi-synthetic
1′S-1′-acetoxychavicol acetate analogs on
MDa-MB-231 breast cancer cells
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
18 September 2017
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su Ki liew1
Abstract: Nine analogs of 1′S-1′-acetoxychavicol acetate (ACA) were hemi-synthesized and
evaluated for their anticancer activities against seven human cancer cell lines. The aim of this study
wastoinvestigatetheanti-proliferative, apoptotic, andanti-migrationeffectsofthesecompoundsand
to explore the plausible underlying mechanisms of action. We found that ACA and all nine analogs
were non toxic to human mammary epithelial cells (HMECs) used as normal control cells, and only
ACA, 1′-acetoxyeugenol acetate (AEA), and 1′-acetoxy-3,5-dimethoxychavicol acetate (AMCA)
inhibited the growth of MDA-MB-231 breast cancer cells with a half-maximal inhibitory concen-
tration (IC50) value of 30.0 µM based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay results, and were selected for further investigation. DNA fragmentation
assays showed that these three compounds markedly induced apoptosis of MDA-MB-231 cells.
Western blot analysis revealed increased expression levels of cleaved PARP, p53, and Bax, while
decreased expression levels of Bcl-2 and Bcl-xL were seen after treatment, indicating that apop-
tosis was induced via the mitochondrial pathway. Moreover, ACA, AEA, and AMCA effectively
inhibited the migration of MDA-MB-231 cells. They also downregulated the expression levels of
pFAK/FAK and pAkt/Akt via the integrin β1-mediated signaling pathway. Collectively, ACA and
its hemi-synthetic analogs, AEA and AMCA are seen as potential anticancer agents following their
abilities to suppress growth, induce apoptosis, and inhibit migration of breast cancer cells.
Keywords: 1′S-1′-acetoxychavicol acetate, ACA, ACA hemi-synthetic analogs, triple-negative
breast cancer, MTT assay, DNA fragmentation, wound-healing assay, Western blot, integrin
β1 signaling pathway
Mohamad nurul azmi2
lionel la in3
Khalijah awang4,5
noor hasima nagoor1,6
1institute of Biological science
(genetics & Molecular Biology),
Faculty of science, University of
Malaya, Kuala lumpur, 2school of
chemical sciences, Universiti sains
Malaysia, Penang, 3Department of
Biotechnology, Faculty of applied
sciences, Ucsi University, 4centre for
natural Product research and Drug
Discovery (cenar), 5Department of
chemistry, Faculty of science, 6centre
for research in Biotechnology for
agriculture (ceBar), University of
Malaya, Kuala lumpur, Malaysia
Introduction
Over the past half-century, natural products presented impressive achievements in
drug discovery. However, major challenges, such as low production yields of natural
products during scale-up efforts,1 inadequate natural resources,2 and their complex
structures that impede improved structural modifications and synthesis of compounds,
have hindered the development of these natural products as pharmaceutical drugs.3
In addition, screening of numerous extracts and purified compounds from a variety
of sources involves substantial expenditure and time. Due to these limitations, it is
imperative to perform structural modifications on potential compounds through organic
hemi-synthesis, as this would allow discovery of analogs with higher efficacy.
1′S-1′-acetoxychavicol acetate (ACA) is a phenylpropanoid which can be found in
the plant Alpinia conchigera (Zingiberaceae).4 It is known to exhibit a broad range of
correspondence: noor hasima nagoor
institute of Biological science (genetics &
Molecular Biology), Faculty of science,
University of Malaya, lembah Pantai,
50603 Kuala lumpur, Malaysia
Tel +60 3 7967 5921
Fax +60 3 7967 5908
email hasima@um.edu.my
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hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
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biological properties such as antiulcer,5 antifungal,6 inhibi- distortionless enhancement by polarisation transfer) and two-
tion of xanthine oxidase,7 inhibition of Epstein–Barr virus dimensional(2D;correlationspectroscopy, nuclearoverhauser
activation,8 and anticancer activity.9–12 Due to its wide range of effect spectroscopy, heteronuclear single-quantum coherence
biological functions, hemi-synthesis of different ACA analogs spectroscopy, heteronuclear multiple-bond connectivity)
was warranted for enhancement purposes.13 However, various nuclear magnetic resonance (NMR) spectra using CDCl3 as
anticancer properties, such as anti-proliferative, apoptotic, and solvent were recorded on Bruker AVN 400 (400 MHz for
anti-migration effects of its analogs, are yet to be reported.
1H NMR, 100 MHz for 13C NMR) spectrometer. Chemical
Breast cancer is the most common cancer in women in shifts were internally referenced to the solvent signals in
many countries, including Malaysia.14 Triple-negative breast CDCl3 (1H, δ 7.26; 13C, δ 77.0), with tetramethylsilane as the
cancer (TNBC) is one of the subgroups of breast cancer, internal standard and mass spectrometry (MS) on a Shimadzu
which lacks expression of estrogen receptor (ER), progester- gas chromatograph-MS spectrometer (HP 6890 Series Mass
one receptor (PR), and HER-2.15 It constitutes roughly 15% Selective Detector and HP 6890 Series GC System).
of diagnosed breast cancers,16 and unfortunately, it does not
respond to the most effective treatments for breast cancer isolation of aca
such as targeted endocrine therapy.15 Moreover, the aggres- The rhizomes of A. conchigera Griff. were collected from Jeli
siveness of TNBC leads to a high number of metastatic cases province of Kelantan, East Coast of Peninsular Malaysia.4
and deaths. Thus, the development of effective anticancer The sample was identified by Prof Dr Halijah Ibrahim
drugs for TNBC treatment is crucial.
from the Institute of Biological Science, Faculty of Science,
In this study, the effects of ACA and its hemi-synthetic University of Malaya. A voucher specimen (KL5049) was
analogs on the proliferation of various cancer cell lines were deposited in the Herbarium of Chemistry Department,
assessed. We also investigated two other major anticancer Faculty of Science, University of Malaya. ACA was isolated
properties, namely apoptosis and anti-migration effects, and following our previously published methods.4
discussed their underlying molecular mechanisms. We dem-
general procedure to obtain
onstrated that ACA, 1′-acetoxyeugenol acetate (AEA), and
1′-acetoxy-3,5-dimethoxychavicol acetate (AMCA) could compounds 2–8
inhibit MDA-MB-231 TNBC cell proliferation by inducing 4-Allyl-2,6-dimethoxyphenol (2), (S)-α-vinylbenzyl alco-
apoptosis through the regulation of vital proteins, namely hol (3), and 4-allyl anisole (5) were purchased from Sigma-
PARP, p53, Bcl-2, Bcl-xL, and Bax. In addition, our results Aldrich Co. (St Louis, MO, USA). Eugenol (4) was purchased
also revealed the potential of these three compounds against from Merck & Co., Inc. (Whitehouse Station, NJ, USA).
tumor metastasis by downregulating the expression of pFAK, Compounds 6, 7, and 8 were synthesized from 2, 3, and 4,
focal adhesion kinase (FAK), pAkt, and Akt via the integrin respectively, by reacting with acetic anhydride (Scheme 1).
β1-mediated pathway. These observations suggested that
general procedure to synthesize
compounds 17–20
Compounds 17–20 were synthesized by the modification of
Lee and Ando’s17 procedure.
ACA, AEA, and AMCA may be potential agents in the
treatment of TNBC.
Materials and methods
general chemistry procedure
Unless otherwise noted, all materials were obtained from aea (17)
commercial suppliers and were used without further purifica- To a solution of vinylmagnesium bromide freshly prepared
tion. Reaction time and purity of products were monitored from magnesium turning (0.24 g, 9.87 mmol), 1 M vinyl
by thin-layer chromatography on Merck silica gel aluminum bromide (6.6 mL, 6.57 mmol) and catalytic amount of iodine
cards (0.2 mm thickness) with fluorescent indicator at 254 nm. crystal in dry diethyl ether (20 mL), a solution of aldehyde 9
Column chromatography was run on silica gel (200–300 (0.50 g, 3.29 mmol) in diethyl ether (10 mL) under ice bath,
mesh) obtained from EMD Millipore (Billerica, MA, USA). were added. The ice bath was removed, and the mixture was
All spectral data were obtained using the following instru- allowed to warm to room temperature. After the reaction mix-
ments: infrared (IR) on a Perkin Elmer RX1 FT-IR spectrom- ture was stored for 2 h at room temperature, saturated aqueous
eter, ultraviolet (UV) on a Shimadzu UV-160A UV–Visible NH4Cl (10 mL) solution was added. The mixture was extracted
Recording Spectrophotometer. One-dimensional (1D; 1H, 13C, with diethyl ether (2×20 mL). The resulting organic extracts
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anticancer effects of aca and its analogs
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Scheme 1 schematic preparation of aca analogs.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; DMaP, 4-dimethylaminopyridine; rT, room temperature.
were combined, and the solvent was removed under reduced mass spectroscopy m/z (electron ionization): 264.0993 (M+•
pressure to yield a crude product. The oily product (intermedi- C14H16O5+• requires 264.0998).
ate 13) obtained by usual workup was acetylated with acetic
anhydride(0.62mL, 6.57mmol)and4-dimethylaminopyridine aMca (18)
(DMAP; 0.81 g, 6.64 mmol) in dichloromethane (15 mL) in an To a solution of vinylmagnesium bromide freshly prepared
ice bath. After consumption of starting material and product from magnesium turning (0.17 g, 7.14 mmol), 1 M vinyl
formation, the reaction was quenched with saturated aqueous bromide (3.6 mL, 3.5 mmol) and catalytic amount of iodine
NH4Cl (10 mL) solution and extracted with dichloromethane crystal in dry diethyl ether (20 mL), a solution of aldehyde
(3×15 mL), dried (NaSO4), and filtered. Evaporation of filtrate 10 (0.30 g, 1.65 mmol) in diethyl ether (10 mL) under
gave a yellow oil, which was purified by column chromatog- ice bath, were added. The ice bath was removed, and the
raphy on silica gel, and eluting with hexane/ethyl acetate (9:1) mixture was allowed to warm to room temperature. After
afforded the desired product as a yellowish oil 17 (0.53 g, the reaction mixture was stored for 1 h at room temperature,
61%). The synthetic analog 17 is the same as the natural analog saturated aqueous NH4Cl (10 mL) solution was added. The
AEA that was previously studied.18,19
mixture was extracted with diethyl ether (2×20 mL). The
The spectral analysis of the yellowish oil: IR (neat) resulting organic extracts were combined, and the solvent
υ
max, cm−1: 1,767, 1,742, 1,647, 1,607, 1,233, 1,198, 857. was removed under reduced pressure to yield a crude product.
1
UV λmax, nm: 299.0. H NMR (CDCl3, 400 MHz), δ: 2.12 The oily product (intermediate 14) obtained by usual workup
(3H, s), 2.31 (3H, s), 3.84 (3H, s), 5.25 (2H, dd, J =10.5), was acetylated with acetic anhydride (0.33 mL, 3.30 mmol)
5.99 (1H, m), 6.24 (1H, d, J =5.8), 6.95 (1H, s), 7.02 (2H, d, and DMAP (0.41 g, 3.36 mmol) in dichloromethane (15 mL)
J =7.8). 13C NMR (CDCl3, 100 MHz), δ: 20.6 (CH3), 21.25 in an ice bath. After consumption of starting material and
(CH3), 55.9 (OCH3), 75.7 (CH), 111.5 (CH), 117.0 (CH2), product formation, the reaction was quenched with satu-
119.6 (CH), 122.8 (CH), 135.9 (CH), 137.6 (C), 139.7 (C), rated aqueous NH4Cl (10 mL) solution and extracted with
151.2 (C), 168.9 (C), 169.7 (C). MS m/z (electron ionization) dichloromethane (3×15 mL), dried (NaSO4), and filtered.
40, 45, 61, 77, 91, 107, 121, 264 (M+•). High resolution Evaporation of filtrate gave a yellow oil, which was purified
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by column chromatography on silica gel, and eluting with bromide (37 mL, 37.0 mmol) and catalytic amount of iodine
hexane/ethyl acetate (9:1) afforded the desired product as a crystal in dry diethyl ether (50 mL), a solution of aldehyde
yellowish oil 18 (0.30 g, 63%).
12 (1.68 g, 12.3 mmol) in diethyl ether (10 mL) under ice bath,
The spectral analysis of the yellowish oil: IR (neat) were added. The ice bath was removed, and the mixture was
1
υ
max, cm−1: 1,761, 1,645, 1,234. UV λmax, nm: 304.5. H allowed to warm to room temperature. After the reaction mix-
NMR (CDCl3, 400 MHz), δ: 2.13 (3H, s), 2.33 (3H, s), 3.81 ture was stored for 3 h at room temperature, saturated aqueous
(6H, s), 5.31 (2H, dd, J =10.0 Hz), 5.98 (1H, m), 6.21 (1H, NH4Cl (10 mL) solution was added. The mixture was extracted
d, J =5.8 Hz), 6.61 (2H, s). 13C NMR (CDCl3, 100 MHz), with diethyl ether (2×30 mL). The resulting organic extracts
δ: 20.4 (CH3), 21.2 (CH3), 56.1 (2OCH3), 76.0 (CH), 104.0 were combined, and the solvent was removed under reduced
(2CH), 117.0 (CH2), 128.4 (C), 135.9 (CH), 137.2 (C), pressure to yield a crude product. The oily product (inter-
152.1 (2C), 168.7 (C), 169.9 (C). HRMS m/z (EI): 294.1107 mediate 16) obtained by usual workup was acetylated with
(M+• C15H18O6+• requires 294.1103).
acetic anhydride (1.51 mL, 16.0 mmol) and DMAP (3.09 g,
25.3 mmol) in dichloromethane (25 mL) in an ice bath. After
consumption of starting material and product formation, the
1′-acetoxy-3,5-dimethoxychavicol (19)
To a solution of vinylmagnesium bromide freshly prepared reaction was quenched with saturated aqueous NH4Cl (10 mL)
from magnesium turning (0.92 g, 38.3 mmol), 1 M vinyl solution and extracted with dichloromethane (3×15 mL), dried
bromide (17 mL, 17.1 mmol) and catalytic amount of iodine (NaSO4), and filtered. Evaporation of filtrate gave a yellow oil,
crystal in dry diethyl ether (30 mL), a solution of aldehyde which was purified by column chromatography on silica gel,
11 (0.98 g, 5.89 mmol) in diethyl ether (10 mL) under ice bath, eluting with hexane/ethyl acetate (95:5) afforded the desired
were added. The ice bath was removed, and the mixture was product as a yellowish oil 20 (1.57 g, 62%).
allowed to warm to room temperature. After the reaction mix-
ture was stored for 2 h at room temperature, saturated aqueous
The spectral analysis of the yellowish oil: IR (neat)
1
υ
max, cm−1: 1,737, 1,515, 1,236. UV λmax, nm: 304.5. H
NH4Cl (10 mL) solution was added. The mixture was extracted NMR (CDCl3, 400 MHz), δ: 2.07 (3H, s), 3.78 (3H, s), 5.28
with diethyl ether (2×30 mL). The resulting organic extracts (2H, dd, J =10.4 Hz), 5.99 (1H, m), 6.22 (1H, d, J =5.8 Hz),
were combined, and the solvent was removed under reduced 6.88 (2H, d, J =6.8 Hz), 7.28 (2H, d, J =6.8 Hz). 13C NMR
pressure to yield a crude product. The oily product (inter- (CDCl3, 100 MHz), δ: 21.3 (CH3), 55.3 (OCH3), 76.3 (CH),
mediate 15) obtained by usual workup was acetylated with 113.9 (2CH), 116.5 (CH2), 128.8 (2CH), 136.4 (CH), 159.5
acetic anhydride (0.60 mL, 6.35 mmol) and DMAP (1.52 g, (C), 169.1 (C). MS m/z (EI) 43, 65, 77, 91, 104, 121, 131,149,
12.4 mmol) in dichloromethane (20 mL) in an ice bath. After 164, 206 (M+•). HRMS m/z (EI): 206.0948 (M+• C12H14O3+•
consumption of starting material and product formation, the requires 206.0943).
reaction was quenched with saturated aqueous NH4Cl (10 mL)
solution and extracted with dichloromethane (3×15 mL), dried cell lines and culture conditions
(NaSO4), and filtered. Evaporation of filtrate gave a yellow A total of seven human cancer cell lines and one normal cell
oil, which was purified by column chromatography on silica line were used in this study. Five cell lines (PC-3 [prostate
gel, and eluting with hexane/ethyl acetate (9:1) afforded the cancer cells], MCF-7 and MDA-MB-231 [breast cancer
desired product as a yellowish oil 19 (0.90 g, 65%).
The spectral analysis of the yellowish oil: IR (CCl4) were cultured in Roswell Park Memorial Institute (RPMI)
max, cm−1: 1,739, 1,602, 1,558, 1,519, 1,459, 1,232. UV λmax
1640 medium, while two cell lines (HSC-4 [oral squamous
cells], EJ-28 and RT-112 [urinary bladder cancer cells])
υ
,
nm: 304.5. 1H NMR (CDCl3, 400 MHz), δ: 2.11 (3H, s), 3.77 cancer cells] and HepG2 [liver cancer cells]) were cultured
(6H, s), 5.31 (2H, dd, J =10.5 Hz), 5.97 (1H, m), 6.16 (1H, d, in Dulbecco’s Modified Eagle’s Medium (DMEM), supple-
J =5.9 Hz), 6.38 (2H, s), 6.49 (1H, s). 13C NMR (CDCl3, mented with 10% (v/v) fetal bovine serum, 100 U/mL peni-
100 MHz), δ: 21.3 (CH3), 55.4 (2OCH3), 76.8 (CH), 100.0 cillin, and 100 µg/mL streptomycin. The human mammary
(CH), 106.6 (2CH), 141.3 (C), 160.9 (2C), 170.0 (C). HRMS epithelial cell line (HMEC) used as the normal cell control
m/z (EI): 236.1045 (M+• C13H16O4+• requires 236.1049).
was cultured in mammary epithelial growth medium. The
cell lines such as PC-3, MCF-7, MDA-MB-231, EJ-28,
HepG2 were obtained from American Type Culture
1′-acetoxy-4-methoxychavicol (20)
To a solution of vinylmagnesium bromide freshly prepared Collection (ATCC, Manassas, VA, USA), RT-112 from
from magnesium turning (2.14 g, 89.2 mmol), 1 M vinyl German Collection of Microorganisms and Cell Cultures
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anticancer effects of aca and its analogs
(Braunschweig, Germany), and HSC-4 from Malaysian concentrations (IC50; 4.8, 9.5 and 29.6 µM, respectively)
Cancer Research Initiative Foundation (CARIF, Selangor, for 24 h. Total DNA was extracted from both untreated and
Malaysia), while HMEC was obtained from Lonza Inc. treated cells using the Suicide Track™ DNA Ladder Isolation
(Allendale, NJ, USA). All cultures were maintained in an Kit (Calbiochem, La Jolla, CA, USA) according to the manu-
incubator with humidified atmosphere (5% CO2) at 37°C.
facturer’s protocol. Analysis of extracted DNA was carried
out on a 1.0% (w/v) agarose gel electrophoresis and stained
with ethidium bromide. Observation of DNA fragmentation
cell viability assay
Cytotoxic effects of ACA and its analogs (Figure 1) on the was performed under UV illumination and visualized using
seven cancer cell lines were determined by using the in vitro a gel documentation system (Alpha Innotech, San Leandro,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- CA, USA).
mide (MTT) assay. All tested compounds were dissolved
anti-migration assay
in dimethyl sulfoxide (DMSO) to a final concentration of
The anti-migration effects of ACA and its analogs were
determined using the wound-healing assay. MDA-MB-231
cells were seeded in six-well plates and cultured until 80%
confluent. After an overnight cultivation, complete medium
was then replaced by serum-free medium treated with
1 µg/mL mitomycin-C, and the cells were further incubated
at 37°C for 2 h to stop cell proliferation. A vertical scratch
was made using a sterile pipette tip across the well, and
cell debris was removed by washing with 1× phosphate-
buffered saline. Cells were treated with control solvent or
ACA and its analogs at their 20% inhibitory concentrations
(IC20; Table S1) in serum-free medium. IC20 were used
10.0 mM. In brief, cells were seeded in triplicates on 96-well
plates at 1.0×104 cells/well. After 24 h, cells were treated with
increasing concentrations (5.0–50.0 µM) of ACA and its ana-
logs for 24 h. A total of 10.0 µL of MTT reagent (5.0 mg/mL)
was added to each well. After incubation in the dark at 37°C
for 2 h, the unreacted MTT dye was removed by aspira-
tion and 200.0 µL of DMSO was added to dissolve MTT
purple formazan. The absorbance was determined at 579 nm
by microtiter plate reader (Tecan Sunrise®, Männedorf,
Switzerland), with a reference wavelength of 650 nm.
Dna fragmentation assay
MDA-MB-231 breast cancer cells were treated with instead of IC50, because IC20 were less toxic on the cells.
ACA, AEA, and AMCA at their half-maximal inhibitory Images of wounded cells were captured using an inverted
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Figure 1 The structures of aca and its analogs (2, 5–8, and 17–20).
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate.
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fluorescence microscope, Nikon Eclipse TS-100 (Nikon Results
Corporation, Tokyo, Japan), at 0 and 24 h after wounding.
effect of hemi-synthetic aca analogs on
The distance between two sides of the wound was analyzed
using Tscratch software, version 1.0 (The Mathworks,
Natick, MA, USA).
the viability of various cancer cell lines
Cytotoxicity evaluation of ACA and its analogs (Figure 1)
in various human cancer cell lines was performed using the
MTT assay method with results expressed as mean IC50 values.
Seven cancer cell lines were used in this study to explore
whether different ACA analogs could exhibit a similar broad
spectrum cytotoxic effect as was reported for ACA on various
cancer types.9–12 All tested analogs indicated weak cytotoxicity
except for compounds ACA, AEA, and AMCA with poten-
cies #30.0 µM (Table 1). Meanwhile, all compounds were
shown to have minimal cytotoxic activity toward HMECs used
asnormalhumanbreastcellcontrol, indicatingnontoxiceffects
against normal cells at therapeutic doses. Solvent control with
DMSO at #1.0% (v/v) showed insignificant effects on cancer
cell viability (data not shown), also indicating that cytotoxicity
was induced as a result of treatment with tested compounds
instead of DMSO, which is known to be cytotoxic at high
concentrations.20 ACA induced cytotoxicity on all cancer cell
lines tested, with highest levels of efficacy being observed in
breast adenocarcinoma (MDA-MB-231) and oral squamous
carcinoma cells (HSC-4) at 24 h post treatment time. Although
AEA and AMCA also induced cytotoxicity, their effects were
found to be selective toward specific cancer cell lines. AEA
was found to induce high levels of cytotoxicity in bladder
adenocarcinoma (EJ-28), hepatocarcinoma (HepG2), and oral
SCC (HSC-4), while AMCA only showed cytotoxic effects
against MDA-MB-231 cells (Table 1).
Western blot assay
MDA-MB-231 cells were treated with ACA, AEA, and
AMCA and incubated for 24 h. Total proteins were extracted
using the NE-PER nuclear and cytoplasmic extraction kit
(Pierce, Rockford, IL, USA) according to the manufacturer’s
protocol. Protein concentrations were determined using
the Bio-Rad DC protein assay (Bio-Rad Laboratories
Inc., Hercules, CA, USA) according to the manufacturer’s
protocol. Equal amounts of protein were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes by semi-dry
transfer system (Bio-Rad Laboratories Inc.). Membranes
were blocked with 5% (w/v) bovine serum albumin and
incubated with primary antibodies against GAPDH, Bcl-2,
Bcl-xL, Bax, p53, PARP, β1 integrin, FAK, pFAK, Akt, and
pAkt (1:1,000 dilutions; Cell Signaling Technology, Dan-
vers, MA, USA). Horseradish peroxidase-linked secondary
antibodies were added, and bound proteins were detected
through enhanced chemiluminescence reagent (Advan-
sta, Menlo Park, CA, USA) and visualized on a Fusion
FX7 imaging system (Vilbert Lonmart, Eberhardzell,
Germany).
statistical analysis
The results were presented as mean ± standard error of the
effect of aca,aea, and aMca on Dna
mean for triplicates of all experiments. One-way ANOVA fragmentation
was used to analyze the statistically significant differences Next, to determine whether their cytotoxic effects are medi-
with a confidence level of p#0.05.
ated through apoptosis, we performed DNA fragmentation
Table 1 ic50 values for aca and its analogs on various cancer cell lines and normal cells
Cell lines
IC50 (µM)
ACA
2
5
6
7
8
AEA
AMCA
19
20
Breast adenocarcinoma (MDa-MB-231)
Breast adenocarcinoma (McF-7)
Bladder carcinoma (rT-112)
Bladder carcinoma (eJ-28)
Prostate carcinoma (Pc-3)
Oral squamous carcinoma (hsc-4)
hepatocyte carcinoma (hepg2)
hMecs
4.8±0.4
30.0±0.3
14.1±3.8
8.2±0.9
26.7±2.3
5.5±0.5
18.0±0.8
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
9.5±0.3
25.2±0.6
10.9±2.4
4.2±2.2
13.8±0.8
5.1±0.2
4.3±0.5
n/a
29.6±5.6
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
Notes: cells were treated with the test compounds for 24 h. Data are reported as mean ± seM (n=3). n/a denotes that cell viability levels were maintained above 50% at
maximum concentration of test compound (50 µM) at 24 h of incubation.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; hMecs, human mammary
epithelial cells; ic50, half-maximal inhibitory concentration; seM, standard error of the mean; n/a, not applicable.
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anticancer effects of aca and its analogs
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by ~30%–50% when compared to untreated cells. The
p53 levels in MDA-MB-231 cells increased by 3.3-, 3.4-,
and 2.3-fold after treatment with ACA, AEA, and AMCA,
respectively (Figure 3E). The three compounds upregulated
the expression of pro-apoptotic Bax by 6.2-, 9.4-, and 2.9-
fold, respectively (Figure 3F). Collectively, this suggests
that PARP, p53, Bcl-2, Bcl-xL, and Bax were involved in
inducing apoptosis via the intrinsic pathway when treated
with ACA, AEA, and AMCA.
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effect of hemi-synthetic aca analogs on
anti-migration of MDa-MB-231 cells
Cell migration is an important step in the metastatic process.22
Therefore, the effects of ACA and its analogs on cell migra-
tion were also examined on breast cancer MDA-MB-231
cells (Figures 4 and S1). This cell line was chosen for
anti-migration study due to its highly aggressive and strongly
metastatic properties among the selected panel of cell
lines.23,24 Our data showed that ACA, AEA, and AMCA and
compounds 5–8 and 19–20 significantly inhibited the migra-
tion rate of MDA-MB-231 cells compared with untreated
cells. Furthermore, results revealed that the difference in
inhibition of cell migration between untreated cells and
cells treated with DMSO was insignificant (p-value .0.05),
suggesting that the anti-migration effects seen were not due
to the solvent.
ꢃꢄꢂ
Figure 2 agarose gel electrophoresis of Dna in MDa-MB-231 cells treated with
aca, aea, and aMca.
Notes: cells were untreated (lane UT) or treated with DMsO 0.08% (v/v), aca,
aea, aMca at their ic50 (4.8, 9.5, and 29.6 µM, respectively) for 24 h. left lane (M)
shows Dna size markers. cellular Dna was extracted and subjected to agarose
gel electrophoresis.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol ace-
tate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMsO, dimethyl sulfoxide;
Dna, deoxyribonucleic acid; ic50, half-maximal inhibitory concentration; UT,
untreated.
assays. As shown in Figure 2, DNA from the untreated
group showed no degradation, whereas DNA laddering was
observed in MDA-MB-231 cells that were treated with ACA,
AEA, and AMCA at their IC50. This typical oligonucleosomal
DNA degradation is one of the hallmarks of apoptotic cell
death, thus suggesting that ACA, AEA, and AMCA induce
cell death through apoptosis.
effect of aca,aea, and aMca on
downregulators of integrin β1
In addition to anti-proliferative effects and induction of
apoptosis, ACA, AEA, and AMCA also showed significant
anti-migration effects on MDA-MB-231 breast cancer cells.
effect of aca,aea, and aMca on ParP,
p53, and Bcl-2 family members
To further verify the occurrence of apoptosis induced Thus, the molecular mechanism related to the anti-migration
by ACA, AEA, and AMCA on MDA-MB-231 cells, we effects of these compounds was also investigated. The effect
performed PARP cleavage analysis, a common marker of these compounds on the expression of integrin β1, which
for induction of apoptosis.21 As shown in Figure 3A, the is a major player in tumor metastasis that mediate the adhe-
cleavage of full-length PARP enzymes (116 kDa) into an sion of cells on extracellular matrix (ECM),25 and some of its
89 kDa subunit protein was observed in the MDA-MB-231 downstream molecules were determined. We examined the
cells that had been treated with ACA, AEA, and AMCA integrin-mediated pathway on MDA-MB-231 breast cancer
at their IC50. Based on densitometry analysis, these three cell line by Western blot analysis and found that the level
compounds increased the cleavage of PARP by 7.8-, 4.8-, of integrin β1 decreased following ACA, AEA, and AMCA
and 1.8-fold compared to untreated cells, respectively treatment (Figure 5A). Upon integrin β1 binding to ECM,
(Figure 3B). ACA, AEA, and AMCA were also found to the downstream signaling molecules such as FAK and Akt
decrease Bcl-2 and Bcl-xL anti-apoptotic protein levels and were phosphorylated26 and participated in the regulation of
increase the expression levels of p53 and pro-apoptotic Bax. cell migration. In analyzing the integrin-induced signaling
As shown in Figure 3C and D, treatment with ACA, AEA, pathway, phosphorylation of FAK and Akt was significantly
and AMCA at their IC50 for 24 h significantly decreased reduced by ACA, AEA, and AMCA after 24 h (Figure 5A).
the expression level of Bcl-2 by ~40%–50% and Bcl-xL AsshowninFigure5B, theintegrinβ1levelsofMDA-MB-231
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Figure 3 effects of aca, aea, and aMca on the apoptotic pathway.
Notes: expression levels of ParP, p53, Bcl-2, Bcl-xl, and Bax proteins from MDa-MB-231 cells were analyzed via Western blot analysis (A). gaPDh was used as a loading
control. The quantitative data (B–F) are presented as mean ± seM from three independent experiments (*p#0.05 by two-tailed unpaired t-test).
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; Bax, Bcl-2-associated X protein;
Bcl-2, B-cell lymphoma 2; Bcl-xl, B-cell lymphoma-extra large; DMsO, dimethyl sulfoxide; gaPDh, glyceraldehyde 3-phosphate dehydrogenase; ParP, poly(aDP-ribose)
polymerase; p53, tumor suppressor p53; seM, standard error of the mean.
cells decreased by 15.5%, 37.1%, and 93.3% after treatment angiogenesis28 and metastasis.29 Wang et al29 showed that
with ACA, AEA, and AMCA, respectively. The reduction ACA exerted cytotoxic effects and inhibited the signal
level of phosphorylated FAK and Akt by ACA, AEA, and transduction pathway related to tumor metastasis, such as
AMCA was about 70%–94% (Figure 5C and D).
signal transducer and activator of transcription 3 (STAT3),
Src homology region 2 domain-containing phosphatase 1
(SHP-1), and matrix metalloproteinase (MMP)-mediated
Discussion
ACA is a natural product that exerts anticancer effects by pathway. In this study, ACA inhibited the growth of MDA-
inducing apoptosis27 and cell cycle arrest4 and inhibiting MB-231 cells with IC50 of 4.8 µM on the breast cancer cell
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anticancer effects of aca and its analogs
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Figure 4 effects of aca and its analogs on the cell migration of MDa-MB-231 cells.
Notes: cells were wounded and treated with DMsO 0.08% (v/v), aca, and nine analogs at ic20 for 24 h. The open wound area at 24 h was quantified using TScratch software
relative to wound area at 0 h. The mean percentage of area migrated ± SEM from three separate experiments is illustrated. Statistically significant differences were compared
between untreated conditions versus treatment groups with (*) denoting p-values #0.05.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMsO, dimethyl sulfoxide;
ic20, 20% inhibitory concentration; seM, standard error of the mean.
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Figure 5 effects of aca, aea, and aMca on the migration pathway.
Notes: expression levels of integrin β1, FaK, pFaK, akt, and pakt proteins from MDa-MB-231 cells were analyzed via Western blot analysis (A). gaPDh was used as a
loading control. The quantitative data (B–D) are presented as mean ± seM from three independent experiments (*p#0.05 by two-tailed unpaired t-test).
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; akt, protein kinase B; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMsO,
dimethyl sulfoxide; FaK, focal adhesion kinase; gaPDh, glyceraldehyde 3-phosphate dehydrogenase; integrin β1, integrin subunit beta 1; pakt, phosphorylated protein kinase B;
pFaK, phosphorylated focal adhesion kinase; seM, standard error of the mean.
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line, which is about 10 times lower than Wang et al’s study. study showed that ACA induced apoptosis via the activation
The significant difference of the results may be due to the het- of caspase-3-like activity.35 Thus, the alteration of PARP by
erogeneity of cancer cells30 and the changes that occurred in ACA, AEA, and AMCA was in agreement with the induction
cells during cultivation in different laboratory conditions.
of apoptosis via the activation of caspase-3. The activation
Even though ACA was seen to induce apoptosis and of the p53 protein is also related to induction of apoptosis36
inhibit migration on some cancer cell types, the apoptotic and regulation of Bcl-2 family members’ expression.37 Anti-
induction and anti-migration mechanisms of hemi-synthetic apoptotic proteins, Bcl-2 and Bcl-xL, act as inhibitors of the
ACA analogs on breast cancer cells have never been reported. mitochondrial intrinsic pathway that block the release of cyto-
In this study, we revealed that not only ACA but also two of chromes and counteract the effects of pro-apoptotic proteins
its analogs were able to induce apoptosis and suppress the such as Bax.38 Pro-apoptotic protein, Bax, redistributes from
migration of MDA-MB-231 breast cancer cells.
the cytosol to mitochondria during apoptotic events39 to
In this study, three compounds (ACA, AEA, and AMCA) cause dysfunction of the mitochondrial membrane.40 In this
possessed 1′-acetoxyl group and showed better cytotoxicity study, the expression of Bax was increased by treatment with
than other compounds. This highlighted the importance of ACA, AEA, and AMCA, while the expression of Bcl-2 and
1′-acetoxyl group in governing anti-proliferative activity. Bcl-xL was decreased. These observations suggested that
The absence of the 1′-acetoxyl group (compounds 2, 5, 6, 8) the induction of apoptosis by ACA, AEA, and AMCA was
could lead to a reduction in inhibitory effects on cancer triggered by the upregulation of pro-apoptotic proteins and
cell growth. These findings are consistent with the study downregulation of anti-apoptotic proteins related to the mito-
by Murakami et al.13 On the other hand, AMCA, which chondrial intrinsic pathway. Furthermore, previous studies
possesses an additional methoxyl group at the 5-position showed that ACA and AEA suppressed the growth of cancer
compared to AEA, exerted a weaker effect against MDA- cells by inhibiting nuclear factor-κB (NF-κB) pathway.19,41–43
MB-231 cells, suggesting that the presence of an additional The expression of the anti-apoptotic proteins, such as Bcl-244
methoxyl group at 5-position may reduce cytotoxic activity. and Bcl-xL,45 was regulated by NF-κB. Hence, the induction
In a study by Xu et al,31 the authors reported the structural of apoptosis by ACA, AEA, and AMCA may also be linked
factors of ACA that regulate tumor cell viability, intracel- to the inactivation of the NF-κB.
lular glutathione level, and glutathione reductase activity in
ACA has also been reported to inhibit metastasis and inva-
Ehrlich ascites tumor cells. They suggested that the acetoxyl sion on different cancer cell types, such as breast cancers,29
group substituted at para position of the benzene ring was oral cancers,43 prostate cancers,28 and lung cancers.46 These
essential, and similarly we found that analog without an investigations point out that ACA may generally suppress cell
acetoxyl group at the 4-position (such as compounds 2 and migration and invasion in various cancer cell types. In this
20) showed insignificant cytotoxic effects.
study, we reported that ACA and its analogs exhibit anti-
MDA-MB-231 was reported as the most sensitive cell migration effects on MDA-MB-231 cells. Although some of
line toward the compounds tested in terms of cytotoxicity the ACA analogs showed poor potency against cancer cell
and was thus selected as a model for further investigation. proliferation, they exhibited strong effects against migration
Another type of breast cancer cell, MCF-7 cell line, was only and thus reignite interest for further studies on the molecular
sensitive to ACA and AEA. Despite both cell lines being mechanisms governing anti-migration activity of ACA and
breast cancer cell lines, MDA-MB-231 is a triple-negative its analogs. In this study, most of the hemi-synthetic ACA
(lack of ER, PR, and HER-2 expression) breast cancer cell analogs were not cytotoxic within the 5.0–50.0 µM dose
line, while MCF-7 is an ER- and PR-positive breast cancer range, but many of them were able to inhibit cancer cell
cell line. Thus, these differences could be the underlying migration with nontoxic concentrations. Hence, the anti-
cause toward response variations and drug sensitivity.32
migration activity of the ACA analogs is not linked to their
DNA fragmentation is a cellular hallmark of apoptosis. direct cytotoxic effects on cancer cells. The inhibition of cell
In apoptotic cells, endonucleases cleave DNA into internu- migration by the compounds occurred at concentrations lower
cleosomal fragments of roughly 180–200 bp.33 Since ACA, than those that inhibited cell proliferation. The difference in
AEA, and AMCA showed active anti-proliferative effects effective concentrations suggested that different mechanisms
and induced internucleosomal DNA fragmentation on MDA- may be involved to promote ACA analogs’ anti-migration
MB-231 cells, induction of apoptosis was confirmed. During effects versus cytotoxic ability.
the apoptotic process, proteolytic cleavage of PARP due to
Cancer cell motility by metastasis is a main step for the
the activation of caspase-334 was seen in this study. Previous progression of malignancy.47 Tumor cells take multisteps
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anticancer effects of aca and its analogs
to metastasize from their initial site to secondary organs by cells by ACA and its analogs. In addition, analogs active in
migration through basement membranes and extracellular anti-proliferation against selected cancer cells were seen to
matrices.48 The migration process is stimulated extracel- possess better anti-migration ability against breast cancer
lularly and initiated by integrins and intracellular signaling cells compared to the natural product itself. Thus, this study
proteins located at the sites of focal adhesion assembly and demonstrates that there is still room for improvement in
disassembly.49 Regulating integrin β1 signaling of FAK designing new analogs with increased anticancer activities.
through modulation of Akt activity could lead to inhibition However, more investigations are required to identify the
of cell migration.50 Many human malignant cancers show different targets of such ACA analogs at the molecular level
increased FAK expression promoted by integrin β1 to result and validate the molecular mechanisms in animal models
in increased metastasis.51 In this study, breast cancer cells for effective treatment of breast cancer. Overall, this study
treated with ACA, AEA, and AMCA suppressed integrin suggested that ACA, along with its hemi-synthetic analogs,
β1 after 24 h (Figure 5), which indicated their ability to AEA and AMCA, are potential combinatory therapeutic
interrupt the integrin signaling pathway. Since integrin acts agents for the treatment of TNBCs.
as the activator of FAK, we examined the effect of these
Acknowledgments
compounds on the activation of pFAK. Upon treatment,
ACA and analogs were prepared by Chin Hui Kee. This study
was supported by the University of Malaya Postgraduate
Research Grant (PV050-2012A, PG100-2012B), the Centre
for Research in Biotechnology for Agriculture (CEBAR)
Flagship Grant (RU005C-2014), Chemistry-HIR Grant
UM.0000091/HIR.C3, RP001-2012A/B, Malaysian Ministry
of Higher Education (MOHE) Fundamental Research Grant
Scheme (FRGS) (FRGS/1/2014/SG05/UCSI/03/1), and the
Biosecurity and Biosafety for Tropical Agricultural Bio-
economy Grant (RU015-2016).
FAK phosphorylation was significantly reduced in MDA-
MB-231 cells. ACA, AEA, and AMCA also decreased
Akt phosphorylation, showing that the activity of pAkt
was correlated with FAK phosphorylation. Thus, rela-
tive intensities of integrin β1, pFAK/FAK, and pAkt/Akt
showed that ACA, AEA, and AMCA showed anti-migration
potency and is dependent, at least in part, on the integrin
β1-mediated signaling pathway. Wang et al29 showed that
inhibition of metastasis by ACA is through interference with
the STAT3 signaling pathway. Their findings support our
current result as STAT3 proteins are known to be interrelated
to integrin β1. Nevertheless, the mechanism(s) in which ACA
and its analogs participate in to achieve their anticancer role
on TNBCs will require further in-depth investigation, such
as the evaluation of key proteins involved in other metastatic
signaling pathways.
Author contributions
SKL carried out the biological experiments and wrote the
manuscript. MNA isolated ACA, synthesized the analogs,
and contributed to drafting the manuscript. LLAI helped
in solving technical experiment problems and editing the
manuscript. KA participated in the design of the analogs.
NHN conceived the entire study and helped in editing the
manuscript. All authors contributed toward data analysis,
drafting and critically revising the paper and agree to be
accountable for all aspects of the work.
In addition, the efficacy of ACA is also being studied for
its use in combination studies with other drugs/biologics. For
example, ACA reduced tumor volume and side effects in vivo
when used in combination with alpha-fetoprotein (AFP).52
While stand-alone doses of the analogs may not seem
clinically relevant at this stage, such structural modifications
of ACA could potentially serve as a platform to find better
candidates which can be used in combination regimens during
preclinical developmental studies. This study demonstrates
that there is still room for improvement in designing modified
ACA analogs with increased anticancer activities.
Disclosure
The authors report no conflicts of interest in this work.
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tion of human breast cancer cells. Overall, these preliminary
results revealed that the anti-proliferative activities are not
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Supplementary materials
Table S1 ic20 values for aca and its analogs on MDa-MB-231 cell line
IC20 (µM)
ACA
2
5
6
7
8
AEA
AMCA
19
20
2.0
48.0
32.0
50.0
31.0
50.0
4.0
12.0
28.0
45.0
Note: ic20 for each analog was used to treat MDa-MB-231 cells for wound-healing assay.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; ic20, 20% inhibitory concentration.
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Figure S1 representative pictures of wound-healing assay of MDa-MB-231 cells after various treatments of aca and its analogs at ic20.
Notes: The open wound area at 24 h was quantified using TScratch software relative to wound area at 0 h. All data are presented as mean ± seM from three independent
replicates, with representative images at 40× magnification shown from each treatment group.
Abbreviations: aca, 1′S-1′-acetoxychavicol acetate; aea, 1′-acetoxyeugenol acetate; aMca, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMsO, dimethyl sulfoxide;
ic20, 20% inhibitory concentration; seM, standard error of the mean.
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