1
Ethanol extract of Hedyotis diffusa Willd up-regulates G0/G1 phase
arrest and induces apoptosis in human leukemia cells by modulating
caspase cascade signaling and altering microarray-assisted pathway
YU‑JUI KUO1, JAI-SING YANG2, CHI-CHENG LU3, SU-YIN CHIANG1,
JAUNG-GENG LIN1, JING-GUNG CHUNG4
1
School of Chinese Medicine-Chinese Herbal Medicine, China Medical University,
Taichung, Taiwan, R.O.C. 2Department of Pharmacology, China Medical University,
Taichung, Taiwan, R.O.C. 3Department of Life Sciences, National Chung Hsing
University, 250, Kuo-Kuang Road, Taichung 402, Taiwan. 4Department of Biological
Science and Technology, China Medical University, Taichung, Taiwan, R.O.C.
Running Title: Hedyotis diffusa Willd induces cell cycle arrest and apoptosis in HL60
cells
Correspondence to: Prof. Jing-Gung Chung, Department of Biological Science and
Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404,
Taiwan. Tel.: +886-4-2205-3366 ext 2161. Fax: +886-4-2205-3764.
E-mail: jgchung@mail.cmu.edu.tw
ABSTRACT: Our previous study has shown that water extract of Hedyotis diffusa
Willd (HDW) promoted immune response and exhibited anti-leukemic activity in
BALB/c leukemic mice in vivo. In the present study, we investigated the
anti-proliferation effects of ethanol extract of Hedyotis diffusa Willd (EEHDW) on
2
lung cancer cell lines (A549, H1355 and LLC), leukemia cell lines (HL-60, WEHI-3)
and a mouse melanoma cell line (B16F10) in vitro. Our results demonstrated that
EEHDW suppressed the cell proliferation of A549, H1355, HL-60, WEHI-3 and
B16F10 cells as well as reduced cell viability in a concentration-dependent manner.
We also found that EEHDW induced cell morphological changes and time- and
concentration-dependently inhibited the cell proliferation of HL-60 cells. In addition,
EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and sub-G1 population
(apoptotic cells). EEHDW provoked DNA condensation and DNA damage in HL-60
cells. The activities of caspase-3, -8 and -9 were elevated in EEHDW-treated HL-60
cells. We also used the DNA microarray to investigate and display the gene levels
related to cell growth, signal transduction, apoptosis, cell adhesion, cell cycle, DNA
damage and repair, transcription and translation. Our findings suggest that EEHDW
may be a potential herbal medicine and therapeutic agent for the treatment of
leukemia.
Key words: Hedyotis diffusa Willd, G0/G1 phase arrest, apoptosis, human leukemia
cells.
Introduction
Hedyotis Diffusa Willd (HDW) has been used in traditional Chinese medicine
(TCM) in Taiwan and China for thousands of years (Lin et al. 2011a). In China, HDW
is used as cancers therapy agent and improved the adverse reactions of chemotherapy
(Lin et al. 2013b),(Chen et al. 2012). The studies of molecular pharmacology showed
that HDW contains major compounds with anti-cancer activities, including
anthraquinones (Liu et al. 2010; Niu and Meng 2013; Wang et al. 2011), polyphenols
(Han et al. 1997), organic acids (Huang et al. 2009),(Cheung et al. 2006),
3
hemiterpenes (Niu and Meng 2013; Wang et al. 2012) and polysaccharides (Cui et al.
2006; Lin et al. 2013c). Several reports showed that the extract of HDW induced cell
cycle and apoptosis in many human cancer cell lines (Lin et al. 2011b; Lin et al. 2012;
Liu et al. 2010; Zhang et al. 2012). The extract of HDW has also proven to target
multiple signaling pathways that inhibit tumor cell proliferation, induce cell cycle
arrest and apoptosis as well as suppress inflammation molecules (Lin et al. 2011a; Lin
et al. 2011b; Lin et al. 2012; Liu et al. 2010; Zhang et al. 2012). It was reported that
injection of HDW could inhibit the proliferation of RPMI 8226 human hematopoietic
cells (Zhang et al. 2012). The anti-cancer mechanisms of HDW might be correlated
with early apoptosis induction, cell cycle arrest, and anti-angiogenesis (Lin et al.
2013b; Lin et al. 2011b; Lin et al. 2012). Furthermore, the studies demonstrated that
the ethanol extract of Hedyotis diffusa Willd (EEHDW) inhibited colorectal cancer
growth in vivo through inhibition of SHH-mediated tumor angiogenesis (Lin et al.
2011b; Lin et al. 2012). Our earlier study demonstrated that water extract of HDW
can reduce the weights of spleen and liver and increased the percentage of CD11b cell
surface marker (monocytes), but it reduced the percentage of CD3 (T-cell) and CD19
(B-cell) markers in WEHI-3 leukemic mice (Lin et al. 2011a). Importantly, no
significant effect on body weight occurred in treated animals (Lin et al. 2011a).
However, the molecular mechanism of the anti-cancer activity of EEHDW remains
unclear and not well investigated. The purpose of the research was to focus on the
molecular mechanisms triggered by EEHDW in leukemia, lung cancer and melanoma
cells, and then to clarify the anti-leukemia mechanism of EEHDW in vitro.
Materials and methods
Chemicals
and
Reagents.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), ethanol, propidium iodide (PI), RNase A, Triton X-100 and
4
proteinase K were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
RPMI-1640 medium, Dulbecco’s modified eagle medium (DMEM), L-glutamine,
penicillin/streptomycin and trypsin-EDTA were obtained from Life Technologies
(Carlsbad, CA, USA). Caspase-3, -8 and -9 activity assay kits were purchased from
R&D Systems Inc. (Minneapolis, MN, USA).
Cell Culture. The leukemia cell lines HL-60 (human promyelocytic leukemia) and
WEHI-3 (mouse myelomonocytic leukemia cell), the lung cancer cell lines A549
(human lung adenocarcinoma cells), H1355 (human lung adenocarcinoma cells), LLC
(mouse Lewis lung adenocarcinoma cells) and B16F10 (mouse melanoma cells) were
purchased from the Bioresource Collection and Research Center (BCRC) of Food
Industry Research and Development Institute (Hsinchu, Taiwan). HL-60, WEHI-3
and B16F10 cells were cultured in RPMI-1640 medium. A549, H1355 and LLC were
cultured in DMEM medium. All media were supplemented with 10% heat-inactivated
fetal calf serum (FCS) (HyClone, Logan, UT, USA), 100 Units/ml penicillin, 100
μg/ml streptomycin and 2 mM L-glutamine at 37 °C in a 5% CO2 humidified
incubator (Lin et al. 2013a; Wen et al. 2010; Yang et al. 2006).
Preparation for Ethanol Extract of Hedyotis Diffusa Willd (EEHDW). Hedyotis
Diffusa Willd (500 g) were extracted with 5000 ml of 50% ethanol using refluxing
method and filtered. The ethanol solvent was then evaporated on a rotary evaporator.
The dried powder of EEHDW was obtained by a spraying desiccation method using a
spray dryer. Stock solutions of EEHDW were prepared by dissolving the powder in
DMSO to a concentration of 100 mg/ml and stored at -20 °C. The working
concentrations of EEHDW were made by diluting the stock solution in the culture
medium. The final concentration of DMSO in the medium was also kept below 0.2%.
5
Cell Viability and Apoptotic Features. The cell viability assay was assessed by the
MTT method (Chiang et al. 2013). All cells were individually cultured in a 96-well
plate at a density of 1x104 cells per well and incubated with 0, 25, 50, 100 and 200
μg/ml of EEHDW for 24 or 48 h. After that, culture medium containing 500 μg/ml
MTT was added to each well, and then incubated at 37 °C for 4 h before the
supernatant was removed. The formed blue formazan crystals in viable cells were
dissolved with isopropanol/0.04 N HCl, followed by measurement of the absorbance
of each well at 570 nm with the ELISA reader. All experiments were performed in
triplicate. The morphological examination in EEHDW-treated HL-60 cells was
determined under a phase-contrast microscope as described elsewhere (Chen et al.
2010; Wen et al. 2010).
Cell Cycle Distribution Analysis. HL-60 cells were seeded in 12-well plates at a
density of 2.5x105 cells/well and incubated with or without 100 μg/ml of EEHDW for
24 h. For determination of cell cycle phase and apoptosis, cells were fixed gently by
putting 70% ethanol in -20 °C overnight, and then re-suspended in 40 μg/ml PI of
PBS, 0.1 mg/ml RNase and 0.1% Triton X-100 in dark room for 30 min. Cell cycle
distribution and apoptotic nuclei were measured by flow cytometry as previously
described (Yang et al. 2004).
DAPI Staining. HL-60 cells in 12-well plates were treated with or without 100 μg/ml
of EEHDW for 48 h. Cells were thereafter fixed in 4% paraformaldehyde and exposed
to 1 μg/ml of 4’,6-diamidino-2-phenylindole (DAPI) (Molecular Probes by Life
Technologies, Eugene, OR, USA) staining solution in darkness. The apoptotic cells
were observed by fluorescence microscopy (Zeiss, Oberköchen, Germany) as
6
previously described (Chen et al. 2013; Huang et al. 2010).
Comet Assay. HL-60 cells were grown in 12-well plates and incubated with 100 μg/ml
of EEHDW for 48 h. Cells were mixed with 150 μl 0.75% low-melting agarose
(Sigma-Aldrich Corp.) held at 37 °C and layered onto a pre-treated slide with 1.5%
regular agarose. After agarose were solidified on a chilled plate, the slides were
transferred to the same lysis buffer, held at room temperature for 4 h and stained with
propidium iodide (PI) as described elsewhere with some modifications (Chiang et al.
2011; Lu et al. 2010).
Assays for Caspase-3, -8 and -9 Activities. HL-60 cells in 12-well plates were
incubated in presence or absence of 100 μg/ml of EEHDW for 24 h. Cells were then
harvested, and cell lysates were assessed in accordance with the manufacturer’s
instruction provided in the caspase-3, -8 and -9 Colorimetric Assay Kits (R&D
System Inc., Minneapolis, MN, USA). Cell lysates containing 50 μg proteins were
incubated for 1 h at 37 °C with individual specific caspase-3, -8 or -9 substrate in the
reaction buffer (provided in the kits) and measured by OD405 of the released DNA as
previously described (Chiu et al. 2011; Huang et al. 2012; Yang et al. 2012).
cDNA Microarray. HL60 cells (1×107 cells) were incubated with or without 100
μg/ml of EEHDW for 24 h. After exposure, cell pellets were collected, and the total
RNA from each treatment was purified using the Qiagen RNeasy Mini Kit (Qiagen,
inc, Valencia, CA, USA). The RNA purity was determined to check the quality at
260/280 nm using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific,
Wilmington, DE, USA). mRNA was amplified and labeled using the GeneChip WT
Sense Target Labeling and Control Reagents kit (Affymetrix, Santa Clara, CA, USA)
7
for expression analysis. The synthesized cDNA was labeled with fluorescence, and
then hybridized d for 17 h utilizing Affymetrix GeneChip Human Gene 1.0 ST array
(Affymetrix) to determine for microarray hybridization following the manufacturer’s
prototols. The arrays were subsequently washed by Fluidics Station 450 (Affymetrix),
stained with streptavidin-phycoerythrin (GeneChip Hybridization, Wash, and Stain kit,
Affymetrix) and scanned on a GeneChip Scanner 3000 (Affymetrix). The localized
concentrations of fluorescent molecules were quantitated and analyzed using
Expression Console Software (Affymetrix) with default RMA parameters as
previously described (Chang et al. 2012; Liu et al. 2013). The gene expression level
of a 5-fold change was considered a difference in EEHDW-treated HL60 cells in
vitro.
Statistical Analysis. All the statistical results were performed as the mean ± standard
error of the mean (S.E.M.) for the indicated numbers of independent experiments.
Statistical analyses of data were performed using one-way ANOVA followed by
Student's t-test, and the levels of *P < 0.05 was considered significant between the
treated and untreated group (Chang et al. 2012).
Results
EEHDW Reduces the Viability of Human Cancer Cell Lines. The lung cancer cell
lines (A549, H1355 and LLC), leukemia cell lines (HL-60, WEHI-3) and a mouse
melanoma cell line (B16F10) were treated with EEHDW (0, 25, 50, 100 and 200
μg/ml) for 24 or 48 h. The viability were determined by MTT assay. The
concentrations 25, 50, 100 and 200 μg/ml of EEHDW treatment significantly
decreased cell viability in A549, H1355 and HL-60 cells in a concentration-dependent
manner (Figures 1A, 1B and 1D). EEHDW at 50, 100 and 200 μg/ml markedly
8
decreased cell viability in WEHI-3 and B16F10 cells in a concentration-dependent
effect (Figures 1E and 1F). However, we found that EEHDW treatment did not
significantly decrease cell viability of LLC cells (Figure 1C). Our results suggested
that EEHDW exhibited the anti-cancer action and was sensitized in leukemia, lung
cancer and melanoma cells in vitro.
EEHDW Induces Apoptosis and Cell Cycle Arrest in Human Leukemia HL-60 Cells.
Cells were exposed to EEHDW (0, 25, 50, 100 and 200 μg/ml) for 24 and 48 h. The
cell viability was determined by MTT assay. The concentrations of 25, 50, 100 and
200 μg/ml of EEHDW treatments significantly decreased cell viability in HL-60 cells
in a concentration- and time-dependent manner (Figure 2A). After exposure to 100
μg/ml of EEHDW for 48 h, apoptotic bodies were visualized in EEHDW-treated
HL-60 cells (Figure 2B). Our studies investigated the possible mechanisms focused
on cell cycle arrest or apoptosis by inhibitory effects in HL-60 cells after EEHDW
incubation. Figure 3 indicates that EEHDW induced sub-G1 phase (apoptotic cell
death) and increased G0/G1 phase arrest with 24-h treatment. Our results imply that
EEHDW induced G0/G1 phase arrest and cell apoptosis in HL-60 cells.
EEHDW Triggers DNA Condensation and Damage in HL-60 Cells. Effects of
EEHDW on nuclear morphological change were examined by DAPI staining and
DNA damage by the Comet assay. The results in Figure 4 demonstrated that HL-60
cells exhibited nuclear shrinkage and DNA condensation after a 48-h treatment with
100 μg/ml of EEHDW (Figure 4A). HL-60 cells after exposure to 100 μg/ml of
EEHDW had an increase in DNA damage from the Comet assay (Figure 4B). These
results hint that EEHDW induced DNA condensation and DNA damage in HL-60
cells.
9
EEHDW-Stimulated Apoptosis Occurs Through the Induction of Caspase-3, -8 and -9
Activities. HL-60 cells were treated with or without 100 μg/ml of EEHDW for 24 h
before being subjected to caspase-3, -8 and -9 activities. Our data as shown in Figure
5 revealed that EEHDW promoted caspase-3, -8 and -9 activities in HL-60 cells.
Based on our findings, it is concluded that the caspase cascade contributed to
EEHDW-induced apoptosis in HL-60 cells.
Microarray Analysis. cDNA microarray was analyzed to examine the expressed genes
in HL-60 cells after treatment with or without 100 μg/ml of EEHDW as listed in Table
1. We showed that the transcripts of 6 genes with fold changes >5.0 were up-regulated,
while these of 97 genes were down-regulated in EEHDW-treated HL-60 cells. Our
results revealed that EEHDW affected the expression of important genes that
regulated cell growth (AKR1C2, AKR1C1, AKR1C1, ALDH3A1, TXNRD1 and
ANKRD11);
signal
transduction
(G
protein-coupled
receptor
109A,
G
protein-coupled receptor 20, EGF-containing fibulin-like extracellular matrix protein
2, mitogen-activated protein kinase 12, G protein-coupled receptor 73, sprouty
homolog 1, antagonist of FGF signaling, estrogen-related receptor beta, Ral-GDS
related protein Rgr, purinergic receptor P2Y, G-protein coupled, 2, fms-related
tyrosine kinase 4, nasal embryonic LHRH factor, lymphocyte antigen 6 complex,
locus E, hypothetical protein MGC4171;mitogen-activated protein kinase 3,
interleukin
6
receptor);
Apoptosis
(clusterin,
hypothetical
protein
MGC13096,scavenger receptor class B, member 1; BCL2-like 11 (apoptosis
facilitator), hypothetical protein FLJ20425); cell adhesion (GTPase activating
RANGAP domain-like 3, bystin-like); cell cycle (cyclin-dependent kinase 4,
mitochondrial topoisomerase I, replication factor C (activator 1) 1, 145kDa); DNA
10
damage and repair (APEX nuclease (multifunctional DNA repair enzyme) 1,
RuvB-like 1, uracil-DNA glycosylase); transcription and translation (nuclear factor
I/A, zinc finger protein 584, zinc finger protein 202, nuclear factor (erythroid-derived
2), 45kDa, D site of albumin promoter (albumin D-box) binding protein,
lymphoblastic leukemia derived sequence 1, SLC2A4 regulator, polymerase (RNA)
III (DNA directed) polypeptide K, 12.3 kDa, polymerase (RNA) I polypeptide C,
30kDa, polymerase (RNA) I polypeptide B, 128 kDa, polymerase (DNA directed),
epsilon 3 (p17 subunit), MYB binding protein (P160) 1a, polymerase (RNA) I
polypeptide B, 128kDa, PEPP subfamily gene 2, v-myc myelocytomatosis viral
oncogene homolog (avian), POU domain, class 4, transcription factor 3, FLJ42986
protein.
Discussion
It has been reported that HDW is one of the most popular traditional herbal medicines
in Taiwan and China (Lin et al. 2011a). It is noteworthy that HDW has various
pharmacological activities such as anti-cancer (Chen et al. 2008; Lin et al. 2011b),
anti-inflammatory (Lin et al. 2002), hepatoprotective (Lin et al. 2002), and
neuroprotective activities (Kim et al. 2001). HDW is used as a therapy to treat
hepatoma (Chen et al. 2012; Lee et al. 2011; Lin et al. 2011a), lung cancer (Lee et al.
2011), colon cancer (Lee et al. 2011; Lin et al. 2010; Lin et al. 2011b; Lin et al. 2012),
Pancreatic cancer and leukemia (Chen et al. 2008; Lin et al. 2011a). Our earlier study
demonstrated that the water extract of HDW caused anti-leukemic effects in the
BALB/c mouse WEHI-3 model (Lin et al. 2011b). This report is the first study to
investigate the anti-lung cancer effects of EEHDW on human A549 and H1355 cells
in vitro. Although Chen et al have demonstrated that water extract of HDW can
inhibit cell growth of HL-60 cells (Chen et al. 2008), the molecular mechanism of the
11
anti-leukemia activity of EEHDW still showed unsatisfactory results. Our study also
provided the first pharmacological evidence for the anti-leukemia effects of EEHDW
on HL-60 cells by DNA microarray.
The previous studies demonstrated that water extract of HDW inhibited HepG2
cell proliferation through G0/G1 phase arrest and induction of S phase delay (Chen et
al. 2012; Shi et al. 2008). In addition, Lin et al have demonstrated that EEHDW
treatment blocked the cell cycle, preventing G1 to S progression, and reduced mRNA
expression of pro-proliferative PCNA, Cyclin D1 and CDK4, but increased p21
(CDKI) in HT-29 human colon cancer cells (Lin et al. 2010). Our results
demonstrated that EEHDW inhibited HL-60 leukemia cell proliferation (Figure 2)
through G0/G1 phase arrest and induction of cell apoptosis (Figure 3). In addition,
EEHDW affected the expression of important genes that regulated cell cycle G0/G1
phase include cyclin-dependent kinase 4, mitochondrial topoisomerase I, replication
factor C (activator 1) 1, 145kDa gene (Table 1). Our present results indicated that
EEHDW-induced anti-proliferation action might be mediated by the way of G0/G1
phase arrest in HL-60 cells.
Apoptosis is one of the major forms of cell death, associated with characteristic
morphological changes including the formation of membrane blebs and apoptotic
bodies, DNA condensation and fragmentation (De la Rosa et al. 2013; Stevens et al.
2013; Wood et al. 2013). It was reported that EEHDW induced DNA fragmentation,
loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential
and activation of caspase-3 and caspase-9 in HT29 colon cancer cells (Lin et al. 2010).
Our results also showed that EEHDW inhibited HL-60 leukemia cell proliferation
(Figure 2) and induced DNA condensation (Figure 4A), DNA damage (Figure 4B)
and apoptotic cell death (Figure 2B). We revealed that EEHDW affected the
expression of important genes that regulated cell proliferation gene (AKR1C2,
12
AKR1C1, AKR1C1, ALDH3A1, TXNRD1 and ANKRD11), Apoptotic cell death
gene (clusterin, hypothetical protein MGC13096, scavenger receptor class B, member
1; BCL2-like 11 (apoptosis facilitator), hypothetical protein FLJ20425) and DNA
damage and repair gene (APEX nuclease (multifunctional DNA repair enzyme) 1,
RuvB-like 1, uracil-DNA glycosylase) (Table 1). Caspases play a vital role in cell
apoptosis. There are two major pathways by which a caspase cascade results in the
activation of effector caspase-3 initiated by the apical caspases such as caspase-8 and
-9 (Fiandalo and Kyprianou 2012; Hensley et al. 2013; Olsson and Zhivotovsky 2011).
Figure 5 showed EEHDW provoked apoptosis through the activation of caspase-3, -8
and -9 in HL-60 cells. These results conclude that EEHDW-induced apoptosis might
be via the caspase-dependent signaling pathway, which has connection with the
activation of caspases-3, -8 and -9 signals. Wang et al has shown that treatment of
U937 cells with 2-hydroxy-3-methylanthraquinone from HDW resulted in induction
of apoptosis and activation of caspase-3 pathway (Wang et al. 2013). Furthermore,
2-hydroxy-3-methylanthraquinone also induces apoptosis through Fas/FasL-mediated
caspase-8 pathway in human leukemic THP-1 cells (Wang et al. 2011). Hence,
2-hydroxy-3-methylanthraquinone show major potential as a selective anti-leukemia
compound by modulating apoptosis in leukemia cells.
In conclusion, EEHDW is likely to induce growth inhibition of A549, H1355,
HL-60, WEHI-3 and B16F10 cells. We also found that EEHDW promoted G0/G1
phase arrest and apoptosis in HL-60 cells. The molecular signaling pathways involved
in these effects on HL-60 cells are summarized in Figure 6. The findings suggest that
EEHDW triggered apoptotic cell death through activation of caspase-3, 8 and 9 in
HL-60 cell. EEHDW may be one of the best TCM to be developed as a novel
medicine against leukemia in the future. In addition, EEHDW demonstrated potent
inhibitory activity against human lung cancer cells and merits further investigation.
13
Acknowledgements
This work was supported by grant CMU101-ASIA-09 from Department of Health,
Executive Yuan, R.O.C (Taiwan).
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Figure legend
FIGURE 1. Effects of EEHDW on cell viability in cancer cell lines. Lung cancer
A549 (A), H1355 (B), LLC cells (C), leukemia HL-60 (D) and WEHI-3 cells (E), and
melanoma B16F10 cells (F) were incubated in the absence or presence of 0, 25, 50,
100 and 200 μg/ml of EEHDW for 48 h. Cell viability was determined using the MTT
assay. The data are presented as mean ± S.E.M. in triplicate by comparing the treated
with untreated control cells. *, p<0.05 compared with the control value.
FIGURE 2. Effects of EEHDW on cell viability and cell morphology in HL-60 cells.
(A) Cells were treated with or without 0, 25, 50, 100 and 200 μg/ml of EEHDW for
24 and 48 h. Cell viability were determined using the MTT assay. The data are
presented as mean ± S.E.M. in triplicate by comparing the treated with untreated
control cells. *, p<0.05 compared with the control value. (B) The morphological
examination was determined under a phase-contrast microscope.
FIGURE 3. Effects of EEHDW on distribution of cell cycle and apoptosis in HL-60
cells. Cells were exposed in the absence or presence of 100 μg/ml of EEHDW for 24
18
h, then harvested for determination the distribution of cell cycle by flow cytometry (A)
and quantitative results (B) were expressed. The data are presented as mean ± S.E.M.
in triplicate by comparing the treated with untreated control cells. *, p<0.05 compared
with the control value. The morphological examination was determined under a
phase-contrast microscope.
FIGURE 4. Effects of EEHDW on DNA condensation and DNA damage in HL-60
cells. Cells were incubated and treated with or without 100 μg/ml of EEHDW for 48 h.
(A) HL-60 cells stained with DAPI to observe DNA condensation. Arrows indicate
cells showing DNA condensation and nuclear fragmentation. (B) HL-60 cells were
examined for DNA damage by Comet assay. Cells were photographed under
fluorescence microscopy (x200).
FIGURE 5. Effects of caspase-3, -8 and -9 activities on EEHDW-treated HL-60 cells.
Cells were incubated in the absence or presence of 100 μg/ml of EEHDW for 24 h,
and the whole-cell lysate were subjected to caspase-3, -8 and -9 activity assay. Each
result is shown as mean ± S.E.M. in triplicate by comparing the treated with untreated
control cells. *, p<0.05 compared with the untreated control value.
FIGURE 6. Proposed a schematic diagram for the mechanisms of EEHDW-mediated
signaling pathways by DNA microarray in HL-60 human leukemia cells.
19
Table 1: List of genes with more than 5.0-fold change in mRNA levels in HLDNA microarray
-treatment identified by
Ratio
Gene_Name
Gene_ID
Panther_Family_Name
Function
Process
23.94
G protein-coupled receptor 109A
hCG2016371.1
G-PROTEIN COUPLED RECEPTOR
G-protein coupled
receptor
Signal
transduction
18.98
POU domain, class 4, transcription
factor 3
hCG1980143
POU DOMAIN TRANSCRIPTION FACTOR
Transcription
factor
mRNA
transcription
CLUSTERIN
Molecular
function
unclassified
Apoptosis
13.66
12.60
clusterin
cytosolic acetyl-CoA hydrolase
hCG17283.4
hCG37739.4
CYTOSOLIC ACYL COENZYME A
THIOESTER HYDROLASE-RELATED
Hydrolase
Lipid, fatty
acid and
steroid
metabolism
6.22
5.60
glial fibrillary acidic protein
calpain 2, (m/II) large subunit
hCG27744.3
hCG38902.3
COILED-COIL INTERMEDIATE FILAMENT
FAMILY MEMBER
CALPAIN FAMILY MEMBER
Cytoskeletal
protein
Cysteine protease
Cell structure
and motility
Protein
metabolism
and
modification
-5.00
solute carrier family 39 (zinc
transporter), member 3
hCG25250.4
ZINC-IRON TRANSPORTER
Transporter
Transport
20
-5.00
folylpolyglutamate synthase
hCG18548.3
FOLYLPOLYGLUTAMATE
SYNTHASE-RELATED
Synthase and
synthetase
Carbon
metabolism
-5.10
sprouty homolog 1, antagonist of FGF
signaling
hCG1778453.2
SPROUTY
Transcription
factor
Signal
transduction
-5.32
estrogen-related receptor beta
hCG21644.4
NUCLEAR HORMONE RECEPTOR
Nuclear hormone
Signal
receptor
transduction
RIBOSOMAL RNA METHYLTRANSFERASE
Transferase ->
Methyltransferase
Nucleoside,
nucleotide
and nucleic
acid
metabolism
GUANINE NUCLEOTIDE DISSOCIATION
G-protein
Signal
STIMULATOR RALGDS
modulator
transduction
-5.35
FtsJ homolog 1
hCG17419.3
-5.38
Ral-GDS related protein Rgr
hCG2043016
-5.67
lymphoblastic leukemia derived
sequence 1
hCG27763.3
SCL FAMILY
Transcription
factor
mRNA
transcription
-5.68
FK506 binding protein 1A, 12kDa
hCG2019616.1
FK506-BINDING PROTEIN
Isomerase
Protein
folding
-5.71
MKI67 interacting nucleolar
phosphoprotein
hCG1750014.3
RNA-BINDING PROTEIN
Nucleic acid
binding
replication factor C (activator 1) 1,
145kDa
hCG38547.4
-5.80
REPLICATION FACTOR C SUBUNIT
DNA-directed
RNA polymerase
Biological
process
unclassified
Cell cycle;
DNA
21
replication
-5.80
phosphoribosyl pyrophosphate
amidotransferase
hCG2027246
AMIDOPHOSPHORIBOSYLTRANSFERASERELATED
Transferase
Monosacchari
de
metabolism
Cell
-5.87
-5.95
dachshund homolog 1
oxysterol binding protein-like 1A
hCG32210.2
hCG2020467
DACHSHUND FAMILY MEMBER
OXYSTEROL BINDING PROTEIN-RELATED
Transcription
factor
Miscellaneous
function
proliferation
and
differentiation
Lipid, fatty
acid and
steroid
metabolism
Amino acid
-5.98
-6.12
ornithine decarboxylase 1
NHP2 non-histone chromosome protein
2-like 1
hCG21951.2
hCG41538.3
ORNITHINE/DIAMINOPIMELATE
PLP-DEPENDENT DECARBOXYLASES
RIBOSOMAL PROTEIN L7AE-RELATED
Lyase ->
Decarboxylase
Nucleic acid
binding ->
Ribosomal
protein
-6.13
phosphatidylethanolamine
N-methyltransferase
hCG31440.2
PHOSPHATIDYLETHANOLAMINE
N-METHYLTRANSFERASE-RELATED
Transferase ->
Methyltransferase
metabolism ->
Amino acid
biosynthesis
Biological
process
unclassified
Lipid, fatty
acid and
22
steroid
metabolism
-6.29
calreticulin
hCG172487.2
CALRETICULIN AND CALNEXIN
Select calcium
binding protein
Protein
folding
Amino acid
-6.32
pyrroline-5-carboxylate reductase 1
hCG38803.3
PYRROLINE-5-CARBOXYLATE
REDUCTASE
Oxidoreductase
-> Reductase
metabolism ->
Amino acid
biosynthesis
-6.50
acetylcholinesterase (YT blood group)
hCG19216.3
ESTERASE-RELATED
Hydrolase ->
Esterase
Neuronal
activities
-6.57
megakaryocyte-associated tyrosine
kinase
hCG23522.2
TYROSINE PROTEIN KINASE
Non-receptor
tyrosine protein
kinase
-6.80
U3 snoRNP protein 4 homolog
hCG39079.3
U3 SMALL NUCLEOLAR
RIBONUCLEOPROTEIN PROTEIN
IMP4-RELATED
-6.81
purinergic receptor P2Y,
G-protein coupled, 2
hCG23327.4
G PROTEIN-COUPLED RECEPTOR
-6.97
IMP cyclohydrolase
hCG16691.5
BIFUNCTIONAL PURINE BIOSYNTHESIS
Nucleic acid
binding ->
Ribonucleoprotei
n
Protein
metabolism
and
modification
Nucleoside,
nucleotide
and nucleic
acid
metabolism
Signal
transduction
Hydrolase ->
Nucleoside,
23
PROTEIN PURH
-7.36
-7.40
stromal antigen 3
lysophospholipase II
hCG2024106
hCG1981746
Other hydrolase
nucleotide
and nucleic
acid
metabolism
Molecular
Biological
PROTEIN
function
unclassified
process
unclassified
ESTERASE-RELATED
Hydrolase ->
Lipase ->
Phospholipase
Lipid, fatty
acid and
steroid
metabolism
STROMAL ANTIGEN CONTAINING
Nucleoside,
-7.41
-7.46
-7.82
splicing factor, arginine/serine-rich 7,
35kDa
pseudouridylate synthase 1
mitochondrial ribosomal protein S7
hCG15878.3
hCG1744445.2
hCG27458.3
SPLICING FACTOR-RELATED
PSEUDOURIDYLATE SYNTHASE
RIBOSOMAL PROTEIN S7
Nucleic acid
binding
Synthase and
synthetase
Nucleic acid
nucleotide
and nucleic
acid
metabolism
Nucleoside,
nucleotide
and nucleic
acid
metabolism
Protein
24
binding ->
Ribosomal
protein
-8.15
glucose 6 phosphatase, catalytic, 3
hCG30307.3
GLUCOSE-6-PHOSPHATASE
Phosphatase
biosynthesis
Biological
process
unclassified
-8.18
-8.33
retinol dehydrogenase 13
(all-trans and 9-cis)
proliferation-associated 2G4, 38kDa
hCG20266.4
hCG2015367
OXIDOREDUCTASE
METHIONINE AMINOPEPTIDASE
Oxidoreductase
->
Dehydrogenase
Biological
process
unclassified
Transcription
factor
Protein
metabolism
and
modification
-8.53
phosphoglycerate mutase 1 (brain)
hCG25778.3
-8.61
opioid receptor, sigma 1
hCG20471.4
-8.65
SLC2A4 regulator
hCG22760.2
-8.74
exosome component 7
hCG1641243.2
Isomerase ->
Mutase
Carbohydrate
metabolism
C-8 STEROL ISOMERASE-RELATED
Receptor
Biological
process
unclassified
PAPILLOMAVIRUS REGULATORY FACTOR
Transcription
mRNA
PRF-1 RELATED
cofactor
transcription
RIBONUCLEASE PH-RELATED
Nucleic acid
binding ->
Nucleoside,
nucleotide
PHOSPHOGLYCERATE MUTASE
25
Nuclease
and nucleic
acid
metabolism
-8.79
polymerase (RNA) III polypeptide K,
12.3 kDa
hCG1787292.2
TRANSCRIPTION FACTOR TFIIS
DNA-directed
RNA polymerase
mRNA
transcription
-9.02
fms-related tyrosine kinase 4
hCG15283.4
IG DOMAIN SUPERFAMILY
Tyrosine protein
kinase receptor
Signal
transduction
Nucleic acid
binding
Nucleoside,
nucleotide
and nucleic
acid
metabolism
-9.28
splicing factor, arginine/serine-rich 7,
35kDa
hCG15878.3
SPLICING FACTOR-RELATED
Non-receptor
-9.38
-9.50
cyclin-dependent kinase 4
bystin-like
hCG40151.2
hCG16772.3
-9.54
hypothetical protein FLJ20425
hCG38080.3
-9.88
exosome component 2
hCG2024976
PROTEIN KINASE
BYSTIN
CELL GROWTH REGULATING NUCLEOLAR
serine/threonine
protein kinase
Molecular
function
unclassified
Cell cycle;
Mitosis
Cell adhesion
Molecular
PROTEIN
function
unclassified
Oncogene
3'-5' EXORIBONUCLEASE RRP4
Nucleic acid
Nucleoside,
26
binding ->
Nuclease
nucleotide
and nucleic
acid
metabolism
Nucleoside,
-10.02
phosphoribosylaminoimidazole
carboxylase
-10.19
peptidylprolyl isomerase
(cyclophilin)-like 1
hCG2027161
PHOSPHORIBOSYLAMIDOIMIDAZOLE-SUC
CINOCARBOXAMIDE SYNTHASE
Synthase and
synthetase
hCG15366.3
CYCLOPHILIN-TYPE PEPTIDYL-PROLYL
CIS-TRANS ISOMERASE FAMILY MEMBER
Isomerase
Nucleic acid
-10.45
-10.53
-10.65
nucleolar protein NOP5/NOP58
dyskeratosis congenita 1, dyskerin
exosome component 5
hCG32478.2
hCG18117.2
hCG22328.3
NUCLEOLAR PROTEIN NOP56-RELATED
TRNA PSEUDOURIDINE
SYNTHASE-RELATED
RIBONUCLEASE PH-RELATED
binding ->
Ribonucleoprotei
n
Centromere
DNA-binding
nucleotide
and nucleic
acid
metabolism
Protein
folding
Nucleoside,
nucleotide
and nucleic
acid
metabolism
Nucleoside,
nucleotide
and nucleic
protein
acid
metabolism
Transferase ->
Biological
27
Nucleotidyltransf
erase
process
unclassified
mRNA
transcription
-10.72
polymerase (RNA) I polypeptide C,
30kDa
hCG18995.3
DNA-DIRECTED RNA POLYMERASE
SUBUNIT D
DNA-directed
RNA polymerase
-11.04
nasal embryonic LHRH factor
hCG28443.3
NASAL EMBRYONIC LHRH FACTOR
Chemokine
-11.18
importin 4
hCG2014018.1
IMPORTIN
Transporter ->
Other transporter
Nuclear
transport
-11.32
polymerase (RNA) I polypeptide B,
128kDa
hCG16259.3
DNA-DIRECTED RNA POLYMERASE BETA
CHAIN
DNA-directed
RNA polymerase
mRNA
transcription
GLYCINE CLEAVAGE SYSTEM H
Hydrolase ->
Amino acid
metabolism ->
PROTEIN-RELATED
Other hydrolase
Amino acid
-11.49
glycine cleavage system protein H
hCG1980047
Signal
transduction
catabolism
-11.71
-11.72
A kinase (PRKA) anchor protein 1
cytochrome P450, family 27, subfamily
B
hCG33378.3
hCG2014568
A KINASE ANCHOR PROTEIN 1
Signaling
molecule
CYTOCHROME
Transferase ->
P450;METHYLTRANSFERASE - RELATED
Methyltransferase
Protein
targeting and
localization
Nucleoside,
nucleotide
and nucleic
acid
metabolism
28
-11.98
mitochondrial ribosomal protein L12
hCG2040013
RIBOSOMAL PROTEIN L7/L12
Nucleic acid
binding ->
Synthetase
-12.07
polymerase (DNA directed), epsilon 3
(p17 subunit)
hCG29189.2
DNA BINDING TRANSCRIPTION FACTOR
Transcription
factor
mRNA
transcription
hCG1985639
KRAB-RELATED C2H2-TYPE
ZINC-FINGERS;KINESIN-RELATED
Cytoskeletal
protein
Protein
targeting and
localization
60S RIBOSOMAL PROTEIN L22
Nucleic acid
binding ->
Ribosomal
protein
Protein
biosynthesis
-12.17
-12.38
-12.56
-12.60
-12.60
MYC-associated zinc finger protein
ribosomal protein L22
spermine oxidase
mitochondrial methionyl-tRNA
synthetase
endothelin converting enzyme 2
hCG2009685
hCG39338.5
FLAVIN MONOAMINE OXIDASE-RELATED
hCG1820508.1
METHIONYL-TRNA SYNTHETASE
hCG2022032
FAMILY NOT NAMED;ZINC
METALLOPROTEASE
Oxidoreductase
Protein
biosynthesis
Biological
-> Oxidase
process
unclassified
Nucleic acid
binding ->
Synthetase
Protein
metabolism
and
modification
Metalloprotease
Protein
metabolism
and
29
modification
-12.62
nucleolar protein 5A
hCG39617.2
NUCLEOLAR PROTEIN NOP56-RELATED
Nucleic acid
binding ->
Ribonucleoprotei
n
-12.90
Down syndrome critical region gene 2
hCG401227.3
LEUCINE RICH PROTEIN
Molecular
function
unclassified
-13.03
MYB binding protein (P160) 1a
hCG32148.3
MYB BINDING PROTEIN P160
Transcription
cofactor
Molecular
-13.30
-13.34
-14.02
lymphocyte antigen 6 complex, locus E
phenylalanine-tRNA synthetase-like,
beta subunit
arginase, liver
hCG1765592.2
hCG2012454
hCG21029.4
UPAR-LY6 DOMAIN CONTAINING PROTEIN
Nucleoside,
nucleotide
and nucleic
acid
metabolism
Cell
proliferation
and
differentiation
mRNA
transcription
Signal
function
unclassified
transduction
PHENYLALANYL-TRNA SYNTHETASE
BETA CHAIN
Synthase and
synthetase
Protein
metabolism
and
modification
ARGINASE-RELATED
Hydrolase ->
Other hydrolase
Amino acid
metabolism ->
Amino acid
30
catabolism
-14.23
uridine monophosphate synthetase
hCG1780791.2
OROTIDINE 5-PHOSPHATE
DECARBOXYLASE-RELATED
Lyase ->
Decarboxylase
Nucleoside,
nucleotide
and nucleic
acid
metabolism
-14.27
-14.71
-15.66
-15.74
solute carrier family 25
glycine cleavage system protein H
polymerase (RNA) I polypeptide B,
128kDa
mitochondrial ribosomal protein L4
hCG37927.2
hCG1980047
hCG16259.3
hCG28479.3
Transfer/carrier
protein
Transport
GLYCINE CLEAVAGE SYSTEM H
PROTEIN-RELATED
Hydrolase ->
Other hydrolase
Amino acid
metabolism ->
Amino acid
catabolism
DNA-DIRECTED RNA POLYMERASE BETA
DNA-directed
mRNA
CHAIN
RNA polymerase
transcription
50S RIBOSOMAL PROTEIN L4
Nucleic acid
binding ->
Ribosomal
protein
Protein
biosynthesis
MITOCHONDRIAL CARRIER PROTEIN
Nucleoside,
-16.30
aspartate transcarbamylase
hCG23710.3
CARBAMOYL-PHOSPHATE SYNTHASE
Synthase and
synthetase
nucleotide
and nucleic
acid
31
metabolism
-16.79
cyclin-dependent kinase 4
hCG40151.2
PROTEIN KINASE
Non-receptor
serine/threonine
protein kinase
Cell cycle
Nucleic acid
-17.63
-17.79
-17.82
-19.30
-19.57
chromosome 1 open reading frame 33
DPH2-like 2 (S. cerevisiae)
sideroflexin 4
RuvB-like 1 (E. coli)
DEAD (Asp-Glu-Ala-Asp) box
polypeptide 51
hCG41080.3
60S ACIDIC RIBOSOMAL PROTEIN P0
hCG25198.3
DIPTHERIA TOXIN RESISTANCE
PROTEIN-RELATED
hCG37954.2
hCG40109.3
hCG96673.3
SIDEROFLEXIN
RUVB-RELATED
DEAD BOX ATP-DEPENDENT RNA
HELICASE
binding ->
Ribosomal
protein
Molecular
function
unclassified
Transfer/carrier
protein
RNA helicase
RNA helicase
Protein
biosynthesis
Immunity and
defense
Biological
process
unclassified
DNA repair
Nucleoside,
nucleotide
and nucleic
acid
metabolism
-19.64
hypothetical protein MGC15429
hCG42593.2
THERMOACIDOPHILIC ALPHA-BETA
HYDROLASE-RELATED
Hydrolase
Biological
process
32
unclassified
FATTY ACID-BINDING PROTEIN
Transfer/carrier
protein
Lipid, fatty
acid and
steroid
metabolism
hCG1796030.1
PPAR GAMMA COACTIVATOR-RELATED
Transcription
cofactor
Biological
process
unclassified
spermidine synthase
hCG24773.3
SPERMIDINE SYNTHASE-RELATED
Synthase and
synthetase
Metabolism
translocase of outer mitochondrial
membrane 40 homolog
hCG1996194
MITOCHONDRIAL IMPORT RECEPTOR
SUBUNIT TOM40-RELATED
-20.19
fatty acid binding protein 5
(psoriasis-associated)
-21.06
peroxisome proliferative activated
receptor
-22.32
-23.92
hCG1984476
Receptor -> Other Mitochondrial
receptor
transport
Cytoskeletal
-23.92
lamin B2
hCG2004338.1
INTERMEDIATE FILAMENT
protein ->
Intermediate
filament
Cell structure
and motility
-24.16
quinoid dihydropteridine reductase
hCG39606.3
DIHYDROPTERIDINE REDUCTASE
Oxidoreductase
->
Dehydrogenase
-25.27
PEPP subfamily gene 2
hCG1740373.2
HOMEOBOX PROTEIN
Transcription
factor
mRNA
transcription
-26.80
similar to CGI-96;polymerase
hCG1988267
40S RIBOSOMAL PROTEIN S2
Chaperone
Nucleoside,
Amino acid
catabolism
33
nucleotide
and nucleic
acid
metabolism
Nucleoside,
-26.87
similar to CGI-96;polymerase
hCG1988267
40S RIBOSOMAL PROTEIN S2
Chaperone
-32.04
uracil-DNA glycosylase
hCG38494.3
URACIL-DNA GLYCOSYLASE
DNA glycosylase
RNA 3'-TERMINAL PHOSPHATE
Nucleic acid
binding ->
-32.54
RNA terminal phosphate cyclase-like 1
hCG1811283.3
CYCLASE-RELATED
-32.84
hypothetical protein MGC15668
hCG1794543.1
4-HYDROXYPHENYLPYRUVATE
DIOXYGENASE
-39.46
solute carrier family 25
hCG37927.2
MITOCHONDRIAL CARRIER PROTEIN
-44.44
solute carrier family 29
hCG19000.3
EQUILIBRATIVE NUCLEOSIDE
TRANSPORTER
nucleotide
and nucleic
acid
metabolism
DNA repair
Pre-mRNA
processing ->
Synthetase
mRNA
splicing
Oxidoreductase
-> Oxygenase
Amino acid
metabolism ->
Amino acid
catabolism
Transfer/carrier
protein
Transporter ->
Other transporter
Transport
Nucleoside,
nucleotide
34
and nucleic
acid
metabolism
-55.57
v-myc myelocytomatosis viral oncogene
homolog
hCG15917.3
MYC PROTO-ONCOGENE
Transcription
factor
mRNA
transcription
-79.62
interferon-related developmental
regulator 2
hCG2039742.2
INTERFERON-RELATED DEVELOPMENTAL
REGULATOR
Glycosidase
Neurogenesis
hCG1983753
PROTEIN KINASE;MITOGEN-ACTIVATED
PROTEIN KINASE-RELATED
Non-receptor
serine/threonine
protein kinase
Signal
transduction
-82.20
mitogen-activated protein kinase 3
35
36
37
38
39
40
41