Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line

Article abstract of DOI:10.2147/DDDT.S102164

Known as naturally occurring biologically active compounds, flavokawain A and B are the leading chalcones that possess anticancer properties. Another flavokawain derivative, (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one

Full text of DOI:10.2147/DDDT.S102164

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O ꢀ ꢁ ꢂ ꢁ ꢃ ꢄ ꢅ ꢀ ꢆ ꢇ ꢆ ꢄ ꢀ ꢈ ꢉ
Flavokawain derivative Fꢅꢇ induced ꢂ2/M arrest
and apoptosis on breast cancer MꢈF-7 cell line
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ꢃorlaily Mohd ꢄli
Abstract: Known as naturally occurring biologically active compounds, flavokawain A and
2
M ꢃadeem ꢄkhtar
B are the leading chalcones that possess anticancer properties. Another flavokawain derivative,
3
(
E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one (FLS)
ꢉuynh Ky
1
1
was characterized with H-nuclear magnetic resonance, electron-impact mas spectrometry,
Kian ꢅam ꢅim
ꢃadiah ꢄbu
1
4
infrared spectroscopy, and ultraviolet ( H NMR, EI-MS, IR, and UV) spectroscopic techniques.
2
FLS cytotoxic efficacy against human cancer cells (MCF-7, MDA-MB-231, and MCF-10A)
ꢇeema Zareen
Wan Yong ꢉo
resulted in the reduction of IC values in a time- and dose-dependent mode with high specificity
5
50
on MCF-7 (IC50 of 36 μM at 48 hours) against normal breast cell MCF-10A (no IC50 detected
up to 180 μM at 72 hours). Light, scanning electron, and fluorescent microscopic analysis of
MCF-7 cells treated with 36 μM of FLS displayed cell shrinkage, apoptotic body, and DNA
fragmentation. Additionally, induction of G2/M cell arrest within 24 hours and apoptosis at
subsequent time points was discovered via flow cytometry analysis. The roles of PLK-1, Wee-1,
and phosphorylation of CDC-2 in G2/M arrest and proapoptotic factors (Bax, caspase 9, and
p53) in promotion of apoptosis of FLS against MCF-7 cells were discovered using fluorometric,
quantitative real-time polymerase chain reaction, and Western blot analysis. Interestingly, the
1
ꢉan Kiat ꢄlan-Ong
6
ꢇheau Wei Tan
4
ꢃoorjahan Banu ꢄlitheen
2
Jamil bin ꢁsmail
6
ꢇwee Keong Yeap
7
Tunku Kamarul
1
Faculty of Medicine and ꢉealth
ꢇciences, Universiti Tunku ꢄbdul
presence of SCH (thiomethyl group) on ring B structure contributed to the selective cytotoxic-
3
2
ꢀ
ahman, ꢇelangor, Department of
ity against MCF-7 cells compared to other chalcones, flavokawain A and B. Overall, our data
suggest potential therapeutic value for flavokawain derivative FLS to be further developed as
a new anticancer drug.
ꢁ
ꢁ
ndustrial Biotechnology, Faculty of
ndustrial ꢇciences & Technology,
Universiti Malaysia Pahang, Pahang,
3
Malaysia; Department of ꢄgriculture
Keywords: (E)-1-(2′-Hydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-
one (FLS), MCF-7, G2/M arrest, apoptosis, cell cycle, PLK-1, p53, caspase
ꢂenetics and Breeding, ꢈollege of
ꢄgriculture and ꢄpplied Biology,
ꢈantho University, ꢈanTho ꢈity,
4
Vietnam; Department of ꢈell
and Molecular Biology, Faculty of
Biotechnology and Biomolecular
ꢇciences, Universiti Putra Malaysia,
Introduction
Breast cancer is the leading cause of cancer death in females worldwide, accounting
for ~25% (1.68 million cases) of the total new cancer cases and ~14% (521,900 deaths)
5
ꢇchool of Biomedical ꢇciences,The
University of ꢃottingham Malaysia
1
,2
6
of the total cancer deaths in 2012. Among the various types of cancer, breast cancer
ꢈampus, ꢁnstitute of Bioscience,
Universiti Putra Malaysia, ꢇelangor,
Tissue ꢆngineering ꢂroup, ꢃational
Orthopaedic ꢈentre of ꢆxcellence for
is the most frequently diagnosed and the most common cancer that affects Malaysian
7
3
women from all ethnicities. According to recent reports, an estimated 232,670
ꢀ
esearch and ꢅearning, Department
new cases of breast cancer have been expected and diagnosed among women and
of Orthopaedic ꢇurgery, Faculty of
Medicine, University Malaya, Kuala
ꢅumpur, Malaysia
4,5
about 2,360 new cases in men in the US during 2014. Although numerous efforts
6
have been made to decrease the mortality rate of breast cancer, to-date, no ultimate
chemotherapeutic drug is available to overcome this disease. Thus, effort in search-
ing alternative or better candidates of antibreast cancer compound from natural phy-
ꢈorrespondence: ꢇwee Keong Yeap
ꢁ
nstitute of Bioscience, ꢅebuh ꢇilicon,
7
tochemical or synthetic chemistry is still ongoing.
Universiti Putra Malaysia, 43400 ꢇerdang,
ꢇelangor, Malaysia
A number of studies have reported the broad spectrum bioactivities of chalcones
including antioxidant, cytotoxic, anti-inflammation, antihistaminic, and antimicrobial
Tel +60 3 894 72153
Fax +60 3 894 72153
8
ꢆmail skyeap2005@gmail.com
effects. Flavokawains (either A, B, or C) are among the well-studied chalcones that
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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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ꢄli et al
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were extracted from Piper methysticum. Flavokawain A mixed in a 500 mL round bottle flask and left for 15 minutes
FLA) constituted as the highest amount of flavokawain at room temperature. The crude product was transferred into
(
found in kava extract, while flavokawain B (FLB) is the most separating funnel (500 mL) and then added with 200 mL dis-
well studied flavokawain. Both flavokawain possess various tilled water. The mixture was then acidified with the addition
level of cytotoxicity against several cancer cell line including of 2–3 mL hydrochloride. The product was extracted with
9–12
colon, bladder, and breast. An ideal cytotoxic compound 100 mL ethyl acetate. The organic layer was then successively
should possess good selectivity against the targeted cancer washed with brine and dried over anhydrous sodium sulfate.
cell while inducing limited or no toxicity against normal The crude product was finally purified by flash column chro-
9
cell. Comparing the cytotoxic effect of FLA and FLB, matography with ethyl acetate and hexane (5:5) as eluants and
these two compounds were recorded with different degrees recrystallized in ethyl acetate and drops of MeOH.
of in vitro cytotoxicity against estrogen-dependent MCF-7
MTT cell viability assay
(
FLA: ~25 μM; FLB: ~33 μM) and estrogen-independent
MCF-7, MDA-MB-231, and MCF-10A cells (American
Type Culture Collection [ATCC], Manassas, VA, USA)
were seeded overnight in 96-well plates at a concentration
MDA-MB-231 (FLA: ~17 μM; FLB: ~12 μM) breast cancer
cell lines. However, both flavokawains also exhibited cyto-
toxicity on normal MCF-10A (FLA: ~95 μM; FLB: ~45 μM)
5
9
,10
of 0.8×10 cells/mL at 37°C CO incubator in Roswell Park
breast cell line. Synthesis of bioactive compounds allows
2
Memorial Institute medium-1640, Dulbecco’s Modified
Eagle’s Medium (DMEM), and DMEM-F12 supplemented
with hydrocortisone (0.5 μg/mL), insulin (10 μg/mL), human
epidermal growth factor (20 ng/mL) (Sigma-Aldrich Co.,
St Louis, MO, USA) medium, respectively. Then, respective
cells were treated with FLS (ranging between 180 and 3 μM)
for 24, 48, and 72 hours. MTT solution (5 mg/mL) was added
to all wells 3 hours before end of incubation time. Absorbance
was read at wavelength of 570 nm (BioTek Instruments,
the possibility of modifying the lead compound for better effi-
7
cacy and desired cytotoxic action. For example, Badisa et al
have shown that synthetic piperidinyl-diethylstilbestrol and
pyrrolidinyl-diethylstilbestrol possessed better selectivity
against breast cancer MCF-7 cell than normal breast MCF-
1
0A cell. In this investigation, a flavokawain derivative,
(
E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(4-(methylthio)
phenyl)prop-en-1-one (FLS) was synthesized via a Claisen–
Schmidt condensation reaction. Thus, the aim of this study
was to compare the selectivity of flavokawain derivative
FLS on breast cancer and normal cell lines. In addition, the
regulation of cell cycle and apoptotic inducing effect of FLS
against MCF-7 were also observed.
13
Winooski, VT, USA). Percentage viability of the tested cell
line was calculated using the following formula:
Cell
Absorbance sample
=
×100%
viability(%) Absorbance untreated control
Materials and methods
IC value indicating concentration that inhibited 50% of
5
0
ꢇynthesis of Fꢅꢇ
the tested cell lines was obtained from the graph plotted with
percentage of cell viability against concentration of FLS.
FLSwassynthesizedviaaClaisen–Schmidtcondensationreac-
tion. Acetophenone, 4,6-dimethoxy-2′-hydroxyacetophenone
3
.0 g (9.0 mmol) and 4-(methylthio)benzaldehyde (1.4 mL, MꢈF-7 cell treatment
10 mmol) were dissolved in methanol. A 40% water solu- Based on the MTT assay (Table 1), reduction in IC values of
50
tion of potassium hydroxide 5.0 g and 50 mL methanol was FLS was observed after treatment at 24–72 hours on MCF-7
Table 1 ꢈomparison values of ꢁꢈ50 of Fꢅꢇ, Fꢅꢄ, and FꢅB in MꢈF-7, MDꢄ-MB-231 and MꢈF-10ꢄ cells
9
10
Cell lines
FLS (µM)
4 hours
FLA (µM)
FLB (µM) (Abu et al)
2
48 hours
72 hours
72 hours
72 hours
MꢈF-7
40.90±4.50
63.64±3.80
.180
36.36±3.10
51.52±4.20
.180
33.30±2.50
48.48±3.40
.180
25.13±3.00
17.49±1.50
.100
33.76±1.35
12.31±1.22
45
MDꢄ-MB-231
MꢈF-10ꢄ
ꢇelective index of MꢈF-10ꢄ/MꢈF-7
ꢇelective index of MꢈF-10ꢄ/MDꢄ
.4.40
.4.95
.5.41
3.98
1.33
.2.83
.3.49
.3.71
5.72
3.66
Notes: ꢄdapted from ꢄbu ꢃ, ꢄkhtar Mꢃ, Yeap ꢇK, et al. Flavokawain ꢄ induces apoptosis in MꢈF-7 and MDꢄ-MB231 and inhibits the metastatic process in vitro. PLoS One.
9
2
014;9:e105244. ꢄdapted from ꢄbu ꢃ, ꢄkhtar M, Yeap ꢇ, et al. Flavokawain B induced cytotoxicity in two breast cancer cell lines, MꢈF-7 and MDꢄ-MB231 and inhibited
10
the metastatic potential of MDꢄ-MB231 via the regulation of several tyrosine kinases in vitro. BMC Complem Altern M. 2016;16:86.
Abbreviations: Fꢅꢇ, (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; FLA, flavokawain A; FLB, flavokawain B.
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ꢈytotoxicity of Fꢅꢇ
verifying that FLS effect is time- and dose-dependent. To ꢈell cycle analysis
standardize the treatment, one concentration of FLS (36 μM) Cell cycle progression was evaluated using BD Cycletest Plus
was used for the remaining assays. MCF-7 cell was seeded kit and subjected to BD FACS Calibur flow cytometer (BD,
5
at 0.8×10 cells/mL overnight in 6-well plate. After reaching Franklin Lakes, NJ, USA) analysis. In brief, harvested cell
8
0%–90% confluence, the cell was treated with 36 μM (IC50 was incubated with 250 μL of trypsin buffer for 10 minutes
at 48 hours) of FLS for 24, 48, and 72 hours. Untreated control followed by 200 μL of trypsin inhibitor with RNase buffer.
was prepared simultaneously. After 24, 48, or 72 hours of incu- The samples were finally stained with 200 μL of PI solution
bation, cell was harvested using TrypLE (Thermo Fisher Sci- and analyzed.
entific, Waltham, MA, USA), washed with phosphate-buffered
saline and subjected to the subsequent assays.
ꢄnnexinV/Pꢁ apoptosis study
Apoptosis of cell was evaluated using BD Annexin
V-fluorescein isothiocyanate (FITC)/PI apoptosis detection
kit. In brief, harvested cell was washed and added with
ꢅight and scanning electron microscopic
examination
Cell was subjected to microscopic examination using inverted 100 μL of 1× binding buffer. The cell was then stained with
light microscope (Nikon Corporation, Tokyo, Japan) and 5 μL of Annexin V-FITC and 5 μL of PI solution, incubated
scanning electron microscope (SEM; JEOL, Tokyo, Japan) for 15 minutes and diluted with 400 μL of 1× binding buf-
after 72 hours of treatment. Prior to SEM observation, cell fer. All samples were analyzed with BD FACS Calibur flow
was seeded on poly-l-lysine coated round coverslips in cytometer (Becton Dickinson).
a 6-well plate. After 72 hours, untreated control and FLS
Quantitative reverse transcription real-
treated MCF-7 cell were fixed in 4% glutaraldehyde, washed
with 0.1 M sodium cocodylate buffer, fixed with 1% osmium time Pꢈꢀ assay
tetroxide and underwent series of dehydration in acetone and RNA was extracted from cell using RNeasy mini plus
dried using CO . Then, the samples were mounted onto the kit (Qiagen NV, Venlo, the Netherlands). The purity and
2
stubbing, coated with gold in sputter and viewed under JEOL concentration of the isolated RNA were quantified prior to
SEM under the 15 kV setting.
DNA conversion using iScript cDNA synthesis kit (Bio-Rad
Laboratories Inc., Hercules, CA, USA). Expression of polo-
like kinase 1 (PLK1), WEE1 G2 checkpoint kinase (WEE-1),
c-Jun proto-oncogene (c-Jun), Glucose transporter (GLUT)
ꢄcridine orange/propidium iodide dual
staining
Harvested cell was stained with 10 μg/mL of acridine orange were quantified by quantitative real-time PCR (qRT-PCR)
and propidium iodide (PI) each. The morphology of cell was using SYBR select master mix (Thermo Fisher Scientific)
observed under fluorescent microscope (Nikon) with combi- on iQ-5 real-time polymerase chain reaction (PCR) machine
nation of excitation filter (450–490 nm) and long pass filter (Bio-Rad) with normalization to three housekeeping genes,
(
520 nm). Random scoring of 200 cells for both untreated and β-actin, 18srRNA, and GAPDH. All primers are listed in
FLS-treated MCF-7 was taken to determine the percentage of Table 2. PCR condition for this experiment was 1 cycle
viable (green intact cell), apoptotic (green cell with condensed of 50°C/2 minutes for Uracil-DNA glycosylase (UDG)
or fragmented nucleus), and necrotic (red cell) cells.
activation; 1 cycle of 95°C/2 minutes for DNA polymerase
Table 2 The accession number and sequence of the primers used in the quantitative real-time Pꢈꢀ assay
Accession number
Gene
Sequence
ꢃM_001101.3
ACTB
F: 5′-ꢄꢂꢄꢂꢈTꢄꢈꢂꢄꢂꢈTꢂꢈꢈTꢂꢄꢈ-3′
ꢀ
: 5′-ꢄꢂꢈꢄꢈTꢂTꢂTTꢂꢂꢈꢂTꢄꢈꢄꢂ-3′
F: 5-ꢂꢂꢄTTTꢂꢂTꢈꢂTꢄTTꢂꢂꢂꢈ-3
: 5-TꢂꢂꢄꢄꢂꢄTꢂꢂTꢂꢄTꢂꢂꢂꢄTT-3
F: 5-ꢂTꢄꢄꢈꢈꢈꢂTTꢂꢄꢄꢈꢈꢈꢈꢄTT-3
: 5-ꢈꢈꢄTꢈꢈꢄꢄTꢈꢂꢂTꢄꢂTꢄꢂꢈꢂ-3
F: 5-ꢈꢈTꢂꢈꢄꢈꢈꢂꢄꢄꢄꢈꢈꢂꢄꢂTTꢄT-3
:5-ꢈꢈꢂTꢈꢄTꢄTTꢈꢂꢄꢈTTTꢂꢂTTꢂꢈ-3
F: 5-ꢂꢂꢂꢄꢄTTTꢂꢄTꢂTꢂꢈꢂꢄꢈꢄꢂ-3
:5-ꢈTTꢈꢄꢄꢂꢈTꢈꢄTꢄꢄTꢈꢄꢈTꢂꢂꢈT-3
F: 5-ꢄꢈꢄꢄꢈꢄꢈTꢂꢂꢄꢂTꢈꢄTꢈꢄꢄTꢂꢈ-3
ꢃM_002046.4
ꢉQ387008.1
GAPDH
18S rRNA
PLK1
ꢀ
ꢀ
ꢃM_005030.3
ꢃM_001143976.1
ꢃM_006516.2
ꢀ
WEE-1
GLUT1
ꢀ
ꢀ: 5-ꢈꢈꢄꢈꢄꢂꢄꢂꢄꢄꢂꢂꢄꢂꢈꢈꢄꢄTꢈꢄ-3
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ꢄli et al
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activation; 40 cycles of 95°C 2 seconds for denature, 60°C for Affymetrix, Santa Clara, CA, USA). The fluorescence inten-
0 seconds for anneal and extend. Normalization of reference sity in active caspase 9 was measured at Ex/Em =540/570 nm
3
genes was carried out using geNorm algorithm.
using microplatefluorometer (Thermo Scientific). On the other
hand, absorbance for human cytochrome c was measured
using ELISA plate reader at wavelength of 450 nm (BioTek
Western blot analysis
Harvested cell was lysed with radioimmunoprecipitation Instruments). Fold change of caspase 9 and cytochrome c were
assay (RIPA) buffer supplemented with phosphatase inhibitor calculated by dividing fluorescence intensity or absorbance of
cocktail (Hoffman-La Roche Ltd., Basel, Switzerland) prior FLS treated MCF-7 with untreated control MCF-7.
to protein quantification using Bradford assay (Sigma-Aldrich
Co.). Next, 100 μg of cell lysate was electrophoresed in 10% ꢇtatistical analysis
sodium dodecyl sulfate polyacrylamide gel electrophoresis The data are presented as average of mean ± SE (standard
(SDS-PAGE) (Bio-Rad), transferred to nitrocellulose mem- error of the mean), one-way analysis of variance was used to
brane using Pierce Fast Semi-Dry Blotter (Pierce, Thermo assess differences between time point groups. Results were
Fisher Scientific, Waltham, MA, USA), blocked with 0.5% considered statistically significant when P,0.05.
skimmed milk and incubated with primary antibodies (anti-
The Ethical Committee for Research in Universiti Putra
CDC2, anti-p-CDC2, anti-p53, anti-Bax, anti-Bcl-2, and anti- Malaysia, decided that ethical approval was unnecessary for
β-actin at dilution 1:1,000) (Abcam, San Francisco, CA, USA) using commercial human cell lines.
for 1 hour. Following that, membrane was washed, incubated
Results
with goat anti-rabbit immunoglobulin G (IgG) and haematoxy-
lin and eosin stain (H&E) conjugated to alkaline phosphatase ꢇynthesis of Fꢅꢇ
(Abcam) in 1:5,000 dilution and developed under chemilu- FLS was the yellow flat crystals with 78.6% yield through
minescence condition (SuperSignal West Pico, Pierce) using the synthesis. IR (CHCl ): 1,642–1,644 (C=O), 1,550–1,568
3
ChemiDoc XRS (Bio-Rad). The bands, intensity was analyzed (C=C–C=O), 1,458–14,728 (C=C, Ar), and 1,200–1,100
−1 1
using Quantity One 1D analysis software (Bio-Rad).
(C–O) cm . HNMR (CDCl , 500 MHz): δ14.30 (chelated
3
OH), 7.86 (IH, J=15.5 Hz, Hβ), 7.53 (d, 1H, J=15.5 Hz,
Hα), 7.50 (br, d, 2H, H-2,6), 7.26 (m, 3H, H-3,4,5), 5.95
(d, J=2.5 Hz, 1H, H-3′), 5.90 (d, 1H, J=2.5 Hz, H-5′), 3.90
ꢈaspase 9 and cytochrome c detection
study
ExtractedproteinfrompreviousWesternblotanalysiswasalso (s, 3H, OMe, C-6′), 3.82 (s, 3H, OMe, C-4′), 2.54 (s, 3H,
subjected to detection of caspase 9 and cytochrome c using S-CH ). Electron-Impact Mas Spectrometry has indicated
3
CaspGLOW Red Active Caspase-9 staining kit (BioVision, that synthesised FLS has molecular weight of 330.32 with
Milpitas, CA, USA) and human cytochrome c platinum molecular formula C H O S. Molecular structures of FLS,
18
18
4
enzyme-linked immunosorbent assay (ELISA) (eBioscience FLA, and FLB are shown in Figure 1.
+ꢀ&2
2&+ꢀ
6&+ꢀ
+ &2
2&+ꢀ
2&+ꢀ
ꢀ
$
%
2
+
2
/6
2+
2
)
)ODYRNDZDLQꢀ$
+ꢀ
&2
2&+ꢀ
2
+
2
)
ODYRNDZDLQꢀ%
Figure 1 The molecular structures of FLS, flavokawain A and flavokawain B.
Abbreviation: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one.
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ꢈytotoxicity of Fꢅꢇ
Fꢅꢇ selectively reduced the viability of
MꢈF-7 cell
FLS provoked a time-dependent cytotoxicity against both
MCF-7 and MDA-MB-231 cells with 50% reduction in
a reducing concentration from 24 to 72 hours. The IC50
value of FLS on MDA-MB-231 was 1.5-fold higher than
on MCF-7 indicating that FLS is more sensitive to MCF-7
cell cycle analysis is summarized in Figure 3. Significant
(
P,0.05) G2/M arrest was noticed after 24 hours of FLS
treatment. Subsequently, 48 and 72 hours of FLS treatment
were found with drastic increase of sub G /G population
0
1
on MCF-7 indicating the possibility of DNA fragmentation
to happen. To confirm the apoptotic inducing effect of FLS
on MCF-7 at 48 and 72 hours, Annexin V/PI apoptosis
flow cytometry was done to evaluate the late and early
apoptosis indicated by externalization of phosphatidyl-
serine with or without loss of membrane integrity (that
allow PI to enter and stain the DNA), respectively. Based
on the Annexin V/PI result, early apoptosis was noticed
with constant increase from 9.18% to 36.72% from 24 to
(
(
IC50 ~33 μM) than to MDA-MB-231 (IC50 ~48.5 μM)
Table 1). On contrary, no cytotoxicity against normal breast
cell MCF-10A can be detected up to 180 μM. Selective index
(
SI) was calculated based on the IC value of FLS on MCF-
50
1
0A comparing to MCF-7 or MDA-MB-231. FLS exhibited
greater selectivity of MCF-7 (SI .5) cell than MDA-MB-231
SI .3) against MCF-10A.
7
2 hours. Besides, 11.86% of late apoptosis was observed
(
after 48 hours and further increased to 17.29% at 72 hours
on FLS treated MCF-7 cell (Figure 3). These results indi-
cated that FLS treatment promoted G2/M cell cycle arrest
in early time point and subsequently induced apoptosis at
later time point.
Morphology observation of MꢈF-7
treated with Fꢅꢇ
Untreated MCF-7 cells were confluent with normal mor-
phology (Figure 2A). FLS (36 μM) reduced the cell num-
ber of MCF-7 and induced cell shrinkage after 72 hours
of treatment. Also, detachment of cell was also observed Fꢅꢇ downregulated the mꢀꢃꢄ
(
Figure 2B). Scanning electron microscopy has provided a
expression of PLK1 and ꢂꢅU1 but
clearer comparison between ultrastructure of untreated and
FLS treated MCF-7 after 72 hours of incubation. Untreated
control MCF-7 was observed with normal cell morphology
with extended structure and microvilli observed indicating
the control cell was healthy and stably attached on the glass
slide (Figure 2C). In contrast, morphological changes related
to apoptosis including cell shrinkage, irregular surface, large
hole, and apoptotic body were observed in the FLS treated
MCF-7 (Figure 2D). In addition, apoptosis, necrosis, and via-
bility of MCF-7 cell treated with FLS were also investigated
under fluorescent microscope after staining with acridine
orange and PI. Viable cell was stained as green intact cell
upregulated the mꢀꢃꢄ expression of
WEE-1 and c-Jun
Differential expression of PLK1, WEE-1, c-Jun, and GLUT
on untreated control and FLS treated MCF-7 cell was quanti-
fied by qRT-PCR assay on the cDNA synthesized from the
extracted RNA. The results indicated that FLS significantly
(
P,0.05) downregulated expression (.2 fold) of PLK1
and GLUT mRNA expression in MCF-7 cell. On the other
hand, it upregulated WEE-1 expression for all evaluated time
points but only significantly increased c-Jun expression after
7
2 hours of treatment (Figure 4).
(
Figure 2E). On the other hand, MCF-7 cell treated with FLS
Fꢅꢇ promoted the level of ꢈDꢈ2
phosphorylation, Bax/Bcl-2 ratio, p53,
active caspases, and cytochrome c
Based on the Western blot analysis, level of tumor suppres-
sor p53 and proapoptotic Bax were upregulated in time-
dependent manner. Furthermore, the level of antiapoptotic
protein Bcl-2 was downregulated in the similar trend as
well (Figure 5). The phosphorylation of CDC2 significantly
increased with drastic reduction in CDC2 total protein
levels at 24, 48, and 72 hours. Similarly, level of activated
caspase 9 and cytochrome c were also detected to be signifi-
was observed with significant increase of apoptotic popula-
tion (P,0.05), which was marked by chromatin condensa-
tion, cell shrinkage and membrane blebbing (Figure 2F).
Based on random scoring of ~200 cells, untreated control
MCF-7 cell was recorded with .95% of viable population.
Unlike control, FLS treatment induced ~26% of apoptosis
and 14.5% of necrosis or late apoptosis after 72 hours of
incubation (Figure 2G).
Fꢅꢇ induced ꢂ2/M cell cycle arrest
followed by apoptosis on MꢈF-7
Cell cycle progression of FLS (36 μM) treated MCF-7 was cantly increased in FLS treated MCF-7 in time-dependent
examined by flow cytometry PI staining assay. Result of manner (Figure 6).
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Figure 2 ꢅight microscopic images of MꢈF-7.
Notes: (A) Untreated (control) and (B) Fꢅꢇ treated (36 μM at 72 hours). ꢇcanning electron microscopy images of MꢈF-7 (C) untreated (control) and (D) Fꢅꢇ treated (36 μM at
2 hours). ꢄcridine orange/propidium iodide (ꢄO/Pꢁ) double staining of MꢈF-7 (E) untreated (control) and (F) Fꢅꢇ treated (36 μM at 72 hours) and (G) Bar chart analysis of the
7
percentage of viable, apoptotic and necrotic MꢈF-7 of untreated (control) and Fꢅꢇ treated (36 μM at 72 hours) via ꢄO/Pꢁ qualitative analysis at each time point. The experiments were
done in triplicate and all data are expressed as mean ± ꢇꢆ with (*P,0.05). Figures shown were representative of at least three independent replicates with similar parameters.
Abbreviations: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; ꢇꢆ, standard error of the mean.
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Dovepress
ꢈytotoxicity of Fꢅꢇ
A
IC (36 µg/mL) of FLS
50
Control
24 h
1
48 h
1
72 h
0 1
Sub G /G : 0.55±0.04
Sub G /G : 0.42±0.10
Sub G /G : 28.95±1.62
Sub G /G : 36.49±0.90
0
1
0
0
G /G : 63.25±0.96
G /G : 17.74±0.25
G /G : 34.38±0.82
G /G : 34.05±0.61
0 1
0
1
0
1
0
1
S: 18.03±1.24
S: 10.22±0.26
S: 14.22±0.50
S: 17.16±0.69
G2+M: 18.01±1.68
G2+M: 61.06±0.94
G2+M: 22.75±0.52
G2+M: 12.34±0.67
2
1
1
40
80
2
40
2
1
40
80
2
1
1
40
80
G /G1
0
0
G /G1
G2+M
0
G /G1
G2+M
180
G2+M
G /G1
G2+M
0
20 Sub G /G1
0
120 Sub G /G1
120
60
0
0
G /G1
20 Sub G /G1
0
0
S
S
S
60
0
S
60
60
0
0
0
200
400
600
0
200
400
600
0
200
400
600
0
200
400
600
DNA content (RFU)
DNA content (RFU)
DNA content (RFU)
DNA content (RFU)
B
7
0.00
0.00
0.00
0.00
0.00
0.00
0.00
*
*
6
5
4
3
2
1
Control
2
4
4 h
8 h
*
*
*
*
72 h
*
0
.00
Sub G /G1
G /G1
S
G2+M
0
0
C
IC (36 µg/mL) of FLS
50
Control
24 h
48 h
72 h
Treated 0.126
Treated 0.150
Treated 0.151
Treated 0.154
04
104
104
103
104
103
1
Late apoptotic/
necrotic:
Late apoptotic/
Late apoptotic/
Late apoptotic/
necrotic:
necrotic:
4.71±0.31
necrotic:
11.86±0.82
0.64±0.04
17.29±0.65
1
1
1
1
03
103
02
102
102
102
Viable:
98.03±0.14
Early apoptotic:
0.85±0.08
Viable:
Early apoptotic:
9.18±0.26
Viable:
Early apoptotic:
22.85±0.90
Viable:
44.81±0.25
Early apoptotic:
36.73±0.40
01
101
85.64±0.38
101
64.74±0.08
101
00
1
100
100
100
00
101
102
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Annexin V FITC (RFU)
Annexin V FITC (RFU)
Annexin V FITC (RFU)
Annexin V FITC (RFU)
1
20.00
D
100.00
Control
24 h
8
6
4
2
0.00
0.00
0.00
0.00
*
4
7
8 h
2 h
*
*
*
*
*
0.00
FITC–/PI+ FITC+/PI+ FITC–/PI– FITC+/PI–
Figure 3 Flow cytometry cell cycle and ꢄnnexin V analyses of control and Fꢅꢇ treated MꢈF-7 cells.
Notes: (A) ꢉistogram analysis of cell cycle and (B) bar chart showing the percentage of cell in each phase of the cell cycle in MꢈF-7 for 24, 48, and 72 hours when treated
with ꢁꢈ50 dose of Fꢅꢇ (36 μM). (C) ꢉistogram analysis of ꢄnnexin V/FꢁTꢈ and (D) bar chart showing the percentage of cell stained with FꢁTꢈ and Pꢁ in MꢈF-7 treated
with ꢁꢈ50 dose of Fꢅꢇ (36 μM) for 24, 48, and 72 hours. The experiments were done in triplicate and all data are expressed as mean ± ꢇꢆ with (*P,0.05). ꢇub-ꢂ /ꢂ is
0
1
sub-ꢂap0/ꢂap1 phase. ꢁꢈ50 represents the concentration of Fꢅꢇ reduced 50% of MꢈF-7 cells viability.
Abbreviations: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; ꢇꢆ, standard error of the mean; FꢁTꢈ+/Pꢁ+, late apoptosis
population; FꢁTꢈ−/Pꢁ−, healthy cell population; FꢁTꢈ+/Pꢁ−, early apoptosis population; S, synthesis; RFU, relative fluorescence unit.
including anticancer activity but at the same time facing
Discussion
Fighting cancer by combining knowledge of phytochem- disadvantages such as lack of accessibility, and not being cost
istry and synthetic chemistry has been utilized to discover or time effective to obtain in large volume. These shortages
potential chemotherapy agents with better selectivity against can be overcome by synthetic compounds and even modi-
7
cancer than normal cell. Phytochemicals isolated from the fied to increase the availability, bioactivities, and selectivity
1
4
natural sources have been proven with multiple bioactivities against cancer cell.
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Figure 4 qPꢈꢀ analysis of apoptosis and cell cycle related genes; PLK1, WEE-1, c-Jun, GLUT in MꢈF-7 treated with Fꢅꢇ (36 μM) for 24, 48, and 72 hours.
Note: The experiment was done in triplicate and the data are expressed as mean ± ꢇꢆ with (*P,0.05).
Abbreviations: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; ꢇꢆ, standard error of the mean; qPꢈꢀ, quantitative real time
polymerase chain reaction.
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Notes: (A) Western blot analysis of cell cycle and apoptotic-related proteins; ꢈDꢈ2, p-ꢈDꢈ2, BꢄX, Bcl-2, and p53 in MꢈF-7 treated with Fꢅꢇ (36 μM) for 24, 48, and 72
hours. Bar chart analysis of related proteins (B) ꢈDꢈ2 and p-ꢈDꢈ2, (C) ratio of BꢄX/Bcl-2 and (D) p53 after normalized against beta actin (ꢄꢈTB). The experiment was
done in triplicate and the data are expressed as mean ± ꢇꢆ with (*P,0.05). White bars with black dots is ꢈDꢈ2 while black bars with white dots is p-ꢈDꢈ2.
Abbreviations: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one; ꢇꢆ, standard error of the mean.
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ꢈytotoxicity of Fꢅꢇ
contribution against maintaining cytotoxicity on MCF-7 cell
butlesstoxictowardnormalMCF-10Acell,whichpossiblydue
to the involvement of electron donating group, makes enrich
scaffold αβ-unsaturated carbonyl moiety to further promote
apoptosis in breast cancer MCF-7 cells but without further
affecting of normal breast MCF-10A cells. Previous report
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18
by Ethiraj et al has shown that electron donating groups in
chalcones have reduced the cytotoxic activity against cancer
cell. FLB, free of electron donating methoxy or thiomethyl
group, was found to be more sensitive to both breast cancer
MDA-MB-231 and normal MCF-10A cell lines comparing
to both FLA and FLS (Table 1). On the other hand, FLS and
FLA with electron donating group possessed the advantage
of less toxic in breast normal MCF-10A cell thus improved
the selectivity against breast cancer.
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Figure 6 Detection of the activation of caspase 9 and cytochrome c in MꢈF-7
treated with Fꢅꢇ (36 μM) for 24, 48, and 72 hours.
Note: The experiment was done in triplicate and the data are expressed as mean ±
ꢇꢆ with (*P,0.05).
Abbreviations: Fꢅꢇ: (ꢆ)-1-(2′-ꢉydroxy-4′,6′-dimethoxyphenyl)-3-(4-methylthio)
phenyl)prop-2-ene-1-one; ꢇꢆ, standard error of the mean.
Since FLS showed better cytotoxic potential against
MCF-7 cell, the mode of cell death and cell cycle arrest
Several studies have shown that chalcone is an important induced and cell cycle arrest effects were evaluated on
class for the anticancer properties against various cancer cell MCF-7 using IC value of FLS at 48 hours (36 μM). As
5
0
14
lines. Previously, we have synthesized FLA and FLB and indicated by the light microscope observation, reduction
investigated their cytotoxic effects antimetastatic effects on of cell number was recorded in FLS treated MCF-7 cell
MCF-7 and MDA-MB-231 breast cancer cell lines in vivo and (Figure 2B). This result was further supported by the cell
9,10
9
10
in vitro. In terms of FLA and FLB (Abu et al), they pos- cycle analysis where G2/M arrest was observed as early as
sessed greater selectivity against MDA-MB-231 (SI: 5.72 for 24 hours post-FLS treatment. This effect might be contributed
FLA and 3.66 for FLB) than MCF-7 (SI: 3.98 for FLA and by the significant upregulation and downregulation of WEE-1
1
.33 for FLB) with IC on normal MCF-10A around 100 and PLK-1 in the treated cell, respectively (Figure 4). PLK1,
50
and 45 μM for FLA and FLB, respectively. Although FLA a mitotic polo-like kinase, is a key regulator of G2/M phase
and FLB showed good cytotoxicity against MDA-MB-231 progression while WEE-1 is the nuclear kinase that inhibits
1
9
cell, their effect on MCF-7 was just marginal with poor SI the entry of cell into mitosis. Overexpression of PLK1 is
especially FLB, which has lower IC value on normal breast always reported in different types of cancer including breast
50
cell line. In this study, flavokawain derivative (FLS) was syn- cancer, while downregulation of PLK1 was found to link with
thesized and subjected to cytotoxicity evaluation. MTT assay G2/M arrest and subsequently apoptosis posttreatment with
9
indicatedthatsyntheticFLSwasmoresensitivetoMCF-7than natural compound such as FLA. Cell cycle kinase subunit
MDA-MB-231 compared to FLA and FLB (Table 1). More (CDC2) that binds to cyclin B1 to regulate progression from
interestingly, FLS did not show IC value (.180) on normal G2 to M transition was also found to be downregulated by
50
breast MCF-10A cell thus giving the SI of 5.4 to MCF-10A/ FLS. FLS suggests that the downregulation of CDC2 protein
MCF-7. However, the SI of FLS against MCF-10A/MDA- level contributes to the increase of CDC2 phosphorylation.
MB-231 was lower than FLS against MCF-10A/MFC-7 since Suppression of PLK1 and upregulation of WEE-1 were
its cytotoxicity against MDA-MB-231 was low (Table 1). reported to contribute to the downregulation of CDC2 via
2
0
Chalcones consist of two aromatic rings (A and B) linked by a CDC2 Tyrosine-15 phosphorylation. Concomitant with
three-carbon unit αβ-unsaturated carbonyl moiety, which acts this, similar trend was observed in FLS treated MCF-7
as a Michael acceptor. Apart from various substitutions on two cell. Cyclin-dependent kinase inhibitor p21, which can be
rings, basic skeleton of chalcone (1,3-diphenyl-2-propenone) upregulated by p53, was promoted in FLA and FLB treated
1,15–17
9,10
is potential in the treatment of human breast cancer.
The MCF-7 cell. In terms of FLS treated MCF-7 cell, although
difference in the structure of the compounds is the presence p53 was found to be upregulated (Figure 5), its role in the
of methoxy (OCH ) in FLA and SCH (thiomethyl group) in regulation of G2/M may be marginal because p21 was not
3
3
FLS, meanwhile no substituent is present on ring B of FLB. significantly upregulated when evaluated using qRT-PCR
The presence of SCH and OCH on ring B showed the (results not shown). Previous study has shown that G2/M
3
3
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arrest can occur with or without the involvement of tumor of the G2/M arrest. On the other hand, mitochondrial p53
suppressor p53. Although the regulation of p21 in MCF-7 pathway was found as the contributor of the FLS induced
treated with FLS was difference comparing to FLA and FLB, apoptosis in MCF-7 cell. The presence of SCH (thiomethyl
3
all of these compounds were found to promote G2/M arrest group) on ring B structure might play a role in cytotoxicity
9,10,19
via deregulation of PLK1.
and apoptosis of MCF-7 cell compared to other chalcones,
Apoptosis was significantly (P,0.05) enhanced in MCF-7 FLA and FLB. Thus, FLS is more potent candidate in selec-
after 48 and 72 hours of FLS treatment in Annexin V/PI tively combating MCF-7 breast cancer cell. Further in vivo
apoptosis study. The result was further supported by the mor- study shall be carried out to evaluate the antibreast cancer
phology observations where FLS treated MCF-7 was found efficacy of FLS comparing to FLA and FLB.
with typical characteristic of apoptosis including cell detach,
cell shrinkage, membrane blebbing, hole, apoptotic body, and Acknowledgments
chromatin condensation (Figure 2). FLS modulated apoptosis This work was jointly supported by University of Malaya
via upregulation of p53, Bax, caspase 9, and cytochrome c. HIR-MoE grant (reference number - UM.C/625/1/HIR/
FLS induced DNA damage associated with tumor suppres- MOHE/CHAN/03, account number - A000003-50001) and
sor p53 upregulation. p53 was reported as a key regulator of University Malaysia Pahang internal grants no (RDU 120373
BCL family where its upregulation was found to promote and RDU 120389).
proapoptotic BAX and suppress antiapoptotic Bcl-2, which
led to secretion of cytochrome c that stimulates the caspase Disclosure
21
cascade activation of cell death. Study has shown that mito- The authors report no conflict of interest in this work.
chondria played a factor in the initiation of apoptotic process
References
by releasing cytochrome c, apoptogenic factor from the outer
1
. American Cancer Society. Global Cancer Facts and Figures. 3rd ed.
Atlanta: American Cancer Society; 2015. Available from: http://www.
cancer.org/acs/groups/content/@research/documents/document/acspc-
mitochondria membrane space into cytosol of the apoptotic
22
cells. Concomitant with this, upon cytochrome c leakage,
caspase 9 was being activated. This suggests that FLS induced
changes in the mitochondrial membrane potential in the early
stages of apoptotic cell death via mitochondrial-dependent
intrinsic pathway. In this study, raised BAX/Bcl-2 ratio, cyto-
chrome c, and caspase 9 in the FLS treated MCF-7 indicated
the involvement of FLS in promoting the intrinsic apoptosis
as detected in flow cytometry and microscopic examinations.
Our previous reports have shown higher sensitivity of FLA
and FLB in mutant type p53 MDA-MB-231 cell than wild-
type p53 MCF-7 cell. In this study, FLS was more potent in
wild-type p53 MCF-7. The higher selectivity in wild-type
p53 MCF-7 cell may be contributed by the exchange of SCH3
thiomethyl group in FLS. Previous study has shown that the
presence of thiomethyl group improved the selectivity of
the compound toward cancer cell and maintained its cyto-
toxicity via drastic upregulation of p53 and Bax status that
subsequently promote apoptosis in leukemia cells. Similarly,
regulation of FLS and this thiomethyl compound on Bcl-2
0
44738.pdf. Accessed January 6, 2016.
2
3
. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A.
Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108.
. Loganathan R, Radhakrishnan AK, Selvaduray KR, Nesaretnam K.
Selective anti-cancer effects of palm phytonutrients on human breast
cancer cells. RSC Advances. 2015;5:1745–1753.
4. US Mortality Data, National Center for Health Statistics, Centers for
Disease Control and Prevention. American Cancer Society, Surveillance
Research. Atlanta, GA: American Cancer Society; 2014.
. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global
cancer statistics. CA Cancer J Clin. 2011;61:69–90.
. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer
J Clin. 2014;64:9–29.
7. Badisa RB, Darling-Reed SF, Joseph P, Cooperwood JS, Latinwo LM,
Goodman CB. Selective cytotoxic activities of two novel synthetic
drugs on human breast carcinoma MCF-7 cells. Anticancer Res.
2009;29:2993–2996.
. Batovska DI, Todorova IT. Trends in utilization of the pharmacological
potential of chalcones. Curr Clin Pharmacol. 2010;5:1–29.
. Abu N, Akhtar MN, Yeap SK, et al. Flavokawain A induces apoptosis in
MCF-7 and MDA-MB231 and inhibits the metastatic process in vitro.
PLoS One. 2014;9:e105244.
0. Abu N, Akhtar M, Yeap S, et al. Flavokawain B induced cytotoxic-
ity in two breast cancer cell lines, MCF-7 and MDA-MB231 and
inhibited the metastatic potential of MDA-MB231 via the regula-
tion of several tyrosine kinases in vitro. BMC Complem Altern M.
5
6
8
9
1
23
was marginal after 24 and 48 hours treatment.
2
016;16:86.
This study reveals that FLS showed good selectivity
against breast cancer MCF-7 than normal MCF-10A cell line.
The cytotoxicity of FLS on MCF-7 was mainly contributed
by G2/M cell cycle arrest and induction of apoptosis. Down-
regulation of PLK1 and upregulation of WEE-1 associated
with phosphorylation of CDC2 were the major regulators
11. Kuo YF, Su YZ, Tseng YH, Wang SY, Wang HM, Chueh PJ.
Flavokawain B, a novel chalcone from Alpinia pricei Hayata with potent
apoptotic activity: Involvement of ROS and GADD153 upstream of
mitochondria-dependent apoptosis in HCT116 cells. Free Radic Biol
Med. 2010;49:214–226.
1
2. Tang Y, Simoneau AR, Xie J, Shahandeh B, Zi X. Effects of the kava
chalcone flavokawain A differ in bladder cancer cells with wild-type
versus mutant p53. Cancer Prev Res (Phila). 2008;1:439–451.
submit your manuscript | www.dovepress.com
1
906
Drug Design, Development andTherapy 2016:10
Dovepress

Dovepress
ꢈytotoxicity of Fꢅꢇ
1
1
3. Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods.
19. Fukasawa K. Oncogenes and tumour suppressors take on centrosomes.
Nat Rev Cancer. 2007;7:911–924.
20. Fletcher L, Cheng Y, Muschel RJ. Abolishment of the Tyr-15 inhibi-
tory phosphorylation site on cdc2 reduces the radiation-induced G(2)
delay, revealing a potential checkpoint in early mitosis. Cancer Res.
2002;62:241–250.
1
983;65:55–63.
4. Abu N, Ali NM, Ho WY, Yeap SK, Aziz MY, Alitheen N. Damnacan-
thal: a promising compound as a medicinal anthraquinone. Anticancer
Agents Med Chem. 2014;14:750–755.
1
1
5. Go ML, Wu X, Liu X. Chalcones: an update on cytotoxic and chemo-
protective properties. Curr Med Chem. 2005;12:481–499.
21. Jiang X, Wang X. Cytochrome c-mediated apoptosis. Ann Rev Biochem.
2004;73:87–106.
6. Hsu YL, Kuo PL, Tzeng WS, Lin CC. Chalcone inhibits the prolifera-
tion of human breast cancer cell by blocking cell cycle progression and
inducing apoptosis. Food Chem Toxicol. 2006;44:704–713.
7. Sawle P, Moulton BE, Jarzykowska M, et al. Structure-activity relation-
ships of methoxychalcones as inducers of heme oxygenase-1. Chem
Res Toxicol. 2008;21:1484–1494.
8. Ethiraj KR, Aranjani J, Khan FN. Potential cytotoxic and apoptosis
inducing agents: Synthesis and evaluation of methoxy-substituted
chalcones against human lung and cervical cancers. Med Chem Res.
22. Looi CY, Arya A, Cheah FK, et al. Induction of apoptosis in human
breast cancer cells via caspase pathway by vernodalin isolated from
Centratherum anthelminticum (L.) seeds. PLoS One. 2013;8:e56643.
23. Fimognari C, Nüsse M, Iori R, Cantelli-Forti G, Hrelia P. The new
isothiocyanate 4-(Methylthio)Butylisothiocyanate selectively affects
cell-cycle progression and apoptosis induction of human leukemia
cells. Invest New Drugs. 2004;22:119–129.
1
1
2
013;22:5408–5417.
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