Genes Nutr (2011) 6:413–427
DOI 10.1007/s12263-011-0216-z
RESEARCH PAPER
Microarray analysis revealed different gene expression patterns
in HepG2 cells treated with low and high concentrations
of the extracts of Anacardium occidentale shoots
Shaghayegh Khaleghi • Azlina Abdul Aziz
Nurhanani Razali • Sarni Mat Junit
•
Received: 23 November 2010 / Accepted: 11 March 2011 / Published online: 29 March 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract In this study, the effects of low and high concentrations of the Anacardium occidentale shoot extracts
on gene expression in liver HepG2 cells were investigated.
From MTT assays, the concentration of the shoot extracts
that maintained 50% cell viability (IC50) was 1.7 mg/ml.
Cell viability was kept above 90% at both 0.4 mg/ml and
0.6 mg/ml of the extracts. The three concentrations were
subsequently used for the gene expression analysis using
Affymetrix Human Genome 1.0 S.T arrays. The microarray
data were validated using real-time qRT–PCR. A total of
246, 696 and 4503 genes were significantly regulated
(P \ 0.01) by at least 1.5-fold in response to 0.4, 0.6 and
1.7 mg/ml of the extracts, respectively. Mutually regulated
genes in response to the three concentrations included
CDKN3, LOC100289612, DHFR, VRK1, CDC6, AURKB
and GABRE. Genes like CYP24A1, BRCA1, AURKA,
CDC2, CDK2, CDK4 and INSR were significantly regulated at 0.6 mg/ml and 1.7 mg but not at 0.4 mg/ml.
However, the expression of genes including LGR5,
IGFBP3, RB1, IDE, LDLR, MTTP, APOB, MTIX, SOD2
and SOD3 were exclusively regulated at the IC50 concentration. In conclusion, low concentrations of the extracts
were able to significantly regulate a sizable number of
genes. The type of genes that were expressed was highly
dependent on the concentration of the extracts used.
Keywords Anacardium occidentale shoots
Methanol extracts Gene expression
cDNA microarray analysis HepG2 cells
S. Khaleghi A. A. Aziz N. Razali S. M. Junit (&)
Department of Molecular Medicine, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia
e-mail: sarni@um.edu.my
Introduction
The cashew plant or Anacardium occidentale L (A. occidentale) has many medicinal properties that are beneficial
to health. Various scientific evidences have linked the
various parts of cashew plant to several biological activities. The stem bark extract had been shown to have
anti-bacterial [1], anti-viral [22], anti-diabetic [32] and
anti-inflammatory [29] activities. Anti-tumour activity was
detected in the cashew gum [30] and nut [42] while antiulcerogenic was reported in the cashew leaf extracts [19].
Antioxidant activities were also detected in the nut skin
extracts [16]. In addition to the medicinal properties, the
fruit of the A. occidentale is a natural whitening agent that
disrupts pigmentation through the inhibition of tyrosinase
[21].
In Malaysia, the young leaves or shoots of the A. occidentale are widely consumed as salads, and the locals
believed its benefits include diabetic control and prevention. The extracts of the shoots were found to have potent
antioxidant activities [35], were able to inhibit the oxidation of LDL and up-regulated LDL receptor activity in
cultured HepG2 cells [39]. The antioxidant activities
observed in the A. occidentale shoot extracts were attributed to the reported presence of phenolic compounds such
as myricetin and quercetin [19, 27]. Intact quercetin
glycosides, the most common flavonoids found in human
diets, were shown to be absorbed at the small intestine
probably through a sodium-dependent glucose transport
pathway [9, 14]. Once absorbed, quercetin circulates in the
plasma in conjugated forms but its antioxidant properties
were maintained [25].
Other active compounds found in the crude extracts of the
A. occidentale leaves include catechin, epicatechin, tetramer
of proanthocyanidin and biflavanoids amentoflavone [19]
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414
and agathisflavone [20]. Agathisflavone was reported to be
able to induce apoptosis in Jurkat cells (acute lymphoblastic
leukaemia cell line) [20] as well as a potent, competitive
inhibitor for the GABAA/benzodiazepine receptor [43].
Scientific molecular studies on the effects of the
A. occidentale shoot extracts on cells are still lacking
despite its reported antioxidant properties and its use in
traditional medicine. We had earlier reported that the
methanol extracts of the A. occidentale contained the
highest total phenolic content compared to ethyl acetate
and hexane extracts [35]. In this study, we explored the
effects of the methanol extracts of the A. occidentale shoots
on the expression of genes which could be associated with
its antioxidant and medicinal properties.
Materials and methods
Chemicals
All reagents and chemicals used in the experiments were of
analytical grade and obtained mostly from Sigma–Aldrich.
Solvents used for extraction of plants were purchased from
Fisher Scientific. Water used was of Millipore quality
(ELGA Purelab Ultra Genetic system).
Preparation of methanol extract of the shoots
of Anacardium Occidentale
In our previous study [35], we reported that the methanol
extract of the A. Occidentale shoots possessed significantly
higher antioxidant activities compared to those of the ethyl
acetate and hexane extracts. The methanol extracts was
subsequently used in this study. Briefly, the shoots were
washed, air-dried followed by complete drying in an oven
at 40°C. The dried shoots were then ground to powder and
then extracted with methanol with a mass to volume ratio
of 1:20 (g/mL), at room temperature for 24 h. The resulting
extract was filtered and roto-evaporated (Rotavapor R-215)
to dryness at 37°C, and the residues were then redissolved
in dimethylsulfoxide (DMSO). For the subsequent cell
culture experiments, the final concentration of DMSO was
kept below 1% to avoid toxicity to the cells.
High-performance liquid chromatography
Acid hydrolyses was conducted on the dried powder of
A. occidentale [3]. Samples (20 mg) were mixed with 50%
methanol containing 1.2 M HCl and 20 mM sodium
diethyldithiocarbamate as an antioxidant, in reactive vials.
The samples were hydrolysed for 2 h at 90°C. Following
hydrolysis, samples were centrifuged at 50009g for 5 min
and diluted with distilled water (pH 2.5) prior to analysis
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Genes Nutr (2011) 6:413–427
on the HPLC. The hydrolysed samples contained both free
flavonoids and aglycones released from conjugated flavonoids following acid hydrolysis.
The HPLC system used for the flavonoid analyses
comprised a Shimadzu system consisting of a system
controller, a binary pump (LC 20AC), a manual injector
(Rheodyne 7725i manual injector), a column oven
(CTO-10AS VP) and a dual channel UV detector (SPD20A UV–VIS). Absorbance of the samples was monitored
at a wavelength of 260 nm. Flavonoids in the samples were
separated using a reversed-phase column (NovaPak C18,
150 9 3.0 mm, i.d 4 lm) (Waters, USA), at a temperature
of 40°C. Separation of flavonoids was conducted using a
gradient system containing 7–40% acetonitrile in water
(pH 2.5) at a flow rate of 0.5 ml/min over 20 min. Standard
solutions containing catechin, epicatechin, rutin, genistin,
myricetin, morin, quercetin, genistein, kaempferol and
isorhamnetin were prepared and injected on the HPLC
under the same conditions.
Cell culture
The human hepatoblastoma HepG2 cell line (ATCC,
Manassas, VA, USA) was grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% foetal
bovine serum (Flowlab, Australia), 1% penicillin (Flowlab,
Australia) and 1% streptomycin (Flowlab, Australia). Cells
were maintained in humidified air with 5% CO2 at 37°C.
Cell viability analysis using the MTT assay
Cell viability of HepG2 cells in response to treatment with
various concentration of the A. occidentale shoot extracts
was analysed using an MTT assay as described by
Mosmann, 1983 [28] with slight modifications [36]. Briefly,
HepG2 cells at a density of 5000 cells per well were seeded
in a 96-well ELISA microplate. The cells were incubated at
37°C in 5% CO2 for 24 h. After 24 h, increasing concentrations of the shoots extracts (0.2–5.0 mg/ml) were added
into the wells, and the cells were further incubated for 48 h.
Following this, MTT reagent (Merck) was added, and the
mixture was incubated for 4 h. Next, the mixture in each
well was removed, and formazan crystals formed were
dissolved in 10 ll of 75% isopropanol. Spectrophotometric
measurement of the mixture was performed in a microplatereader (Bio-Rad) at 590 and 620 nm wavelengths. A linear
plot of cell viability (%) against the concentrations of plant
extracts was constructed.
Treatment of HepG2 cells for the microarray analysis
For the gene expression analysis, 80–95% confluent HepG2
cells maintained in DMEM were treated with shoot extracts
Genes Nutr (2011) 6:413–427
at 0.4, 0.6 and 1.7 mg/ml. The cells were then incubated at
37°C for 24 h. As a control, cells were incubated in fresh
DMEM, in the absence of the extracts. All experiments
were performed in triplicate. After 24 h, cells were
trypsinized and then precipitated by centrifugation at
2619g for 5 min. Following this, cells were washed with
PBS twice before total cellular RNA (tcRNA) was
extracted from the treated and untreated cells.
Extraction of total cellular RNA (tcRNA)
tcRNA from both treated and untreated (control) HepG2
cells was isolated and then purified using RNAEasy kit
and RNase-free DNAse set (Qiagen) according to the
manufacturer’s instructions. The quality of the tcRNA
was estimated by measuring the absorbance ratio of
260–280 nm while its integrity was analysed using denaturing gel electrophoresis. An A260/A280 ratio above 1.8
indicated that the tcRNA was of good quality. The integrity
of the tcRNA was indicated by the presence of two distinct
bands corresponding to the ribosomal 28S and 18S subunits, with the intensity of the larger, 28S band approximately twice than that of the smaller, 18S band.
Microarray analysis
Affymetrix Human Gene 1.0 S.T (sense target) arrays were
used for the gene expression analysis according to the conventional Affymetrix eukaryotic RNA labelling protocols
(Affymetrix). Briefly, freshly extracted tcRNA (100 ng)
isolated from the treated and untreated HepG2 cells was
reversed transcribed to single-stranded sense strand DNA
(cDNA) in two cycles using the Whole Transcript (WT)
cDNA synthesis, amplification kit and sample clean-up
module. The sense strand cDNA was then cleaved into small
fragments using a mixture of UDP and apurinic/apyrimidinic
endonuclease 1 or APE1. Following this, the fragments were
end-labeled with biotinylated dideoxynucleotides using the
WT Terminal Labeling kit. The biotinylated fragments
(5.5 lg) were then hybridized to the Affymetrix Human
Gene 1.0 S.T array at 45°C for 16 h in hybridization Oven
640. After hybridization, the arrays were stained and then
washed in the Affymetrix Fluidics Station 450 under standard conditions. The stained arrays were scanned at 532 nm
using an Affymetrix GeneChip Scanner 3000, and CEL files
for each array were generated using the Affymetrix GeneChipÒ Operating Software (GCOS). The data were preanalyzed using Affymetrix Expression Console software.
415
list of up-regulated and down-regulated genes. Probeset
IDs without any annotation in the Partek software were
filtered out. The filtered, whole gene list was then subjected
to a one-way analysis of variance (ANOVA) in the Partek
Genomic software, to determine significantly expressed
sets of genes which was set according to P values less than
0.01 (P \ 0.01) instead of P \ 0.05 to avoid false positive
results. Significantly expressed genes were then re-filtered
to include only those with fold change difference of equal
to or greater than 1.5. Additional information on the biological functions of the genes and the genes products was
determined from the Gene Ontology (GO) Enrichment tool
in the Partek Genomic Suite Software. Information on
function of genes can be derived from the Gene Ontology
database which provides a structured annotation of genes
with respect to molecular function, biological process and
cellular component. Further information on GO could be
retrieved from http://www.geneontology.org/.
Validation of the DNA microarray data
using qRT–PCR
The microarray data were verified using real-time relative
quantitative RT–PCR (qRT–PCR) which was performed in
TM
a StepOne Real-Time PCR System (Applied BioSystem).
The same cDNA and primer pairs for the selected
up-regulated and down-regulated genes as well as a
housekeeping gene, GADPH, as listed in Table 1 were
used. The PCR amplification was carried out in 0.2 ml
MicroAmpÒ Optical 8-tube strips in a final volume of 20 ll
containing a mixture of cDNA (30 ng), reverse and forward primers (1 lM), pre-prepared Power SYBRÒ Green
PCR master mix containing SYBRÒ Green 1 dye, AmpliTaq GoldÒ DNA Polymerase dNTPs, dUTP, Passive Reference 1 and optimized buffer components. The PCR
parameters consisted of 40 cycles of amplification with
initial denaturation at 95°C for 15 s, annealing of primers
and elongation of the newly synthesized strands at 60°C for
60 s. The PCR mixture was initially held for 10 min at
95°C for AmpliTaq GoldÒ DNA polymerase activation.
The Comparative CT Method (DDCT) was chosen for the
relative quantitation of gene expression. Each sample type
was run in triplicate. mRNA levels of the selected genes
were normalized against that of GADPH.
Results
Cell viability analysis
Microarray data normalization and analysis
The CEL files generated were converted to text files and
exported to Partek Genomic Suite software to get the whole
MTT assays were performed to measure the viability of the
cells in response to the treatment with different concentrations of the shoot extracts. The dose–response curve of
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Genes Nutr (2011) 6:413–427
Table 1 Primer sequences for
the selected genes used for
validation of the microarray
data using real-time relative
quantitative PCR (qRT–PCR)
Gene name (Genebank ID)
Primer sequences
Product size (bp)
DHFR (NM_000791)
Forward: 50 CATGGTCTGGATAGTTGGTGGC 30
108
Reverse: 50 GTGTCACTTTCAAAGTCTTGCATG 30
Forward: 50 ATCAAGGGATCCACAAATGA 30
TYMS (NM_001071)
205
Reverse: 50 GGTCAACTCCCTGTCCTGAA 30
Forward: 50 CAAGTGCCCTTGGACAAAGC 30
LIPC (NM_000236)
0
Reverse: 5 TGACAGCCCTGATTGGTTTCT 3
CYP24A1 (NM_000782)
130
0
Forward: 50 CTCATGCTAAATACCCAGGTG 30
300
Reverse: 50 TCGCTGGCAAAACGCGATGGG 30
Forward: 50 TGCGGTGCATGCAGTGTAAGAC 30
PLAUR (NM_002659)
183
Reverse: 50 TCAAGCCAGTCCGATAGCTCAG 30
PLCXD1 (NM_018390)
Forward: 50 ACGAGTACCTGGTCGCCTGTAT 30
117
Reverse: 50 CATAGGAGACGATGACCTGTTGG 30
SQSTM1 (NM_003900)
Forward: 50 CCAGTGACGAGGAATTGACAA 30
156
Reverse: 50 CATCGCAGATCACATTGGGG 30
HPLC analysis
Percentage of Cell Viability
100
90
HPLC analyses of the hydrolysed samples revealed the
presence of quercetin and kaempferol (Fig. 2a) and the
absence of catechin, epicatechin, rutin, genistin, genistein,
myricetin, morin and isorhamnetin. The quercetin and
kaempferol peaks were confirmed by comparing retention
times of the peaks with the standards (Fig. 2b) which was
run under the same conditions as the samples. The presence
of kaempferol has not been reported previously.
80
70
60
50
40
30
IC50
20
10
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Concentration of extracts of the Anacardium occidentale
shoots (mg/ml)
Fig. 1 A dose–response curve of percentage viability of HepG2 cells
(5,000 cells/well) treated with various concentrations of the extract of
the A. occidentale shoots (0.2–5.0 mg/ml). Analysis was done in
triplicate, and the results were expressed as % of HepG2 cell
viability ± std dev. From the plot, the IC50 was found to be 1.7 mg/ml
the viability of HepG2 cells in response to the treatment
with different concentrations of the methanol extract of
A. occidentale shoots is shown in Fig. 1. The graph shows
a tri-phasal response. In the first phase, cell viability was
maintained above 90% until 0.6 mg/ml of the extract
concentration was reached. In the second phase, cell viability decreased steeply to below 20% from 0.6 to 3 mg/ml
extract concentrations. In the third phase, the cells barely
survived (viability below 10%) beyond 5 mg/ml extract
concentration. The concentration of the shoot extract that
reduced cell viability by 50% (IC50) was 1.7 mg/ml. Low
concentrations of 0.4 and 0.6 mg/ml as well as the IC50
concentration were subsequently chosen for the gene
expression analysis.
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Microarray analysis: normalization
and visualization of data
Figure 3 shows the principal component analysis (PCA)
plot of the microarray data for each of the array for the
control and samples treated with 0.4, 0.6 and 1.7 mg/ml of
the shoot extracts. The three arrays for the control samples
were grouped together but separately from those of the
treated samples. Figure 4 shows hierarchical clustering of
the microarray data generated from each of the arrays
where the reproducibility pattern correlates with that
shown by the PCA plot.
Venn diagram in Fig. 5 shows the number of genes that
were significantly regulated (P \ 0.01) by at least 1.5-fold.
The total number of genes regulated by 0.4, 0.6 and
1.7 mg/ml of the methanol extracts of A. occidentale were
246, 696 and 4,503, respectively. An increase in concentration by 1.5-fold from 0.4 to 0.6 mg/ml was able to
increase the number of regulated genes by threefold (65%).
In addition, increasing the concentration from 0.4 to
1.7 mg/ml increased the number of regulated genes by
18-fold (95%). Figure 5 also shows that a total of 94 genes
were mutually regulated in response to the three
Genes Nutr (2011) 6:413–427
417
(a)
(b)
2
1
1
2
20.0
21.0
22.0
23.0
24.0
mins
20.0
22.5
mins
Fig. 2 Gradient reverse phase HPLC analysis of flavonoids in the
shoots of A. occidentale and the flavonoid standards. HPLC analysis
was performed on the hydrolysed samples of A. occidentale (Fig. 2a)
and the flavonoid standards (Fig. 2b). Flavonoids were separated on a
NovaPak C18 reversed-phase column (150 9 3.0 mm i.d, 4 lm),
using a linear gradient system of 7–40% acetonitrile in water (pH
2.5), at a flow rate of 0.5 ml/min. Absorbance was measured at a
wavelength of 260 nm. 1:quercetin; 2:kaempferol
concentrations where 5 were up-regulated and 89 were
down-regulated. Ninety-eight genes were mutually regulated in response to the 0.4 and 0.6 mg/ml extracts. On the
other hand, 178 were mutually regulated in response to the
0.4 and 1.7 mg/ml extracts. A larger number of genes, with
a total of 571, were regulated when cells were treated with
0.6 and 1.7 mg/ml of the extracts.
Mutually regulated genes in response to the three concentrations included CDKN3, LOC100289612, DHFR,
VRK1, CDC6, AURKB, CYP2S1 and GABRE (Table 2).
Genes like CYP24A1, CDH2, E2F5, BRCA1, BRCA2,
AURKA, CDC2 (CDK1), CDK2, CDK4, CHECK1,
CCNA2, ACAT, IGFBP1, DUSP5 and INSR were significantly regulated at 0.6 and 1.7 mg/ml but not at 0.4 mg/ml.
In addition, the expressions of genes such as the LGR5,
IGFBP3, RB1, IDE, LDLR, MTTP, APOB, SCP2, MTIX,
SOD and SOD3 were exclusively regulated at the 1.7 mg/
ml dose. Amongst the highly significantly suppressed
genes were CYP24A1, LGR5, CDH2 and DHFR by 27.8-,
16.4-, 15.5-, 10.0-fold, respectively. On the other hand,
amongst the highly induced genes were the DUSP5,
IGFBP 3, IGFBP1, LDLR and INSR by 9.1-, 8.0-, 4.2-,
3.6- and 2.6-fold, respectively (Table 2).
Other genes that were being significantly regulated in
response to the 1.7 mg/ml shoot extracts were those associated with cell cycle check points either directly or indirectly. These included the CDK5, CDK6, CCNB1 and 2,
CCNE1 and 2, CCNH, CDKN1A (p21/Cip1), CDKN1C
(p57/Kip2), CDKN2B (p15), CDKN2D (p19), CDKN3,
RBL1, RBL2, NSUN6, NOP2, DAPK1, PAK2, HDAC2 and
G2E3. Genes coding for other ubiquitin ligase isoforms,
UBE2C and UBE3B, were also aberrantly expressed. In
addition, genes associated with the Wnt/b-catenin signalling pathway including the Wnt6, FZDs 1, 4, 6, 7, 8, 9 and
10, CDH1, CTNNA2, DKK1, APC, NUCKS1, CSNK1G2,
CSNK1G3 and TCF were all aberrantly expressed. In
addition, genes associated with cancers, BRIP1, BAP,
BRCC3, RAS, SOS1, STAT2, were all down-regulated (data
not shown).
Fig. 3 A principal component
analysis (PCA) plot derived
from biological replicates
(n = 3) of HepG2 cells grown
in 0.4, 0.6 and 1.7 mg/ml of the
extracts of the A. occidentale
shoots
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Genes Nutr (2011) 6:413–427
1.7 mg/ml vs control
Array 1
Array 2
1.7
mg/ml
Array 3
Array 1
0.6
mg/ml
Array 2
Array 3
Array 1
0.4
mg/ml
Array 2
Array 3
Array 1
Control
Array 2
Array 3
Fig. 4 Hierarchical cluster analysis of genes showing the differential
expression of genes in HepG2 cells in response to the treatment with
0.4, 0.6 and 1.7 mg/ml of extracts of A. occidentale shoots
Gene ontology (GO): biological interpretation
Gene ontology analysis of the products of the significantly
regulated genes in response to the 3 concentrations of the
A. occidentale shoot extracts is shown in Figs. 6, 7 and 8.
The data are presented according to the following categories; biological process (Fig. 6), molecular function
(Fig. 7) and cell component (Fig. 8). Selected significantly
down-regulated and up-regulated genes in each of the
category are shown in Table 3.
Figures 6, 7 and 8 show that most of the genes (and the
subsequent gene products) are involved in cellular processes, biological regulations and metabolic processes in
response to the three concentrations of the extracts. A total of
18 genes that are involved in localization, growth, locomotion and pigmentation were regulated in response to the
0.6 mg/ml but not the 0.4 mg/ml extracts. As the concentration was increased from 0.6 to 1.7 mg/ml, the number of
genes rose from 18 to 88 in the same 3 subcategories. In
addition, 3 genes that are involved in reproduction were
regulated only in the presence of 1.7 mg/ml of the extracts.
Genes like CYP24A1 and DHFR are involved in cellular as
well as metabolic processes (Table 3A). In addition, under
the ‘‘Molecular function’’ category, the majority of genes
that were regulated in response to the 3 concentrations were
involved in binding, followed by catalytic activity and
transcriptional regulation activity (Fig. 7). Table 3B lists
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0.4 mg/ml vs control
0.6 mg/ml vs control
Fig. 5 A Venn diagram illustrating the number of genes in HepG2
cells that were mutually regulated at 0.4, 0.6 and 1.7 mg/ml of the
shoot extracts. The total numbers of genes regulated at 0.4, 0.6 and
1.7 mg/ml of the extracts of A. occidentale were 246, 696 and 4503
respectively. An increase in concentration by 1.5-fold from 0.4 to
0.6 mg/ml was able to increase the number of regulated genes by
threefold (65%). Increasing the concentration from 0.4 to 1.7 mg/ml
increases the number of regulated genes by 18-fold (95%). A total of
94 genes were mutually regulated in response to the 3 concentrations,
5 were up-regulated and 89 were down-regulated. Ninety-eight genes
were mutually regulated in response to the 0.4 and 0.6 mg/ml
extracts. On the other hand, 178 were mutually regulated in response
to the 0.4 and 1.7 mg/ml extracts. A larger number of genes of
571 were mutually regulated when cells were treated with 0.6 and
1.7 mg/ml extracts
selected genes such as INSR and LDLR that are involved in
binding, MAOB in catalytic activity and RB1 in transcription
regulator activity. The majority of the gene products were
found as cell part, in extracellular region/part and in synapses (Fig. 8), and the selected genes are listed in Table 3C.
Validation of the microarray data using qRT–PCR
The microarray data were validated by quantitating selected significantly regulated genes, PLAUR, PLCXD1,
SQSTM1, CYP24A1, DHFR, TYMS and LIPC using realtime RT–PCR (qRT–PCR). All data were normalized to the
reference gene, GAPDH. As shown in Fig. 9, the expressions of the PLAUR, PLCXD1 and SQSTM1 genes were all
up-regulated while those of CYP24A1, DHFR, TYMS and
LIPC were down-regulated. The expression patterns
obtained through qRT–PCR analysis were consistent with
those of the microarray results.
Discussion and conclusion
In Malaysia, the young leaves or shoots of the A.occidentale are widely consumed as salads and the locals believed
Genes Nutr (2011) 6:413–427
419
Table 2 Selected significantly expressed genes in HepG2 cells in response to treatment with 0.4, 0.6 and 1.7 mg/ml of the extracts of
A. occidentale shoots
Gene ID
Gene name
Gene product
Fold change
(0.4 mg/ml)
Fold change
(0.6 mg/ml)
Fold change
(1.7 mg/ml)
NM_005192
CDKN3
Cyclin-dependent kinase inhibitor 3
-2.7
-2.6
-10.5
AY605064
LOC100289612
Arsenic transactivated protein 1
-3.0
-3.6
-10.2
AK293146
NM_003384
DHFR
VRK1
Dihydrofolate reductase
Vaccinia related kinase 1
-2.4
-2.3
-2.4
-2.6
-10.0
-6.7
NM_001254
CDC6
Cell division cycle 6 homolog (S. cerevisiae)
-2.4
-2.7
-4.3
NM_030622
CYP2S1
Cytochrome P450, family 2, subfamily S, polypeptide 1
?1.8
?2.3
?3.5
NM_004961
GABRE
Gamma-aminobutyric acid (GABA) A receptor, epsilon
?1.6
?2.1
?2.7
NM_004217
AURKB
Aurora kinase B
-1.6
-1.6
-2.2
NM_000782
CYP24A1
Cytochrome P450, family 24, subfamily A, polypeptide 1
NR
-2.9
-27.8
NM_001792
CDH2
Cadherin 2, type 1, N-cadherin (neuronal)
NR
-1.8
-15.5
NM_001951
E2F5
E2F transcription factor 5, p130-binding
NR
-1.9
-5.5
NR_027676
BRCA1
Breast cancer 1, early onset
NR
-2.4
-5.1
NM_000236
LIPC
Lipase, hepatic
NR
-3.2
-4.7
NM_198433
AURKA
Aurora kinase A
NR
-1.5
-4.4
NM_001786
CDC2
Cell division cycle 2, G1 to S and G2 to M
NR
-2.5
-4.1
NM_001798
CDK2
Cyclin-dependent kinase 2
NR
-2.0
-3.2
NM_001237
CCNA2
Cyclin A2
NR
-1.7
-3.1
NM_000059
NM_000019
BRCA2
ACAT1
Breast cancer 2, early onset
Acetyl-coenzyme A acetyltransferase 1
NR
NR
-2.0
-1.9
-2.7
-2.6
NM_000075
CDK4
Cyclin-dependent kinase 4
NR
-1.8
-2.6
NM_001274
CHEK1
CHK1 checkpoint homolog (S. pombe)
NR
-1.8
-2.3
NM_000208
INSR
Insulin receptor
NR
?2.3
?2.6
NM_000596
IGFBP1
Insulin-like growth factor binding protein 1
NR
?3.7
?4.2
NM_004419
DUSP5
Dual specificity phosphatase 5
NR
?3.3
?9.1
NM_003667
LGR5
Leucine-rich repeat-containing G protein-coupled receptor 5
NR
NR
-16.4
NM_000321
RB1
Retinoblastoma 1
NR
NR
-7.6
NM_031966
CCNB1
Cyclin B1
NR
NR
-5.7
-3.7
NM_000253
MTTP
Microsomal triglyceride transfer protein
NR
NR
NM_002979
SCP2
Sterol carrier protein 2
NR
NR
-3.5
NM_004969
IDE
Insulin-degrading enzyme
NR
NR
-2.1
NM_000384
APOB
Apolipoprotein B
NR
NR
-2.0
NM_001013398
IGFBP3
Insulin-like growth factor binding protein 3
NR
NR
?8.0
NM_000527
LDLR
Low-density lipoprotein receptor
NR
NR
?3.6
NM_005952
NM_003102
MTIX
SOD3
Metallothionein 1X
Superoxide dismutase 3, extracellular
NR
NR
NR
NR
?2.8
?2.1
Details of the GenBank accession number, name of the gene and its respective gene product, fold change difference between treated and nontreated cells and p values are included. NR Not regulated
its benefits include diabetes control or prevention. The
methanol extracts of the shoots were found to have potent
antioxidant activities [35]. Bioactive compounds found in
the A. occidentale shoot extracts that could be linked to
the antioxidant activities and other medicinal properties
included myricetin and quercetin [19, 27], amentoflavone
[19] and agathisflavone [20]. Dietary antioxidants could be
absorbed through the intestine, albeit in small quantities.
Once in the circulation, they are quickly metabolized
[14, 44] but the antioxidant properties were retained [25].
Our group had reported that low concentration of crude
extract of antioxidant-rich T. indica (0.3 mg/ml) was able
to significantly regulate a sizable number of genes in
HepG2 cells [36]. In this study, based on the MTT assays,
cells showed more than 90% viability at 0.4 and 0.6 mg/ml.
The IC50 concentration was found to be 1.7 mg/ml. This
study was aimed to (1) investigate the effects of low and
high concentration of the A. occidentale shoot extracts on
123
420
Genes Nutr (2011) 6:413–427
Fig. 6 Gene ontology analysis under the ‘‘Biological process’’ category, of significantly regulated genes (P \ 0.01, fold change of at least ±1.5)
in HepG2 cells in response to 0.4 mg/ml (a), 0.6 mg/ml (b) and 1.7 mg/ml (c) of the extracts of A. occidentale shoots
the expression of genes in liver HepC2 cells and (2)
identify genes that could be associated with the medicinal
properties of the shoot extracts.
HPLC analyses showed the presence of quercetin in the
extracts of the A. occidentale shoots that confirmed earlier
findings by other researchers [19, 27]. In addition, we also
detected the presence of kaempferol which has not been
reported previously. Kaempferol possessed significant
antioxidant activities [34] and has cancer chemopreventive
properties towards several tumour cell lines including lung
and leukaemic cell lines [8, 31].
In this study, cDNA microarray analysis showed that the
extracts of the A. occidentale shoots at a low concentration
of 0.4 mg/ml was able to significantly up-regulated
(P \ 0.01) a total of 248 genes by at least 1.5-fold.
Amongst the down-regulated genes were those encoding
123
arsenic transactivated protein (LOC100289612), vacciniarelated kinase 1 (VRK1), dihydrofolate reductase (DHFR),
cell division cycle 6 homolog (S.cerevisiae) (CDC6),
cyclin-dependent kinase inhibitor 3 (CDKN3) and aurora
kinase B (AURKB). Increasing the concentrations from 0.4
to 0.6 mg/ml led to an increase in the number of regulated
genes from 248 to 696. Amongst the 696 genes were
CYP24A1, CDH2, E2F5, BRCA1, BRCA2, AURKA, CDC2
(CDK1), CDK2, CDK4, CHECK1, CCNA2, ACAT,
IGFBP1, DUSP5 and INSR. These genes were also
expressed in response to the IC50 concentration, but as
expected, the fold change was much larger compared to that
of the 0.6 mg/ml. At an IC50 concentration of 1.7 mg/ml, a
total of 4286 genes were significantly regulated. In addition,
the expressions of genes such as the LGR5, IGFBP3, RB1,
IDE, LDLR, MTTP, APOB, SCP2, MTIX, SOD and SOD3
Genes Nutr (2011) 6:413–427
421
Fig. 7 Gene ontology analysis under the ‘‘Molecular function’’ category, of significantly regulated genes (P \ 0.01, fold change of at least
±1.5) in HepG2 cells in response to 0.4 mg/ml (a), 0.6 mg/ml (b) and 1.7 mg/ml (c) of the extracts of A. occidentale shoots
were exclusively regulated at the 1.7 mg/ml dose. Interestingly, for all three concentrations, the down-regulated
genes were three times as many as those that were
up-regulated (full data are not shown) as indicated in the
hierarchical analysis. Mutually regulated genes in response to
the three concentrations included CDKN3, LOC100289612,
DHFR, VRK1, CDC6, AURKB, CYP2S1 and GABRE
(Table 2). Amongst the highly significantly suppressed genes
were CYP24A1, LGR5, CDH2 and DHFR by 27.8-, 16.4-,
15.5-, 10.0-fold, respectively. On the other hand, amongst
the highly induced genes were the DUSP5, IGFBP 3,
IGFBP1, LDLR and INSR by 9.1-, 8.0-, 4.2-, 3.6- and
2.6-fold, respectively.
CYP24A1 gene codes for the hepatic enzyme, CYP24A1
or 24-hydroxylase which is the rate limiting enzyme in the
catabolism of the active form of vitamin D, 1a,25-(OH)2D3
or calcitriol [18, 23]. 1a, 25-(OH)2 D3, synthesized in the
kidney by the CYP27B1, promotes dietary absorption of
calcium and phosphate as well as maintain the levels of the
two minerals. There are increasing evidences that individuals with low serum vitamin D have a higher risk of
developing various types of cancers [18, 23] and myocardial diseases [33]. In addition, 1a, 25-(OH)2 D3 is also
important in regulating cell cycle check points as well as
controlling multiple signalling pathways including those of
the MAPK/ERK, PI3 K/AKT, Wnt and TGF-b [5]. The
CYP24A1 gene has been reported to act as an oncogene and
its overexpression was detected in cancers of the colon,
ovary and lung [2, 18, 23]. In vivo studies have indicated
that exposing cancer cells to a high concentration of the
active metabolites of vitamin D stopped the cells from
progressing. This occurred via a mechanism affecting cell
cycle and increasing apoptosis, ultimately slowing or
stopping growth of the tumour [45]. The shoot extracts of
123
422
Genes Nutr (2011) 6:413–427
Fig. 8 Gene ontology analysis under the ‘‘Cellular component’’ category, of significantly regulated genes (P \ 0.01, fold change of at least
±1.5) in HepG2 cells in response to 0.4 mg/ml (a), 0.6 mg/ml (b) and 1.7 mg/ml (c) of the extracts of A. occidentale shoots
the A. Occidentale were able to highly suppress the
CYP24A1 gene; hence, it has the potential to be used
synergistically with vitamin D to maintain the bioavailability and bioactivity of the latter.
Apart from the CYP24A1 gene, LGR5 and CDH2 were
also highly suppressed, by 16- and 15-fold respectively
at the IC50 concentration of the shoot extracts. The leucinerich repeat-containing G-protein-coupled receptor 5
(LGR5) belongs to the G-protein-coupled receptor (GPCR)
superfamily [15]. LGR5 had been reported to be a marker
of adult stem cells where the LGR5 gene transcription is
under the control of the canonical or beta-catenin Wnt
signalling pathway [11]. This pathway, which is involved
in embryogenesis and normal physiological processes, is
critical in the regulation of adult stem cells [11, 37].
Dysregulation of this pathway is linked to cancers [37]. An
overexpression of LGR5 was observed in many types of
123
cancers including those of the colon [26, 46], oesophagus
[4], ovary [26] and the hepatocytes [47]. In this study,
genes associated with the Wnt/b-catenin signalling pathway were also regulated at the IC50 concentration, albeit
modestly. These included the Wnt6, FZDs 1, 4, 6, 7, 8, 9
and 10, CDH1, CDH2, CTNNA2, DKK1, APC, NUCKS1,
CSNK1G2, CSNK1G3 and TCF (data not shown).
One of the most interesting observations of this study
was the fact that the shoot extracts were able to directly
regulate a spectrum of genes involved in the G1 as well as
G2 cell cycle check points. These included the CDK1
(CDC2), CDK2, CDK4, CDK5, CDK6, CCNA2, CCNB1
and 2, CCNE1 and 2, CCNH, CDKN1A (p21/Cip1),
CDKN1C (p57/Kip2), CDKN2B (p15), CDKN2D (p19),
CDKN3, RB1, RBL1, RBL2, NSUN6, NOP2, AURKA,
AURKB, DAPK1, PAK2, E2F5, HDAC2, VRK1, CREB and
G2E3. Genes coding for other ubiquitin ligase isoforms,
Genes Nutr (2011) 6:413–427
423
Table 3 Gene ontology analysis of selected significantly regulated genes
(A) Gene ontology Selected down-regulated genes
(Biological
process)
Selected up-regulated genes
Cellular process
CYP24A1, DHFR, CDH1, CDH2, CDC2, CDK2, CDK4, CDK5, DUSP5, INSR, IRS2, SOD2, SOD3, LDLR
CDK6, CDKN3, CCNA2, CCNB1, CCNB2, CCNE1, CCNE2,
CHEK1, RB1, AURKA, AURKB, BRCA1, BRCA2, IDE, LIPC,
MTTP, SCP2, APOB, ACAT1
Biological
regulation
CDH1, CDC2, CDK2, CDK4, CDK5, CDK6, CDKN3, CHEK1,
CDH4, CDKN2B, CDKN1A, APC2, IRS2, WNT6,
CCNA2, CCNB1, CCNB2, CCNE1, CCNE2, E2F4, AURKA,
FZD1, FZD7, FZD8, FZD9, FZD10, IGFBP1,
RB1, CKS1B, PAK2, BRCA1, BRCA2, CSNK1G3, FZD4, FZD6, IGFBP2, IGFBP3, IGFBP6, INSR, LDLR, SOD2
LIPC, APOB, APOBEC3F, APOH, MTTP, IDE
Metabolic process CDH1, CCNH, CHEK1, CDK5, E2F4, CYP24A1, RB1, DHFR,
BRCA1, LIPC, MAOB, MTTP, SCP2, ACAT1, APOB
APC2, SOD2, SOD3, INSR, LDLR
Response to
stimuli
Establishment of
localization
BRCA1, BRCA2, CHEK1, CDK5
IRS2, CDKN2D, SOD2, SOD3, CDKN2B,
CDKN1A, MT1X
LDLR
Developmental
process
CDH2, RB1, CCNB2, BRCA1, SCP2, FZD6, CDK6, BRCA2,
CCNF, DKK1, CDK5 E2F4, IDE, APOB, CDH1, FZD4
FZD7, CDK5R1, FZD9, IRS2, IGF2, CDKN2D,
FZD10, CDKN1C, SOD2, FZD8, WNT6, FZD1,
INSR
Multi-organism
process
RB1
INSR, LDLR
Biological
adhesion
CDH2, FZD6, CDK5, CDH1
CDH4
Reproductive
process
SCP2, BRCA2, CHEK1, APOB
Multicellular
organismal
process
APOH, ACAT1, CDK5, E2F4
FZD9, LDLR
Rythmic process
CDK4
–
Growth
CCNB2, BRCA2
–
Pigmentation
–
SOD2
LIPC, MTTP, SCP2, APOB
(B) Gene ontology
(Molecular function)
Selected down-regulated genes
Selected up-regulated genes
Binding
CYP24A1, CDH2, CDKN3, RB1, DHFR, CCNB1,
CCNB2, BRCA1, LIPC, AURKA, CDC2,
BRCC3, MTTP, SCP2, CDK2,CCNA2, APOH,
CDK6, RBL2, BRCA2, ACAT1, CDK4, CHEK1,
CCNE1, AURKB, CDK5, E2F4, IDE, APOB,
CDH1, FZD4, CSNK1G2, CCNE2
CYP24A1, CDKN3, DHFR, BRCA1, LIPC,
AURKA, CDC2, MAOB, SCP2, CDK2, CDK6,
BRCA2, ACAT1, CCNH, CDK4, CHEK1,
AURKB, CDK5, IDE
CDH4, IGFBP2, IRS2, FZD10, SOD2, FZD8,
WNT6, SOD3, IGFBP6, FOXO3, FZD1, INSR,
MT1X, LDLR, IGFBP3
Catalytic activity
SOD2, SOD3, INSR, DUSP5
Transcription regulator activity
RB1, BRCA1, BRCA2, E2F4, CDH1
FOXO3
Structural molecular activity
Molecular transducer activity
–
FZD6, CDK5, IDE, FZD4
WNT6
FZD7, FZD9, IRS2, FZD10, FZD8, WNT6, FZD1,
INSR, MT1X, LDLR
Enzyme regulator activity
CDH1, CCNE1
IGFBP3
Transporter activity
LIPC, MTTP, APOB
LDLR
Electron carrier activity
CYP24A1, MAOB
–
Antioxidant activity
–
SOD3
123
424
Genes Nutr (2011) 6:413–427
Table 3 continued
(C) Gene ontology
(Cellular component)
Selected down-regulated genes
Selected up-regulated genes
Cell part
CYP24A1, LGR5, CDH2, RB1, CCNB1, CCNB2,
E2F5, TYMS, BRCA1, LIPC, ATG4C, SOS1,
AURKA, G2E3, CDC2, CHEK2, BRCC3,
MAOB, MTTP, SCP2, GAS2, MTHFD1, CDK2,
CCNA2, APOH, CKS1B, RBL1, UBE2C, FZD6,
BARD1, PAK2, CDK6, RBL2, BRCA2, CCNF,
ACAT1, CCNH, CDK4, PAK1IP1, NUCKS1,
DKK1, STAT2, CHEK1, CCNE1, AURKB,
CDK5, E2F4, DAPK1, IDE, TOP2B, BCCIP,
CSNK1G3, APOB, CDH1, TET1, APOBEC3F,
FZD4, ACAT2, CSNK1G2, CCNE2, CDK7,
LRP1
FZD7, CTNNA2, CDK5R1, CDH4, APC2, FZD9,
IRS2, CDKN2D, FZD10, CDKN1C, AQP6,
SOD2, FOLR3, FZD8, AQP12A, SOD3, FOXO3,
FZD1, CDKN2B, CDKN1A, LRP12, INSR,
LDLR, AQP3, EGLN3, IGFBP3, DUSP5
Extracellular region
LIPC, ATG4C, APOH, DKK1, APOB
APC2, IGFBP2, IGF2, FOLR3, WNT6, SOD3,
IGFBP6, IGFBP4, IGFBP1, IGFBP3
Synapse
Extracellular region part
CDH2, CDK5
LIPC, APOH, IDE, APOB
CDK5R1
APC2, IGFBP2, IGF2, WNT6, SOD3, IGFBP4,
IGFBP1, IGFBP3
Macromolecular complex
–
APC2
Synapse part
SOS1
–
Genes were categorized according to their involvement in biological process (A), molecular function (B) and as cell component (C)
Fig. 9 Validation of the microarray data using semi-quantitative
RT–PCR (qRT–PCR). A few genes that were significantly regulated
in HepG2 cells in response to treatment with extracts of the
A. occidentale shoots were selected namely PLAUR, PLCXD1,
SQSTM1, CYP24A1, DHFR, TYMS and LIPC using real-time
RT–PCR. All data were normalized to the reference gene, GAPDH.
The expressions of the PLAUR, PLCXD1 and SQSTM1 genes were all
up-regulated while those of CYP24A1, DHFR, TYMS and LIPC were
down-regulated. The expression patterns obtained through real-time
RT–PCR were consistent with the microarray results
123
UBE2C and UBE3B, was also aberrantly expressed. VRK1
is involved in the regulation of DNA replication through
the phosphorylation of CREB leading to the regulation of
CCND1 gene expression [17].
Aurora kinases comprise three members, Aurora A,
Aurora B and Aurora C. Aurora-A is transcriptionally
regulated by E2F3 during a cell cycle. E2F3 induces
Aurora-A expression by binding directly to Aurora-A promoter and subsequently stimulates the promoter activity
[12]. Both could thus be an important target for cancer
intervention [12]. In addition, aurora-B has been shown to
be overexpressed in many cancers including breast cancers
[10]. The methanol extracts of the A. occidentale shoots at
the IC50 concentration, suppressed the Aurora A and Aurora B, but not Aurora C, by fourfold and twofold respectively. An RNA methyltransferase, NSUN2 had been
shown to be a novel substrate for Aurora B, which contained a NOL/NOP/sun domain [38]. In this study, the
extracts were able to suppress both Aurora A and Aurora B
as well as NOP2 and NSUN6, suggesting its potential as an
anti-cancer agent.
Insulin-like growth factor binding protein-3 (IGFBP3)
inhibits the growth of non-small cell lung cancer (NSCLC)
cells. IGFBP3 overexpression inhibits the phosphorylation
of Akt and glycogen synthase kinase-3 beta and the activity
of MAPK, all three are activated by IGF-mediated signalling pathways that have mitogenic and anti-apoptotic
properties and have been implicated in the development of
Genes Nutr (2011) 6:413–427
lung cancer [24]. Nuclear IGFBP3 induces apoptosis and
is targeted to ubiquitin/proteosome-dependent proteolysis.
IGFBP3 degradation is dependent on active ubiquitin-E1
ligase [41].
The shoot extracts also up-regulated LDLR gene which
correlated with findings by Salleh et al. [39] who reported
that the cashew shoots were able to increase LDLR
activity in cultured HepG2 cells. Other genes associated
with lipid metabolism including LIPC, ACAT1, MTTP,
APOB and SCP2 were down-regulated. LDL-R is
responsible for the internalization of cholesterol-rich
lipoprotein, LDL, from the blood circulation through the
recognition of its ApoB by the receptor [6]. SCP2 gene
expression was reported to be enhanced by oxidized LDL
[13] which could be ingested by macrophages leading to
the formation of foam cells, a critical step in atherosclerosis. ACAT is responsible to convert free cholesterol to
cholesteryl ester in tissues. ACAT1 is the main isoenzyme
in the neuronal brain [7], and its presence is associated
with certain forms of Alzheimer disease. MTTP encodes
microsomal triacylglycerol transfer protein (MTP) which
is required for the assembly of nascent chylomicrons and
VLDL while hepatic lipase encoded by LIPC does not
only hydrolyses triacylglycerols and phospholipids in circulating plasma lipoproteins, it also regulates lipoprotein
uptake by cells [40]. Except for the ACAT1, the expression
of the LDLR, MTTP, SCP2 and APOB genes was only
observed in response to the IC50 concentration, not those
of 0.4 and 0.6 mg/ml suggesting a concentration-dependent expression of the genes.
The anti-diabetic properties of the shoot extracts could
be linked to the up-regulation of the genes coding for the
insulin receptor and the down-regulation of the insulindegrading enzyme (IDE).
The hypolipidaemic, anti-diabetic and anti-cancer
properties could be attributed to the presence of quercetin
and kaempferol in the shoot extracts of the A. occidentale.
Quercetin and kaempferol are normally present in nature as glycosides. Free and glycoside forms of the two
flavonoids are absorbed in the intestine (9, 14, 48) and are
found conjugated with glucuronides or sulphates in the
blood circulation. The concentrations of the extracts
selected in the study were based on MTT assays whereby
cell viability was maintained above 90% at the extract
concentrations of 0.3 and 0.4 mg/ml. The concentrations
used were probably not a true reflection of the flavonoids
levels in the plasma as it was reported that the levels of
free and conjugated flavonoids such as kaempferol in
blood was in the range of 1.7–6.1 lg/ml after oral ingestion of 25–50 mg/kg body weight [48]. However, when
using an in vitro model like HepG2 cells, sometimes it is
necessary to use higher concentrations of extracts to see
significant changes in gene expression [36]. Nevertheless,
425
this in vitro study is still useful to provide preliminary
information on the possible molecular mechanisms in
relation to the medicinal properties of the shoot extracts of
A. occidentale. Further confirmation using an in vivo
model would need to be carried out to corroborate the in
vitro findings.
Conclusion
In conclusion, low concentrations of the shoot extracts of
A. occidentale were able to significantly regulate a sizable
number of genes in HepG2 cells. However, the type of the
expressed genes was highly dependent on the concentration
of the extracts used. Amongst the genes that were significantly regulated were those involved in regulating cell
cycle check points, apoptosis and cell proliferation, lipoprotein metabolism and insulin signalling suggesting the
potential of the plant to act as anti-cancer, hypolipidaemic
and anti-diabetic agents.
Acknowledgments This study was funded by the following
research grants: E-Science Fund (12-03-02-2061) from the Ministry
of Science, Technology and Innovation Malaysia (MOSTI), FRGS
(FP004/2003C) from the Ministry of Higher Education Malaysia
(MOHE), the UMRG (RG014/09AFR) and Research University
Grants (SF076-2007A & FR113/2007A) from the University of
Malaya. We would like to thank Mr Siah Eng Tian for the technical
assistance on the microarray analysis.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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