Synthesis of an anthraquinone derivative (DHAQC) and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line

Article abstract of DOI:10.2147/DDDT.S65468

Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the FriedelCrafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC₅₀ on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC₅₀ dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line.

Full text of DOI:10.2147/DDDT.S65468

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Open ꢄccess FullText ꢄrticle
O ꢀ ꢁ ꢂ ꢁ ꢃ ꢄ ꢅ ꢀ ꢆ ꢇ ꢆ ꢄ ꢀ ꢈ ꢉ
ꢇynthesis of an anthraquinone derivative (DꢉꢄQꢈ)
and its effect on induction of ꢂ2/M arrest
and apoptosis in breast cancer MꢈF-7 cell line
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
17 February 2015
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ꢇweeKeong Yeap
Abstract: Anthraquinones are an important class of naturally occurring biologically active
compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic
acid (DHAQC) (2) was synthesized with 32% yield through the Friedel–Crafts condensation reac-
tion. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were
further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity
in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal.
DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability)
value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of
the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2)
was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7
cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2)
arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1
genes.Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome
c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexinV/propidium
iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic,
and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which
demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line.
Keywords: cytotoxic, selective index, cell cycle
Muhammad ꢃadeem
2
ꢄkhtar
3
Kian ꢅam ꢅim
ꢃadiah ꢄbu
Wan Yong ꢉo
4
,5
6
2
ꢇeema Zareen
1
Kiarash ꢀoohani
4
ꢉuynh Ky
1
ꢇheau Wei Tan
ꢃordin ꢅajis
7
1
,4
ꢃoorjahan Banu ꢄlitheen
1
ꢁ
nstitute of Bioscience, Universiti
Putra Malaysia, ꢇelangor Darul
ꢆhsan, Malaysia; Faculty of ꢁndustrial
ꢇciences and Technology, Universiti
Malaysia Pahang, Kuantan, Pahang,
Malaysia; Faculty of Medicine and
ꢉealth ꢇciences, Universiti Tunku
ꢄbdul ꢀahman, ꢇelangor Darul ꢆhsan,
Malaysia; Faculty of Biotechnology
2
3
4
and Biomolecular ꢇciences, Universiti
Putra Malaysia, ꢇelangor Darul
ꢆhsan, Malaysia; Bright ꢇparks Unit,
Introduction
5
Cancer is one of the most major health problems, according to any frame of reference,
and, unfortunately, is not likely to lose that qualification any time soon. The breast is
one of the four major cancer sites, and, of all cancers, breast cancer has the highest
University of Malaya, Kuala ꢅumpur,
Malaysia; ꢇchool of Biomedical
6
ꢇciences, University of ꢃottingham
Malaysia ꢈampus, ꢇelangor Darul
1
7
incidence rate and is the second highest cause of death in females. Thus, research in
ꢆhsan, Malaysia; ꢇcientific ꢈhairs Unit,
Taibah University, Medina, ꢇaudi ꢄrabia
medicinal chemistry has been focused on the discovery of novel anticancer compounds,
including natural compounds that can specifically target cancer cells and minimize the
2
possible side effects on normal cells. Anthraquinones are potential natural anticancer
compounds that can be isolated from Damnacanthus and Morinda spp. Among the
natural anthraquinones, damnacanthal has been shown to possess potential cytotoxic,
ꢈorrespondence: ꢃoorjahan Banu
ꢄlitheen
Faculty of Biotechnology and
Biomolecular ꢇciences, Universiti Putra
Malaysia, UPM 43400 ꢇerdang, ꢇelangor
Darul ꢆhsan, Malaysia
3
4
immunomodulatory, and anticancer activities. These cytotoxic and apoptotic activi-
ties against breast cancer cell lines were found to be regulated by damnacanthal via
5
activation of p53 and p21 genes. Other naturally occurring anthraquinones have also
been shown to exhibit similar anti-inflammatory, antibiotic, antiviral, and antineoplastic
Tel +60 3 8946 7471
6
–8
activities. However, due to the lack of accessibility, the time and cost of preparation,
and the limited quantity of naturally occurring anthraquinones, chemically synthesized
Fax +60 3 8946 7510
ꢆmail noorjahan@upm.edu.my
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License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
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Yeap et al
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compounds have garnered a great deal of attention as a in KBr disk: 3450-, 3230 (OH), 2973 (CH), 1646 (H-C
=O),
means of complementing the natural isolated compounds in 1670 (non-chelated C
=
O), 1466 (C
1
=
C, aromatic), 1244,
9
anticancer drug discovery. Based on this connection, we syn- 1262, 1230, 1132, 710. H-NMR (500 MHz, acetone-d ):
6
thesized the compound 1,3-dihydroxy-9,10-anthraquinone- δ 12.21 (s, OH), 8.22 (d, 1H, J=7.5 Hz, H-8), 8.09 (d, 1H,
1
0
2
-carboxylic acid (DHAQC) (2) by Jones oxidation similar J=7.5 Hz, H-5), 7.75–7.72 (m, 2H, H-6, and H-7), 7.70 (s,
+
to the previously synthesized compound, 2-hydroxymethyl- 1H, H-4), MS m/z (relative intensity): 284.20 (M , 242),
1
,3-dimethoxy-9,10-anthraquinone (1).
281 (45), 226 (56), 254 (79), 206 (7), 196 (24), 180 (57),
One of the major obstacles in anticancer compound 77, 65, 54.
discovery is that the candidate compounds could have
nonspecific toxicity against both cancerous and normal
ꢈell culture
1
1
Human erythromyeloblastoid leukemia K562 cells,
estrogen-dependent breast cancer MCF-7 cells, estrogen-
independent breast cancer MDA-MB-231 cells, and normal
breast MCF-10A cells were purchased fromAmerican Type
Culture Collection ([ATCC] Manassas, VA, USA). K562
and MCF-7 cells were cultured in Roswell Park Memorial
Institute (RPMI)-1640 (Sigma-Aldrich Co, St Louis, MO,
USA) and supplemented with 10% fetal bovine serum (FBS)
cells. Thus, identification of a novel compound with high
selectivity against cancerous cells rather than normal cells
is one of the major objectives of this type of study. The cur-
rent study aimed to evaluate and compare the selectivity of
DHAQC (2) to damnacanthal on cancerous and normal cell
lines. Moreover, the cell cycle arrest and apoptotic effects of
DHAQC (2) on MCF-7 cells was also evaluated by quantita-
tive polymerase chain reaction (PCR), Western blot, and flow
cytometry analysis.
(
GE Healthcare, Little Chalfont, UK), while MDA-MB-231
cells were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) (Sigma-Aldrich Co) supplemented with 10% FBS.
MCF-10A cells were cultured in DMEM/F-12 (Sigma-Al-
drich Co) supplemented with 10% FBS, hydrocortisone (0.5
µg/mL), insulin (10 µg/mL), and human epidermal growth
factor (20 ng/mL) (Sigma-Aldrich Co). All cell lines were
Materials and methods
ꢇynthesis of DꢉꢄQꢈ (2)
Damnacanthal was synthesized according to the methods
1
2
of Akhtar et al. Compound (1) (2-hydroxymethyl-1,3-
dimethoxy-9,10-anthraquinone) was synthesized as described
1
2
kept in a 37°C incubator with 5% CO2.
in our previous publication. Briefly, the phthalic anhydride
and 1,3-dihydroxy-2-methylbenzene were mixed in a molten
3
-(4,5-Dimethylthiazol-2-yl)-2,5-
mixture ofAlCl /NaCl.Then, the product was acetylated with
3
diphenyltetrazolium bromide (MTT)
acetic anhydride and potassium carbonate. Next, the product
underwent methylation with K CO /(CH ) SO , which was cell viability assay
2
3
3
2
4
further brominated with Wohl-Ziegler’s reaction. Finally, MCF-7, MDA-MB-231, and MCF-10A cells were seeded
5
the product was hydrolyzed in acetic acid and water (8:2) in 96-well plates (0.8×10 cells/mL) and cultured overnight.
to yield compound (1). For the synthesis of compound (2), Then, starting at 30 µg/mL, serial 1:1 dilutions of the exam-
DHAQC (2), compound (1) (500 mg, 1.7 mmol) was dis- ined compounds were added to triplicate wells on the seeded
5
solved in 30 mL of acetone in a round bottle flask equipped plates. The nonadherent K562 cells were loaded (0.8×10
with a CaCl drying tube.The reaction mixture was stirred at cells/mL) on the 96-well plate, which contained the appro-
2
0°C–5°C in an ice bath. Jones reagent was prepared by mixing priate dilutions of compounds. All plates were incubated for
CrO + H SO at 0°C in acetone.After 2 hours of storage in a 48 hours at 37°C, 5% CO .After the incubation period, 20 µL
3
2
4
2
refrigerator, 5 mL of the Jones reagent was diluted with water of MTT (5 mg/mL) solution (Sigma-Aldrich Co) was added
and added slowly under nitrogen atmosphere until the yellow to all wells and incubated in darkness for 3 hours. Then,
solution turned greenish. The product was treated with 5% 170 µL of solution was discarded and 20 µL of dimethyl
solution of sodium bisulfite, extracted with ethyl acetate, and sulfoxide (DMSO) was added to each well to solubilize the
purified by column chromatography.The compound DHAQC purple crystals. The plates were read using a microplate
(
(
2) was analyzed by electron ionization mass spectrometry reader at 570 nm (BioTek Instruments, Inc., Winooski, VT,
EI-MS), infrared (IR), and nuclear magnetic resonance USA). IC (inhibitory concentration with 50% cell viabil-
5
0
spectroscopic (NMR) techniques. Compound (2) was ity) values, indicating the concentration of DHAQC (2) or
obtained as a yellow-green amorphous powder and purified damnacanthal that inhibited 50% of cell viability compared
-1
by column chromatography. The yield was 32.0%. IR (cm ) to untreated control, were obtained, and selective index was
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ꢈytotoxicity of DꢉꢄQꢈ
Table 1 Quantitative real-time polymerase chain reaction primers
designed for quantification of FOXM1, WEE1, p21, and PLK1 genes
calculated by dividing the IC value of MCF-10A cells by
5
0
the IC value of MCF-7 or MDA-MB-231 cells.
5
0
Accession
number
Gene
Sequence
ꢈell preparation
MCF-7 cells were seeded overnight and treated with the IC
ꢃM_001220778.1 p21
F: 5-TꢂTꢈꢈꢂTꢈꢄꢂꢄꢄꢈꢈꢈꢄTꢂꢈ-3
5
0
ꢀ: 5-ꢄꢄꢄꢂTꢈꢂꢄꢄꢂTTꢈꢈꢄTꢈꢂꢈTꢈ-3
value of DHAQC (2) for 24 hours for quantitative real-time
PCR (qRT-PCR) and with IC25 (inhibitory concentration
that inhibits 25% of cell viability) (3.50 µg/mL) and IC50
ꢃM_005030.3 PLK1
F: 5-ꢈꢈTꢂꢈꢄꢈꢈꢂꢄꢄꢄꢈꢈꢂꢄꢂTTꢄT-3
ꢀ:5-ꢈꢈꢂTꢈꢄTꢄTTꢈꢂꢄꢈTTTꢂꢂTTꢂꢈ-3
ꢃM_001243089.1 FOXM1 F: 5-ꢄTꢄꢈꢂTꢂꢂꢄTTꢂꢄꢂꢂꢄꢈꢈꢄꢈT-3
: 5-TꢈꢈꢄꢄTꢂTꢈꢄꢄꢂTꢄꢂꢈꢂꢂTTꢂ-3
F: 5′-ꢄꢂꢄꢂꢈTꢄꢈꢂꢄꢂꢈTꢂꢈꢈTꢂꢄꢈ-3′
: 5′-ꢄꢂꢈꢄꢈTꢂTꢂTTꢂꢂꢈꢂTꢄꢈꢄꢂ-3′
18srRNA F: 5-ꢂTꢄꢄꢈꢈꢈꢂTTꢂꢄꢄꢈꢈꢈꢈꢄTT-3
: 5-ꢈꢈꢄTꢈꢈꢄꢄTꢈꢂꢂTꢄꢂTꢄꢂꢈꢂ-3
F: 5-ꢂꢂꢄTTTꢂꢂTꢈꢂTꢄTTꢂꢂꢂꢈ-3
: 5-TꢂꢂꢄꢄꢂꢄTꢂꢂTꢂꢄTꢂꢂꢂꢄTT-3
ꢀ
(
7.20 µg/mL) of DHAQC (2) for 48 hours for flow cytom-
ꢃM_001101.3
ꢉQ387008.1
ꢃM_002046.4
ACTB
etry, Western blot, and caspase-9 analysis. Untreated control
cells were prepared simultaneously. After the treatment
period, cells were harvested, washed, and subjected to the
following assays.
ꢀ
ꢀ
GAPDH
ꢀ
Note: ACTB, 18srRNA, and GAPDH were housekeeping genes used for normalization
of the target genes.
Abbreviations: F, forward; ꢀ, reverse.
Flow cytometry cell cycle Pꢁ (propidium iodide) assay
Treated and untreated MCF-7 cells were stained with BD
TM
Cycletest Plus DNA reagent kit (BD, Franklin Lakes, NJ,
target genes (p21, PLK1, and FOXM1) were generated from
the amplification of the serially diluted RNA samples isolated
from the control MCF-7 cell line to optimize the assay. Each
of the, treated and untreated samples were run in triplicate and
non-templatecontrolwasincluded. Normalizationofreference
USA) according to the manufacturer’s protocol. The cell
cycle phase was analyzed using BD CellQuest Pro software
by FACSCalibur™ (BD, Franklin Lakes, NJ, USA).
Flow cytometry annexinV/Pꢁ assay
13
genes was done using geNorm.
Treated and untreated MCF-7 cells were mixed with 100
µL of 1× binding buffer. Then, 5 µL of annexin V and PI,
each from the Annexin V-FITC apoptosis detection kit (BD
Biosciences, San Jose, CA, USA), were added to the cells
and incubated for 15 minutes.Afterwards, stained cells were
washed and mixed with 500 µL of 1× binding buffer, and the
apoptosis of the cells was analyzed using BD CellQuest Pro
software by FACSCalibur.
Western blot analysis
Protein was extracted from IC and IC DHAQC (2)-treated
25
50
and untreated control cells using radioimmunoprecipitation
assay buffer, and the protein concentration was determined
using Bradford protein assay (Thermo Fisher Scientific).The
cell homogenates were electrophoresed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
(Bio-Rad Laboratories, Inc., Hercules, CA, USA), transferred
qꢀT-Pꢈꢀ assay
RNA from the treated and untreated MCF-7 cells was onto nitrocellulose membrane, blocked with Tris-buffered
extracted using the RNeasy Plus Mini Kit (Qiagen NV, Venlo, saline–Tween buffer containing 5% skimmed milk powder
the Netherlands) according to the manufacturer’s protocol.The for 1 hour, and incubated with primary antibodies (anti-p53,
RNA quality was quantified using a NanoDrop spectrophotom- anti-BAX, anti-Bcl-2, anti-cytochrome c, and anti-β-actin at
eter (Thermo Fisher Scientific,Waltham, MA, USA).The RNA dilution 1:1000) (Abcam plc, Cambridge, UK) for another
TM
was converted to complementary (c)DNA using an iScript
hour. Then, membranes were washed and incubated with
cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, the anti-rabbit secondary antibodies-conjugated with alka-
USA). Expression of p21, PLK1, and FOXM1 were evaluated line phosphatase (1:5000 dilution) for 1 hours, followed by
®
by qRT-PCR with an SYBR Select Master Mix (Thermo washing with Tris-buffered saline–Tween buffer, and finally
®
Fisher Scientific, Waltham, MA, USA) on an iQ-5 Real Time visualized by chemiluminescence with CDP-Star reagent
®
PCR (Bio-Rad Laboratories Inc.) using the primers as stated in (NEB [UK], Hitchin, UK) using a BioSpectrum system
Table 1 and the following PCR conditions: one cycle of 50°C (UVP, Upland, CA, USA). The size of the targeted proteins
for 2 minutes for UDG (uracil-DNA glycosylase) activation; was determined by protein marker (PageRuler Prestained
one cycle of 95°C for 2 minutes for DNA polymerase activa- Protein Ladder; Thermo Fisher Scientific), and the density
tion; 40 cycles of 95°C for 2 seconds for denature; and 60°C results obtained from this targeted protein were analyzed using
®
for 30 seconds for annealing and extension. Standard curves VisionWorks LS analysis software (Version 7.1 RC3.54;
for housekeeping genes (β-actin, 18srRNA, and GAPDH) and UVP) by normalizing against β-actin.
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Caspase-9 fluorescence assay and viability
in the presence of general caspase inhibitor
Table 2 The ꢁꢈ50 values of DꢉꢄQꢈ (2) and damnacanthal in
K562, MꢈF-7, MDꢄ-MB-231, and MꢈF-10ꢄ cell lines after 48
hours of treatment
MCF-7 cells were pretreated with or without cell perme-
able general caspase inhibitor (Z-VAD-FMK) (Promega
Corporation, Fitchburg, WI, USA) at 10 µM before treatment
with IC and IC of DHAQC (2) for 48 hours. After the treat-
Cell line
DHAQC (2) Damnacanthal
(
μg/mL)
9.10±1.16
.20±1.00
(μg/mL)
K562
6.20±1.32
4.30±0.67
7.13±1.38
6.51±1.65
1.51
25
50
MꢈF-7
7
ment period, viability of DHAQC (2)-treated MCF-7 cells with
or without Z-VAD-FMK was quantified using MTT assay as
described above.Then, protein was extracted from IC and IC
50
MDꢄ-MB-231
MꢈF-10ꢄ
9.94±1.25
24.15±1.13
ꢇelective index of MꢈF-10ꢄ/MꢈF-7 3.35
25
ꢇelective index of MꢈF-10ꢄ/MDꢄ-
MB-231
2.43
0.91
DHAQC (2)-treated and untreated control cells with or without
Z-VAD-FMK using radioimmunoprecipitation assay buffer,
and the protein concentration was determined using Bradford
protein assay. Levels of activated caspase-9 were detected using
CaspGLOW Red Active Caspase-9 Staining Kit (BioVision,
Note: ꢇelective indexes on breast cancer cells were calculated based on ꢁꢈ50 of
MꢈF-10ꢄ normal cells versus MꢈF-7 and MDꢄ-MB-231 cells.
Abbreviations:DꢉꢄQꢈ,1 ,3-dihydroxy-9,10-anthraquinone-2-carboxylicacid;ꢁꢈ ,
5
0
inhibitory concentration with 50% cell viability.
Inc., Milpitas, CA, USA) according to the manufacturer’s contributor to the cytotoxicity properties.The planar tricyclic
protocol. The fluorescence intensity in DHAQC (2)-treated structure of anthraquinone is essential for cytotoxic activi-
15
and untreated MCF-7 cells was measured at excitation (Ex)/ ties and has been widely investigated for cancer therapy.
emission (Em) =540/570 nm using a microplate fluorometer Anthraquinone derivatives such as doxorubicin, daunorubi-
Arzoskan Flash; Thermo Fisher Scientific).
cin, and carminomycin are highly effective in the treatment
(
1
6
of tumors. Cytotoxicity of DHAQC (2) on K562, MCF-7,
and MCF-10A cell lines was evaluated using the MTT assay,
ꢇtatistical analysis
Data were presented as mean ± standard deviation from three and IC value of these cell lines after 48 hours of treatment
5
0
experiments in three different biological replicates, and the were obtained (Table 2). The selectivity of DHAQC (2)
significance (P,0.05) was evaluated by one-way analysis and damnacanthal on normal and cancerous cell lines was
of variance (ANOVA) with Duncan test as post hoc analysis calculated by dividing the IC on normal MCF-10A cells
5
0
using SPSS software (v 14; SPSS Inc., Chicago, IL, USA).
by that of either estrogen-dependent MCF-7 or estrogen-
independent MDA-MB-231 cells. Both DHAQC (2) and
damnacanthal showed the highest cytotoxicity against MCF-7
Results and discussion
Chemical modifications in the side chain are one common compared to the other cell lines. Further, the IC values of
5
0
approach in drug discovery for improving the therapeutic DHAQC (2) and damnacanthal in MCF-7 and K562 were
potential of a certain compound. Our main interest was to lower compared to the precursor, compound (1) with IC50
1
2
improve the yield of damnacanthal using different kinds of values of 12.80 and 14.00 µg/mL, respectively. Previously,
oxidizing agents, such asAgO/HNO ,Vilsmeier reagent, and we demonstrated the contribution of methoxy, CHO groups,
3
14
Lewis acid. None of the methods gave a reasonable yield and and OH group to the cytotoxicity of anthraquinones against
1
4
12
most gave oxidative demethylated products. However, the tested cancerous cell lines. DHAQC (2) possesses two OH
oxidation approach of a previously synthesized compound, groups and one carboxylic acid (COOH) group, which gives
1
2
2
-hydroxymethyl-1,3-dimethoxy-9,10-anthraquinone (1),
it a greater hydrophilic character compared to damnacanthal.
with Jones reagents resulted in a 32% yield of DHAQC (2) The presence of hydroxyl and acidic groups in DHAQC (2)
Figure 1).
increases the solubility and might facilitate absorption or
Both damnacanthal and DHAQC (2) contain the diffusion across the cellular membrane, as revealed by the
10
(
anthraquinone planer basic skeleton, which is the major theoretical membrane-interaction quantitative structure–
activity relationship (MI-QSAR) studies of 188 drug-like
1
7,18
O
O
OH
compounds. The better selectivity of DHAQC (2) toward
cancer cell lines is possibly a result of its hydrophilic charac-
ter, which might fulfill Lipinski’s rule of five and have drug-
O
OCH3
COOH
OH
CH OH
2
+
0
.25 mole% CrO / H
3
OCH3
O
19
2
like properties. Many drugs, for instance, mitoxantrone,
1
possessing 1,4 hydroxyl groups have demonstrated potent
antitumor activity and have been used widely in clinic since
Figure
1
1
ꢇynthesis of DꢉꢄQꢈ (2) from 2-hydroxymethyl-1,3-dimethoxy-9,
0-anthraquinone (1).
2
0,21
Abbreviation: DꢉꢄQꢈ, 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid.
1980. Moreover, the hydrophilic property of DHAQC (2)
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ꢈytotoxicity of DꢉꢄQꢈ
A
B
C
4
.48%±3.72%
2ꢀ.3ꢀ%±3.18%
G2+M
3
7.66%±4.11%
G2+M
G0/G1
.38%±0.73%
G0/G1
0.13%±1.36%
G2+M
G0/G1
20.48%±2.ꢀ1%
1
1
SubG0/G1
SubG0/G1
SubG0/G1
S
S
S
2
7.45%±2.66%
1
1.76%±1.31%
21.33%±1.74%
0
200
400
600
0
200
400
600
0
200
400
600
DNA content
DNA content
DNA content
D
E
F
Late apoptosis:
16.36%±3.72%
Late
apoptosis:
Late
apoptosis:
1.28%±1.ꢀ3%
8.1ꢀ%±2.17%
Early
apoptosis:
Early
apoptosis:
2ꢀ.35%±3.81%
Early
apoptosis:
1.44%±1.36%
27.81%±3.14%
10
0
10
1
10
2
10
3
10
4
10
0
1
10
2
10
3
10
4
10
10
0
10
1
10
2
10
3
10
4
Annexin V-FITC
Annexin V-FITC
Annexin V-FITC
Figure 2 ꢈell cycle progression (A–C) and apoptosis (D–F) of DꢉꢄQꢈ (2)-treated (ꢁꢈ25 and ꢁꢈ50 values) and untreated MꢈF-7 cells after 48 hours of incubation.
Notes: (A–C) indicate cell cycle phases of untreated control, ꢁꢈ25 of DꢉꢄQꢈ (2)-treated MꢈF-7 cells, and ꢁꢈ50 of DꢉꢄQꢈ (2)-treated MꢈF-7 cells, while (D–F) indicate
apoptosis of untreated control, ꢁꢈ25 of DꢉꢄQꢈ (2)-treated MꢈF-7 cells, and ꢁꢈ50 of DꢉꢄQꢈ (2)-treated MꢈF-7 cells by annexin V/propidium iodide apoptosis assay,
respectively.
Abbreviations: DꢉꢄQꢈ, 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid; FITC, fluorescein isothiocyanate; IC , inhibitory concentration with 25% cell viability; ꢁꢈ ,
25
50
inhibitory concentration with 50% cell viability.
may also be involved in it’s chelating with magnesium and cells, further evaluation on cell cycle regulation, apoptosis,
1
5
zinc ions in DNA synthesis and the repair process. Added gene expression, and apoptosis-related protein levels were
to this, the hydrophilicity or introduction of acidic groups evaluated. The cell cycle regulation and apoptotic effects
increased the hydrophilicity of the rings and facile to bind were evaluated using flow cytometry after 48 hours of
with receptor. It is well known that the polycyclic (aromatic) treatment. Many cytotoxic agents arrest the cell cycle
structure of anthraquinone can intercalate with DNA in
between base pairs, either covalently or electrostatically.This
3
DNA intercalation with anthraquinone compounds possibly
DHAQC (2) IC50
2
1
2
2
inhibits the DNA replication in cancer cell lines. Previous
research found that the position but not the total amount
of hydroxyl in anthraquinone played an important role in
determining its toxicity against normal and cancerous cell
lines in vitro. Thus, modification of the hydroxyl group in
DHAQC (2) might reduce its toxicity to normal cells while
maintaining its apoptosis-inducing effect on cancer cells,
thus leading to greater selectivity of DHAQC (2) compared
0
1
p21
−
−
2
−3
−4
23
to that of damnacanthal. As a result, the slight modification
in natural compounds, as in DHAQC (2), could be useful for
the development of new prototypes of anthraquinone-type
cytotoxic agents.
Genes of interest
Figure 3 ꢆxpression of p21, PꢅK1, and FOXM1 after 24 hours of ꢁꢈ50 DꢉꢄQꢈ
2)-treated compared to untreated MCF-7 cells quantified by quantitative real-time
(
polymerase chain reaction assay.
Notes: ꢄll data are mean ± standard deviation of triplicate. *Indicates significant fold
change (.twofold) compared to control, P-value ,0.05.
Abbreviations:DꢉꢄQꢈ,1 ,3-dihydroxy-9,10-anthraquinone-2-carboxylicacid;ꢁꢈ ,
Since DHAQC (2) showed the highest sensitivity and
selectivity on hormone-dependent breast cancer MCF-7
5
0
inhibitory concentration with 50% cell viability.
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A
DHAQC (2)
IC25 IC50
M
C
3
0 kDa
0 kDa
Bcl-2
BAX
~26 kDa
2
5
0 kDa
0 kDa
~
~
48k Da
53 kDa
4
6
0 kDa
0 kDa
p53
5
Cytochrome c
~12 kDa
5
0 kDa
β-actin
~47 kDa
4
0 kDa
B
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
DHAQC (2) IC25
DHAQC (2) IC50
p53
Cytochrome c
BAX
Bcl-2
Targeted proteins
C 10
9
8
7
6
5
4
3
2
1
0
Control
DHAQC (2) IC25
DHAQC (2) IC50
Groups
Figure 4 (A) ꢀepresentative plots of Bcl-2, BꢄX, p53, cytochrome c, and β-actin (ꢄꢈTB) of untreated, ꢁꢈ25 DꢉꢄQꢈ (2)-treated, and ꢁꢈ50 DꢉꢄQꢈ (2)-treated MꢈF-7
cells after 48 hours. (B) Fold change of ꢁꢈ25 DꢉꢄQꢈ (2)-treated and ꢁꢈ50 DꢉꢄQꢈ (2)-treated MꢈF-7 cells compared to untreated MꢈF-7 cells. (C) ꢀatio of BꢄX/Bcl-2 for
untreated, ꢁꢈ25 DꢉꢄQꢈ (2)-treated, and ꢁꢈ50 DꢉꢄQꢈ (2)-treated MꢈF-7 cells after 48 hours. *Indicates significant fold change (P,0.05) compared to control.
Abbreviations: ꢈ, control; DꢉꢄQꢈ, 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid; M, protein marker; ꢁꢈ , inhibitory concentration with 25% cell viability; ꢁꢈ ,
25
50
inhibitory concentration with 50% cell viability.
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ꢈytotoxicity of DꢉꢄQꢈ
of cancerous cells at the S and G2/M phases, followed apoptosis results were similar to those of our previous study,
2
4
by the induction of apoptosis. DHAQC (2) arrested wherein damnacanthal was found to induce G2/M arrest,
5
MCF-7 at the S and G2/M phases (21.33%±1.74% and DNA fragmentation, and apoptosis toward MCF-7 cells.
3
7.66%±4.11%, respectively) as compared to the control
11.76%±1.31% and 4.48%±%3.72%, respectively) MCF-7 cells, expression of p21, PLK1, and FOXM1 genes
after 48 hours of incubation. DHAQC (2) also induced involved in G2/M arrest was evaluated using qRT-PCR. PLK1
0.48%±2.91% of the MCF-7 cell population to enter the is a key regulator of G2/M phase progression, while forkhead
Since cell cycle arrest was observed in DHAQC (2)-treated
(
2
subG0 phase, which indicated the occurrence of DNA boxM1(FOXM1)isadirectbindingpartnerofPLK1,FOXM1
fragmentation in the DHAQC (2)-treated cells. This result plays an important role in regulating the mitotic entry and
25
was further supported by the annexin V/PI apoptosis study, expression of clusters of G2/M target genes. Downregulation
wherein DHAQC (2) was able to induce 16.36%±3.72% of PLK1 and FOXM1 expression are always associated with
26
of cells to undergo late apoptosis and 29.35%±3.81% to G2/M arrest and apoptosis (Kim et al, 2012). On the other
undergo early apoptosis (Figure 2). These cell cycle and hand, upregulation of p21 was found to arrest the cell cycle
A
1
20
00
x
x
Control
1
+Z-VAD-FMK
y
y
8
6
4
2
0
0
0
0
0
zz
z
Control
DHAQC (2) IC25
DHAQC (2) IC50
Groups
B
3
c
Control
2.5
+Z-VAD-FMK
b
b
2
a
1
0
.5
1
a
a
.5
0
Control
DHAQC (2) IC25
DHAQC (2) IC50
Groups
Figure 5 ꢈell viability (A) and active caspase-9 fold change (B) of ꢁꢈ25 DꢉꢄQꢈ-treated and ꢁꢈ50 DꢉꢄQꢈ-treated MꢈF-7 cells with or without general caspase inhibitor
Z-VꢄD-FMK.
Note: Different lowercase letters indicate significant fold change (P,0.05) of standard error of the mean ± standard deviation compared to control.
Abbreviations: DꢉꢄQꢈ, 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid; ꢁꢈ , inhibitory concentration with 25% cell viability; ꢁꢈ , inhibitory concentration with 50%
25
50
cell viability.
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in the G2/M phase as reported in damnacanthal-treated and cytochrome c, but had no dose-dependent effect on Bcl-2
5
MCF-7 cells. Based on the qRT-PCR results, DHAQC (2) (Figure 4). The ability of DHAQC (2), as an anthraquinone, to
was able to inhibit the expression of PLK1 significantly. bind and chelate the DNA may contribute to enhancement of
28
Further, expression of p21 and FOXM1 were up- and down- p53 levels. Furthermore, this DNA binding ability may be fur-
regulated, respectively, although it is not significant (Figure 3). therenhancedbytheincreasedhydrophilicpropertyofDHAQC
Depletion of PLK1 gene was also reported to subsequently (2). p53 is a tumor suppressor protein that plays an important
27
induce apoptosis in cancer cells (Liu and Erikson, 2003).
role in regulating G2/M arrest and apoptosis via upregulation of
Thus, significant PLK1 downregulation by DHAQC (2) con- p21, transcriptional target of p53, and multidomain proapoptotic
tributed to the G2/M arrest and apoptosis in MCF-7 cells as protein BAX. BAX is essential for the onset of mitochondrial
observed in the flow cytometry analysis.
permeabilization and apoptosis via the intrinsic pathway. The
Besides regulation of G2/M arrest-related genes, effects balance between expression levels of antiapoptotic Bcl-2 and
of DHAQC (2) on apoptosis-related proteins, including Bcl-2, proapoptotic BAX is crucial for cell survival or death, wherein
BAX, p53, and cytochrome c, were also evaluated by Western increase of the BAX/Bcl-2 ratio will activate the release of
blot analysis. After 48 hours of treatment, both IC and IC
50
cytochrome c from mitochondria and subsequently activate the
25
29
of DHAQC (2) were able to enhance the levels of p53, BAX, intrinsic apoptotic pathway. Induction of BAX increased the
O
O
OH
Activated
COOH
OH
Inhibited
DHAQC (2)
Up-regulated
Down-regulated
p53
p21
BAX/Bcl-2
PLK 1
FOXM1
Cytoc c
3
ꢂ.66±4.11ꢁ
Casp 9
G0/G1
0.4ꢀ±2.91ꢁ
G2/M
2
SubG0/G1
Late appotosis:
6.36±3.ꢂ2ꢁ
1
S
21.33±1.ꢂ4ꢁ
Early
appotosis:
29.35±3.ꢀ1ꢁ
SC
10
4
0
200
400
600
DNA content
10
0
10
1
10
2
10
3
Annexin V-FITC
Count
Figure 6 Proposed mechanisms of DꢉꢄQꢈ (2)-induced ꢂ2/M phase arrest and apoptosis in MꢈF-7 cells.
Abbreviations: ꢈasp9, caspase-9; ꢈytoc c, cytochrome c; DꢉꢄQꢈ, 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid; FITC, fluorescein isothiocyanate.
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ꢈytotoxicity of DꢉꢄQꢈ
BAX/Bcl-2 ratio, which subsequently enhanced the release of data. All authors took part in either drafting the article or
cytochrome c from the mitochondria (Figure 4) and increased revising it critically for important intellectual content. All
the level of activated caspase-9 (Figure 5), which indicates authors gave final approval of the version to be published.
24
the activation of the intrinsic apoptotic pathway. Similarly to
30
another anthraquinone derivative, dantron, stimulation of acti- Disclosure
vated caspase-9 and reduction of cell viability was suppressed The authors report no conflicts of interest in this work.
in the presence of general caspase inhibitor Z-VAD-FMK.
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