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Cell type-dependent effects of andrographolide


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Article in Life sciences April 2012
Impact Factor: 2.7 DOI: 10.1016/j.lfs.2012.04.009 Source: PubMed

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Life Sciences 91 (2012) 751760

Contents lists available at SciVerse ScienceDirect

Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Cell type-dependent effects of andrographolide on human cancer cell lines


Myra T.W. Cheung, Rajkumar Ramalingam, Kenneth K.K. Lau, Michael W.L. Chiang, S.K. Chiu,
H.Y. Cheung , Y.W. Lam
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong

a r t i c l e

i n f o

Article history:
Received 20 December 2011
Accepted 3 April 2012
Keywords:
Andrographis paniculata
Andrographolide
Proteomics
Cell cycle
Cancer cell lines

a b s t r a c t
Aims: Andrographolide (ANDRO) is emerging as a promising anti-tumour compound. While it causes apoptosis in most cancer cells, andrographolide induces cell cycle arrest in hepatocellular cancer lines. In this study,
we studied the effect of andrographolide on hepatocellular cancers and other cancer types, and elucidated
the possible hepatoma-specic features of andrographolide toxicity.
Main methods: We compared the responses of a panel of human cell lines to andrographolide treatment by
using ow cytometry, cell synchronisation and time-lapse microscopy. We have also examined their expression of cell cycle-related proteins and proteome changes after andrographolide treatment.
Key ndings: Andrographolide exerts its effect on hepatocellular cancer cells through cell cycle arrest and not
apoptosis. In HepG2 cells, it blocks G2 cells from entering mitosis and prevents mitosis from completion. This
might be due to the disruption of mitotic spindle during metaphase. Despite the dramatic differences in their
responses to andrographolide, HepG2 and HeLa cells display similar biochemical consequences. Andrographolide induces DNA damages, as indicated by the expression of phospho-H2AX in both cell lines. Proteomic
experiments show that heme oxygenase 1 and heat shock protein 70 are among the proteins induced by
andrographolide, which indicate the possible role of oxidative stress in the anti-cancer mechanism of this
drug.
Signicance: Andrographolide can invoke different cellular responses depending on the biochemical and
physiological context in different cell and cancer types, and reveal an additional dimension of the therapeutic
applications of this compound.
2012 Elsevier Inc. All rights reserved.

Introduction
Andrographis paniculata (A. paniculata) has been used for centuries
as a folk remedy for a variety of chronic and infectious diseases. In the
Indian Pharmacopoeia, this herb is a prominent component in at least
26 Ayurvedic formulas (Akbarsha and Murugaian, 2000) which is
used to remove body heat and dispel toxins from the body (Deng,
1978; Zoha et al., 1989). The main constituent of A. paniculata is a
diterpene lactone called andrographolide (ANDRO) (Gorter, 1911;
Chan et al., 1963; Cava et al., 1965), which exhibits pharmacological
effects on various systems, including hepatic functions, and inammatory activity (Akbarsha et al., 1990; Rana and Avadhoot, 1991;
Shen et al., 2000). ANDRO was recently identied as an antagonist
of nuclear transcription factor-kappaB (NF-Kb) activity which functions as a master regulator of gene expression and exercises
Correspondence to: H.Y. Cheung, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong. Tel.: + 852
34427746.
Correspondence to: Y.W. Lam, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong. Tel.: + 852
34426347; fax: + 852 27844091.
E-mail addresses: bhhonyun@cityu.edu.hk (H.Y. Cheung), yunwlam@cityu.edu.hk
(Y.W. Lam).
0024-3205/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2012.04.009

widespread control over multiple signalling pathways (Perkins,


2007) through a direct modication of the p50 subunit (Xia et al.,
2004).
In recent years, a plethora of studies have focused on the effects of
ANDRO on apoptosis. ANDRO appears to suppress apoptosis in differentiated cells (Chen et al., 2004; Burgos et al., 2005; Lee et al., 2010a,
2010b, 2010c; Roy et al., 2011; Lee et al., 2010a, 2010b, 2010c). However, in all human cancer cells studied so far, ANDRO induces apoptosis either on its own (Cheung et al., 2005; Kim et al., 2005; Zhou et al.,
2006; Harjotaruno et al., 2007; Li et al., 2007a, 2007b, 2007c; Ji et al.,
2007; Zhao et al., 2008; Yang et al., 2010; Pratheeshkumar et al.,
2011) or in combination with other treatments (Zhou et al., 2008;
Yang et al., 2009; Zhou et al., 2010; Hung et al., 2010). It is not yet
clear why ANDRO can be anti-apoptotic in some cells and proapoptotic in others. Recent studies have pointed out that ANDRO
can activate the pro-apoptotic JNK pathway (Ji et al., 2007; Zhou et
al., 2008) while suppressing the anti-apoptotic AKT and ERK pathways (Tsai et al., 2004; Lee et al., 2010a, 2010b, 2010c). Interestingly,
ANDRO also down-regulates the expression of the epidermal growth
factor receptor (Tan et al., 2010) and hypoxia-inducible factor-1a (Lin
et al., 2011), factors that are important for tumour growth and survival. The fact that ANDRO alone can simultaneously act on multiple targets and pathways may explain its highly divergent effects. The net

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M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

outcome of ANDRO treatment on a particular tissue is likely the consequence of the combined effect of this compound on these diverse
targets, amalgamated with the genetic and physiological backgrounds
of that cell type. Exploration of how ANDRO interacts with this holism
of factors will produce a better understanding of ANDRO-mediated
chemotherapy, and help to establish an improved cancer therapeutic
strategy with less potential side effects.
We and others have recently described an alternative anti-cancer
mechanism of ANDRO (Li et al., 2007a, 2007b, 2007c; Shen et al.,
2009): in human hepatocellular cell lines, ANDRO induces a prolonged cell cycle arrest at the G2/M phase, followed by a caspaseindependent cell death. These observations led us to hypothesise
that ANDRO can exert different growth inhibitory effects, either cell
cycle arrest or apoptosis, on different cancer types, possibly depending on their physiological background and histological origins. In
this study, we characterise this cell type-dependent effect of ANDRO
in details. To our knowledge, this study is the rst detailed comparison of the molecular consequences of ANDRO treatment on different
cell and cancer types.
Methods
Cell culture and chemicals
HepG2, HeLa, Hep3B, PLC, and Sk-Hep1cells were obtained from
ATCC (Gibco, USA), and cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS),
100 unit/mL penicillin and 100 g/mL streptomycin at 37 C and 5%
CO2. HCT116 p53 wild-type and HCT116 p53 null cells (Bunz et al.,
1998) were supplied by Dr. B. Vogelstein (Johns Hopkins University
School of Medicine, Baltimore, MD). ANDRO (Sigma) was dissolved
in dimethyl sulfoxide (DMSO, Sigma) at 0.1 M as the stock solution.
IKK-2 inhibitor V (Calbiochem) was prepared in DMSO at 0.1 M as
the stock solution.
Cell viability assay
Cells were incubated with 100 M of ANDRO or an equivalent volume of DMSO for 12, 24, 36 and 48 h. After that, the cells were
washed with 1 phosphate buffered saline (PBS, Gibco, USA) and incubated with 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT,
Sigma) for 1 h at 37 C followed by the addition of 100 L of DMSO
per well to stop the reaction. The optical densities of the resulting
mixtures were measured at 570 nm by using a microplate reader
(Molecular devices, SPECTRAmax). The quantity of viable cells was
expressed by a comparison between the control and treated cells,
and GI50 values were calculated from concentrationeffect curves.
Cell cycle analysis and apoptosis detection
The DNA content of the cells was determined by propidium iodide
staining and ow cytometry as described previously (Li et al., 2007a,
2007b, 2007c). For the detection of apoptosis, the cells were stained
with Annexin V, Alexa Fluor 488 conjugate (Invitrogen, USA) in an
annexin-binding buffer (10 mM 4-(2-hydroxyethyl)-1- piperazine
ethane sulfonic acid (HEPES), 140 mM sodium chloride and 2.5 mM
calcium chloride for 15 min. The cells were co-stained with
0.05 mg/mL of propidium iodide immediately prior to analysis. The
uorescence signals were measured with a ow cytometer (FACSCalibur, Becton Dickinson, CA), and the data analysed with CellQuest
software (Becton Dickinson, CA).
Detection of mitochondrial membrane potential
The cells were treated with or without 100 M of ANDRO for 24 h.
After treatment, 2 M of JC-1 (Invitrogen) was added and the cells

were incubated at 37 C for 15 min. After trypsinisation, the labelled


cells were washed with warm-PBS twice before analysis. The uorescence signals are measured with a ow cytometer (FACSCalibur, Becton Dickinson, CA), and the data analysed with CellQuest software
(Becton Dickinson, CA).
Transmission electron microscopy
The HepG2 cells were treated with or without 100 M of ANDRO
for 24 h followed by cell harvesting. The cells were xed for 2 h at
25 C in a primary xative (2.5% glutaraldehyde, EMS), 0.05% CaCl2
(Sigma) and 0.2 M sodium cacodylate (EMS) in double-distilled
water (ddH2O). After primary xation, the cells were rinsed with
0.1 M sodium cacodylate and then mixed with 2% agar (USB). The
agar blocks were washed with 0.1 M sodium cacodylate. A secondary
xative which contained 2% osimium tetroxide (EMS) was added for
2 h at 25 C in the dark. Then, the blocks were rinsed stepwise with
0.1 M sodium cacodylate three times for 5 min each, followed by
0.05 M and 0.025 M sodium cacodylate for 10 min each. Ultrapure
water was used for the last wash twice for 10 min each before dehydration. The samples were dehydrated by using different concentrations (30%, 50%, 70%, 80%, 90%, and 95%) of ethanol for 5 min each
and washed twice with 100% ethanol for 10 min. The samples were
washed with an ethanol/acetone mixture (3:1, 1:1, and 1:3 ratio)
for 10 min each. Finally, 100% acetone was used to wash the samples
twice for 15 min each. Prior to 2-day embedding, the cells were inltrated with a 100% acetone/Spurr's resin (EMS) mixture overnight.
This was then replaced with several changes of pure Spurr's resin
over 2 days. The blocks were trimmed and sectioned. After staining
with uranyl acetate (EMS) and Reynolds lead citrate (EMS), the sections were imaged on an electron microscope (Philips Tecnai 12
BioTWIN).
Western blotting
The cells were washed with PBS and lysed in 2% sodium dodecyl
sulfate (SDS) which contained 1 ethylene diamine tetraacetic acid
(EDTA)-free protease inhibitor (Roche, USA). Following sonication
for a few minutes, protein concentrations were measured by a Bradford assay. Aliquots of cell lysates were subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membranes were blocked
with 5% non-fat milk (Nestle, Switzerland) in Tris buffered saline
(TBS) that contained 0.05% Tween 20 (USB) and probed with antibodies directed against phospho-cdc2 (Tyr15), CDK2, and CDC2
(Cell Signaling, USA), cyclin A, cyclin B, and cyclinD3 (BD Biosciences,
San Jose, CA), p53 (Invitrogen, USA), p21 (Santa Cruz Biotechnology),
beta-actin (Sigma), and phospho-H2Ax (Millipore, Massachusetts)
followed by a horseradish peroxidase-linked secondary antibody
(GE Healthcare, UK). The antigens were then visualised by enhanced
chemiluminescence (Amersham).
Immunouorescence
The cells were seeded onto coverslips one day before xation in 4%
paraformaldehyde-PBS. The xed cells were then permeabilised for
10 min with 1% Triton X100 in PBS. The coverslips were blocked
with 50 mM glycine for 30 min at 25 C. After blocking, a diluted primary antibody (anti -tubulin or anti phosphor-H2AX) was added on
the coverslips for 45 min at room temperature. The primary antibody
was removed by rinsing with PBS. A corresponding diluted secondary
antibody and 4,6-Diamino-2-phenylindole dihydrochloride (DAPI,
Millipore) were added for 20 min. After incubation, the coverslips
were mounted on the glass slides with a mounting medium. The images were taken by using a confocal microscope (Leica TCS SPE).

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

Time-lapse imaging
The cells were cultured in 35-mm glass bottom dishes (SPL). In
some of the experiments, the cells were stained with 400 ng/mL of
Hoechst 33342 (Sigma) for 5 min at 37 C. Prior to starting the recording, the cultures were incubated in a closed, temperaturecontrolled chamber (WeatherStation environmental chamber). Live
images were obtained on an inverted microscope (Leica, TCS SP5)
by using a camera and running LAF software. During acquisition, images were taken every 10 min during a time period of 48 h. The
movies were processed in AVI format with the LAF software.
Cell synchronisation
For G1/S synchronisation, 2 mM thymidine (Sigma) was added to
the cells for 16 h at 37 C. After removing the thymidine and washing
with PBS, the medium was refreshed for 10 h at 37 C. Finally, the medium was replaced by one that contained 2 mM thymidine for another 16 h at 37 C. After synchronisation, the cells were released
followed by the addition of 100 M ANDRO for another 24 h. The control and treated cells were xed, stained with propidium iodide, and
analysed with a ow cytometer as described above.
Quantitative proteomics
Before making lysates, the cells were metabolically labeled by culturing for ve rounds of cell division in stable isotope labeling with amino
acids in cell culture (SILAC) DMEM medium supplemented with 10%
dialysed fetal calf serum (Biowest, Nuaille, France) to ensure that all
the cellular proteins were labeled until saturation (Ong et al., 2002).
All labeled amino acids were obtained from the Cambridge Isotope Lab
(Andover, MA). The cells used for ANDRO treatment were labeled with
DMEM that contained 13C615N4 L-arginine (R10) and 13C615N2 L-lysine
(K8), whereas the cells used as control were labeled cultured in
DMEM that contained 12C614N4 L-arginine (R0) and 12C614N2 L-lysine
(K0). For whole cell lysate, SILAC-labeled cells were cultured and directly lysed in 2% SDS lysis buffer as described above.
Protein concentrations of the lysates were determined by a Bradford assay (Thermo Fisher). Both types of whole cell lysates (R0K0
and R10K8) were mixed in a 1:1 ratio and the mixed protein samples
were separated by electrophoresis as described above. The gel was
stained with colloidal Coomassie Blue (Invitrogen) according to the
manufacturer instructions. The gel lane was excised into 14 slices.
Each gel slice was destained, reduced, alkylated and trypsindigested as previously described (Shevchenko et al., 2006). The
resulting peptides were separated by high-performance liquid chromatography (HPLC, Dionex, Sunnyvale, CA) on a commercial C18 reverse phase column (Dionex) over an 80-minute gradient (Mobile
phase A: 0.1% uoroacetic acid in 2% acetonitrile (ACN) in Milli-Q
water; mobile phase B: 0.1% uoroacetic acid in 98% ACN) and then
analysed by a micrOTOF-Q II ESI-Qq-TOF mass spectrometer (Bruker
Daltonik GmbH, Bremen, Germany).
The peak list was generated by using Data Analysis software, Version 4.0 (Bruker Daltonics). The mass spectrometry (MS) data were
searched by using the National Center for Biotechnology Information
human protein database for homo sapiens (05-07-2010 release,
178,730 sequences searched), MASCOT search engine, version 2.2
(Matrix Science) (Rudnick et al., 2005). The xed modication was
carbamidomethyl (C) and variable modications used were oxidation
(M), as well as appropriate SILAC modications. Trypsin specicity
was used, allowing for two missed cleavages, and a mass tolerance
of 0.08 Da was used for MS precursors and 0.15 Da for fragment
ions. Peptide charges of +2 and + 3 were selected. Individual ions
with mascot scores higher than 20 were used (a threshold commonly
used for condent protein identication from tandem MS data)
(Bindschedler et al., 2008). SILAC quantitation was performed by

753

using WarpLC software (Bruker Daltonics), which measures the averaged MS peak heights of isotopic pairs. Proteins with heavy/light isotopic ratios less than 0.01 were discarded as they mostly represented
environmental contaminants.

Results
Andrographolide induces G2/M arrest, not apoptosis, in HepG2 cells
ANDRO causes widespread cytotoxicity in human cancer cells.
NCI60 drug screening data (Shoemaker, 2006) show that the presence of ANDRO at 40 M for 48 h is enough to induce a 50% growth
inhibition in most of the cell lines tested (Suppl Fig. 1A). Since the
NCI60 panel does not represent all common human cancer types,
we tested the cytotoxicity of ANDRO in 6 additional cell lines, covering cancer types such as hepatoma, prostate, lung and cervical epithelial carcinoma (Suppl Fig. 1B). ANDRO also induced effective growth
inhibition in those cell lines. However, the ANDRO-induced growth
inhibition of HepG2 follows a distinct kinetics from that of other
cells tested (Fig. 1A) compared to HeLa and two HCT116 derivatives
(p53 wild-type and p53 null). The effect of ANDRO on HepG2 viability
was apparent only after 20 h of treatment (Fig. 1A).
To investigate this delayed response of HepG2 to ANDRO, we compared the behaviours of HepG2 and HeLa cells during the rst 24 h of
ANDRO treatment (Fig. 1). We observed that: (1) compared to HeLa
cells, Annexin V staining was less pronounced in ANDRO-treated
HepG2 cells (Fig. 1B), (2) measurement of mitochondrial membrane
potential by JC1 dye indicated a signicant lower extent of mitochondrial depolarisation in HepG2 cells than in HeLa cells after ANDRO
treatment (Fig. 1C), (3) ow cytometry analysis did not detect subG1 DNA peaks in ANDRO-treated HepG2 cells, which is readily detectable in treated HeLa cells (Fig. 1D), and (4) transmission electron microscopy of ANDRO-treated HepG2 did not show any classical signs of
apoptosis, such as chromatin condensation and membrane rufing
(Fig. 1E). Taken together, the delayed cytotoxicity of ANDRO towards
HepG2 cells during the initial stage of treatment is possibly due to a
lower extent of apoptosis in this cell line, compared to HeLa cells.
To test whether the effect of ANDRO on HepG2 and HeLa is dosedependent, we examined the level of apoptosis and/or cell cycle arrest in these two cell lines treated with ANDRO at various concentrations. Low concentrations of ANDRO did not induce any detectable
apoptosis in HeLa cells (Suppl Fig. 2). However, no cell cycle arrest
was observed at these concentrations either, which suggests that
cell cycle arrest in ANDRO-treated HepG2 is not sub-lethal response
to ANDRO, but represents a specic phenotype exhibited by this cell
line.

The p53 background is irrelevant to the G2/M arrest induced by


andrographolide
It is unlikely that the difference in the response of HepG2 cells and
other cancer cell lines to ANDRO treatment is related to the p53 status, as both p53 dysfunctional (HeLa) and p53 wild-type cell lines
(HCT116) displayed similar ANDRO growth inhibitory kinetics
(Fig. 1A). Indeed, the p53-null HCT116 variant also showed a similar
prole in response to ANDRO as p53 wild-type HCT116 (Fig. 1A). Consistent with previous reports (Zhou et al., 2008), ANDRO treatment
induced the expression of p53 in HepG2 and HCT116 (Fig. 2).
ANDRO exerted different responses in p21 expression in these cell
lines. It induced the p21 level in HCT116, and to a lesser extent, in
p53 null HCT116, but not in HepG2 and HeLa (Fig. 2B). Despite
these diverse p53 backgrounds and p21 responses in the cell lines
tested, HepG2 exhibited a unique response to the ANDRO treatment,
which suggests that the G2/M arrest of this cell line by ANDRO

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M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

Fig. 1. A. Growth kinetics of four human cell lines in the presence of ANDRO (100 M). BD. Properties of HepG2 and HeLa cells treated with ANDRO (100 M, 24 h) or the equivalent
volume of DMSO. B. Annexin V staining. C. Mitochondrial membrane potential measured by JC1 dye. The increase of green uorescence (JC1 monomer) represents membrane depolarisation. D. Cell cycle stage distribution measured by propidium iodide staining. E. Transmission electron micrograph of HepG2 cells treated with ANDRO (100 M, 24 h) Bars:
2 m.

treatment is likely related to the histological origin of HepG2, rather


than its p53 background.

Andrographolide-induced G2/M arrest is common to hepatocellular


carcinoma
We previously reported that HepG2 undergoes G2/M arrest upon
ANDRO treatment, and apoptotic DNA ladders are not detectable
until after 96 h of treatment (Li et al., 2007a, 2007b, 2007c). We
asked if cell cycle arrest and delayed apoptosis also occurred in
other heptocellular cell lines treated with ANDRO. Four human hepatoma cell lines, HepG2, Hep3B, Sk-Hep1 and PLC, were treated with
ANDRO (100 M, 24 h) and their cell cycle stage distributions were
analysed by ow cytometry (Fig. 3A). ANDRO promptly induced apoptosis in the HeLa cells, as indicated by the occurrence of a sub-G1
DNA peak. However, sub-G1 peaks were not evident in any of the
hepatoma cell lines tested. Instead, ANDRO-treated hepatoma cells
displayed various extents of G2/M accumulation. This conrms our
earlier observation of G2/M arrest in ANDRO-treated HepG2 cells,
and suggests that this phenomenon is a general response by the hepatoma cells to ANDRO. This sets hepatocellular carcinoma apart from
a vast number of cell lines of diverse histological origins, which are induced to apoptosis on ANDRO treatment (Cheung et al., 2005; Kim et
al., 2005; Zhou et al., 2006; Harjotaruno et al., 2007; Li et al., 2007a,

Fig. 2. Expression of p53 and p21 in HepG2, HeLa, HCT116 p53 wild-type and HCT116
p53-null cells treated with ANDRO (100 M) for 0, 2, 7.5 and 24 h. Protein loadings are
normalised by -actin abundance.

2007b, 2007c; Ji et al., 2007; Zhao et al., 2008; Pratheeshkumar et


al., 2011; Yang et al., 2009).
Andrographolide induces aberrant mitosis in hepatocellular carcinoma
To clarify the nature of ANDRO-induced G2/M arrest in hepatoma,
we examined the nuclear morphology of ANDRO-treated HepG2 cells.
Hoechst dye labelling showed a progressive accumulation of HepG2
cells with condensed chromatin upon ANDRO treatment (Fig. 3B
and C, arrow), which suggests that ANDRO caused a mitotic arrest
in this cell line. Since cells with condensed chromatin are sometimes
mistaken as apoptotic cells, we used time-lapse microscopy to examine the mitotic process of ANDRO-treated HepG2 (Fig. 3D). All the
mock-treated HepG2 cells observed could successfully complete mitosis. However, ANDRO-treated mitotic cells appeared to be arrested
at metaphase. A close inspection of the chromatin morphology of
ANDRO-treated HepG2 indicated abnormal chromatin alignment
and/or segregation (Fig. 3B and E). Mitotic spindles also appeared to
be malformed (Fig. 3E). The failure of ANDRO-treated HepG2 cells
to complete mitosis is conrmed by the accumulation of cyclin B
(Fig. 3F), which is normally degraded on the onset of mitosis. Accumulation of cyclin B was not observed in ANDRO-treated HeLa.
Cell type- and cell cycle stage dependent effects of andrographolide
Although ANDRO-treated HepG2 cells could not complete mitosis,
ANDRO did not appear to arrest all HepG2 cells at mitosis, even after
prolonged treatment (see below). The mitotic index of ANDROtreated HepG2 cells did not exceed 14% (Fig. 3C), which suggests
that the effects of ANDRO on HepG2 may not be restricted to mitotic
arrest, and ANDRO may exert multiple effects on different parts of the
cell cycle. Interestingly, ANDRO induced the accumulation of Cyclin
D3 in HepG2, but not in HeLa (Fig. 3F), suggesting a possible

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

755

Fig. 3. G2/M arrest of hepatocellular carcinoma treated with ANDRO. A. Cell cycle stage distribution of HeLa cells and four hepatoma cell lines, HepG2, SK-HepG1, Hep3B and PLC,
treated with 100 M ANDRO (24 h) or equal volume of DMSO. B. HepG2 cells, treated with ANDRO (100 M) for 0 h and 7 h, labelled by cell permeate DNA dye Hoechst 33342. C.
Percentage of normal and aberrant mitosis in ANDRO-treated HepG2 for different durations. D. Time-lapse microscopy of Hoechst 33342-stained HepG2 cells treated with either
ANDRO (100 M) or DMSO. E. Representative mitotic HepG2 cells treated with DMSO or ANDRO (100 M), stained with Hoechst 33342 (blue) and -tubulin (red). F. Expression
of cyclins in HepG2 and HeLa cells treated with ANDRO (100 M) for 0, 2, 7.5 and 24 h.

deregulation in the interphase (Zimmet et al., 1997). To delineate the


effects of ANDRO on cells at different cell cycle stages, we followed
the fates of HeLa and HepG2 cells after ANDRO treatment by using
time-lapse microscopy. Before the addition of ANDRO (or DMSO),
we rst imaged populations of HeLa and HepG2 cells for 24 h in
order to obtain information on the cell cycle status of each cell. We
then added ANDRO (100 M) or the equivalent volume of DMSO to
the cells and continued imaging for 40 h. This approach allows for
systematic tracking of individual cells at various cell cycle stages in
asynchronous cultures. For example, we analysed daughter cells
exited from mitosis within an hour before the addition of ANDRO.
These represent early G1 cells. In DMSO controls, almost 100% of
these early G1 HeLa and HepG2 cells successfully entered mitosis
within about 39 h (Fig. 4A). In ANDRO-treated cultures, the fate of
these cells signicantly differed. Early G1 HeLa cells died, presumably
of apoptosis, as early as 5 h after treatment. By 35 h post-treatment,
90% of these cells had died (Fig. 4B), as judged by their rounding up
and detachment. Remarkably, none of the ANDRO-treated HepG2
cells died, or entered mitosis, even after 40 h of treatment.
Next, we analysed the response of HeLa and HepG2 cells that were
in mitosis at the time of ANDRO addition (Fig. 4C and D). In the DMSO
control, 100% of both cell lines completed mitosis within 5 h (Fig. 4C).
Neither mitotic HeLa nor HepG2 cells could complete mitosis in the
presence of ANDRO, and died after a prolonged mitotic arrest. Mitotic
HeLa cells started to die after 5 h of arrest, while HepG2 died after
12 h (Fig. 4D). Therefore, ANDRO appears to cause mitotic catastrophe (Castedo et al., 2004) in both cell lines. HepG2 are more resistant

to the ANDRO-induced mitotic catastrophe, which possibly explains


the apparent accumulation of the mitotic index when treated with
ANDRO (Fig. 3C).
Andrographolide blocks mitotic entry of HepG2 cells
The time-lapse experiments indicated that G1 HepG2 cells did not
die upon ANDRO treatment, but could not reveal whether these cells
were arrested at G1 or allowed to proceed to G2. We used synchronised cell cultures to further investigate the fate of these cells. HeLa
and HepG2 cells were reversibly arrested at the G1/S phase by a double thymidine block (Fig. 5), and then release to resume the cell cycle
in the presence of ANDRO or DMSO. The DMSO-treated cells proceeded through the cell cycle (data not shown). The ANDRO-treated
HeLa cells could not proceed, with the majority of the cells still
trapped in the S phase after 24 h. Prominent apoptosis was also
detected. However, ANDRO-treated HepG2 cells were able to complete DNA replication and reached the G2 phase in the presence of
ANDRO (Fig. 5).
Andrographolide-induced G2/M arrest of HepG2 cells not caused by
inhibition of NF-kB activity
ANDRO is known to directly block the DNA binding of the NF-kB
transcription complex (Xia et al., 2004). The inhibition of NF-kB will
lead to profound changes in cell growth and proliferation, as both oncogenes and tumour suppressing genes can be affected. The observed

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M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

Fig. 4. Tracking the fate of individual HepG2 and HeLa cells exposed to ANDRO. A. G1 cells formed from mitosis within an hour before the addition of DMSO followed for 40 h by
time-lapse microscopy. The cumulative percentage of G1 cells that have reached mitosis was counted. Black line: HepG2, grey line: HeLa. B. The same experimental design, except
that ANDRO (100 M) is added. In this case, none of the G1 cells reach mitosis. For HeLa cells, the fate of these cells is death (judged by the rounding up and oating off). For HepG2,
neither death nor mitosis is observed. C. Mitotic cells at the time of DMSO addition followed by time-lapse imaging. The cumulative percentage of cells completing mitosis was
counted. D. ANDRO treatment. None of the mitotic cells complete mitosis. The cumulative percentage of death was counted. Right panels: representative images from the timelapse movies.

cell type-dependent effect of ANDRO may be the manifestation of NFkB inhibition in different genetic or physiological contexts. If this is
the case, the inhibition of the NF-kB pathway by non-ANDRO inhibitors should also cause G2/M arrest in HepG2 cells. To test this hypothesis, we treated HepG2 and HeLa cells with IKK-2 inhibitor V at 1 M
for up to 48 h. This dosage is known to inhibit the NF-kB pathway
(McElwee et al., 2009). As shown in Fig. 6A, IKK-2 inhibitor V does
not induce G2/M arrest in HepG2 cells, suggesting that the ANDROinduced phenotype in this cell line is caused by an NF-kBindependent mechanism.
We have previously shown that ANDRO interferes with the redox
homeostasis of cultured cells (Li et al., 2007a, 2007b, 2007c; Zhang et
al., 2008). The resulting oxidative stress could lead to cellular damages that activate cell cycle checkpoints. Treatment with ANDRO appears to cause DNA damages in both HeLa and HepG2 cells, as
indicated by an increase of nuclear foci labelled by an antibody
against phosphorylated H2AX (Fig. 6B). Western blotting analysis
also shows a dramatic increase in H2AX phosporylation (Fig. 6C).
Responses of HeLa and HepG2 cells to andrographolide revealed by
comparative proteomics
To investigate the molecular basis of the distinct effects of ANDRO
on different cell lines, we used SILAC-based quantitative proteomics
(Ong et al., 2002) to compare the proteomic responses of HepG2
and HeLa cells to ANDRO treatment. For each cell line, heavy
isotope-encoded cells were treated with ANDRO at 100 M for 48 h,
while an equal number of unlabeled cells were treated with an equivalent volume of DMSO for the same duration. The two cell populations were mixed, lysed and processed for MS analysis. For each
identied peptide, the peak intensity of the labelled peptide (derived

from ANDRO-treated cells) was compared to that of its unlabelled


counterpart (from mock-treated cells). The ratio between these two
isotopomers measures the relative abundance of the protein represented by this peptide (Fig. 7A), and thus reects the change in abundance of this protein after ANDRO treatment. This experimental
design allows for the direct comparison of protein expression in
HepG2 and HeLa cells in response to ANDRO treatment.
We quantitated the abundance changes of 65 proteins in both
HeLa and HepG2 cells after ANDRO exposure (Fig. 7B and Suppl
Table I). Most of the detected proteins did not exhibit discernible
changes in abundance in response to ANDRO, which suggests that
ANDRO may exert its toxicity through the perturbation of a relatively
small number of specic proteins instead of the global deregulation of
protein expression. Some proteins responded differently to ANDRO in
the two cell lines. Two histones, H2A.2 and H3, were signicantly
down-regulated in HepG2 but not in HeLa. Of the proteins that
were signicantly affected by ANDRO treatment, some were upregulated in both HeLa and HepG2 cells. The expression of heme oxygenase 1 and heat shock protein 70 increased in both cell lines. This is
consistent with our earlier observation in which ANDRO binds to glutathione (Zhang et al., 2008) and induces the accumulation of reactive
oxygen species in the cells (Li et al., 2007a, 2007b, 2007c), an effect
known to stimulate the expression of these two proteins
(Schiaffonati and Tiberio, 1997; Tacchini et al., 1993; Li et al., 2007a,
2007b, 2007c). ANDRO treatment caused the down-regulation of calcium binding protein S100A4 in both cell lines. This protein plays a
role in cell motility and adhesion, and has been used as a prognostic
marker for tumour metastasis (Boye and Maelandsmo, 2010). The
down-regulation of S100A4 in response to ANDRO treatment supports the observed role of ANDRO as an anti-metastasis inhibitor
(Chao et al., 2010; Pratheeshkumar et al., 2011).

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

757

Fig. 5. HepG2 and HeLa cells are arrested at the G1-S junction by a double thymidine block, and then released by a wash-out. ANDRO (100 M) is then immediately added. The cells
were collected 2, 4, 6 and 24 h after ANDRO addition. The cell cycle stage distribution was analysed by propidium iodide staining and ow cytometry.

758

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

Fig. 6. A. Effect of NF-kB inhibitor (IKK-2 inhibitor V) on the cell cycle stage distribution of HepG2. B. Phospho-H2AX (Ser-139) staining of HepG2 and HeLa cells treated with DMSO
or ANDRO (100 M, 24 h). C. Detection of phospho-H2AX in HepG2 and HeLa cells treated with DMSO, 50 M and 100 M ANDRO (24 h). Protein loadings are normalised by -actin
abundance.

Discussion
In this study, we have conrmed and extended our previous observation that ANDRO treatment causes cell cycle arrest in human hepatocellular carcinoma HepG2. Unlike other cancer cell lines that
undergo prompt apoptosis upon exposure to ANDRO, HepG2 exhibits
a G2/M delay, with relatively little apoptosis in the initial phase
(Figs. 1 and 2). This phenomenon occurs in all hepatoma cell lines
tested so far (Fig. 2A), and is therefore likely to be a specic hepatocellular response. Time-lapse microscopy and cell synchronisation
analysis (Fig. 4) depict a complex picture of ANDRO-induced growth
inhibition in hepatoma (Fig. 8). In cell lines such as HeLa, ANDRO

invokes apoptosis regardless of cell cycle stages. This is consistent


with the ow cytometry data that show no obvious cell cycle arrest
in HeLa cells prior to the detection of apoptosis (Figs. 1D and 2A). In
HepG2 cells, however, ANDRO does not affect the viability of cells at
G1, which can proceed through the S phase to G2 (Figs. 4B and 5).
The entry of G2 cells into mitosis, however, is blocked by ANDRO
(Fig. 4B). HeLa and HepG2 cells that have already entered mitosis at
the time of ANDRO addition cannot complete mitosis, possibly due
to the lack of proper chromosome segregation and malformation of
mitotic spindles (Fig. 3E), and eventually die of mitotic catastrophe
(Figs. 3D and 4D). Compared to HeLa, HepG2 cells appear to withstand a longer period of mitotic arrest (Fig. 4D). The net outcome of

Fig. 7. Effect of ANDRO on HepG2 and HeLa proteomes by quantitative proteomics. A. Experimental design. B. SILAC ratios plotted in log2 scale of proteins identied in both HepG2
and HeLa cells.

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

Fig. 8. Response of HepG2 and HeLa cells at different cell cycle stages to ANDRO
treatment.

these events is the initial arrest of HepG2 at G2 and an accumulation


of mitotic cells. The mitotic index decreases as mitotic catastrophe
starts after about 12 h of blockage (Fig. 4D).
The growth arrest induced by ANDRO in HepG2 during the rst
24 h is primarily caused, not by apoptosis, but by a G2/M block and
mitotic catastrophe (Fig. 8). This is inconsistent with an earlier report
that suggests ANDRO induces apoptosis in HepG2, HCT116 and HeLa
cells within 24 h with equal effectiveness (Zhou et al., 2008). In that
study, the levels of apoptosis in these cell lines were compared
based on DAPI staining and end-point microscopy analyses, which
identied cells with apoptotic chromatin morphology. This method
can lead to erratic results, as we have shown here (Fig. 3B) that
many ANDRO-treated HepG2 cells are at the state of aberrant mitosis,
with an appearance quite similar to that of apoptotic cells. By the use
of time-lapse microscopy, we were able to show that these cells are
indeed mitotic, and not apoptotic.
ANDRO is a rare example of compounds that cause different
growth suppression phenotypes in different cancer types. We used
SILAC-based quantitative proteomic experiment to compare the biochemical responses of HeLa and HepG2 cells to ANDRO. ANDRO appears to cause perturbations of a similar set of proteins in HepG2
and HeLa cells. In both cell lines, ANDRO treatment dramatically upregulates the expression of heme oxygenase I, a protein known to
be induced by oxidative stress (Gozzelino et al., 2010). We and others
have observed that ANDRO induces the accumulation of reactive oxygen species in cells, and can directly modulate the glutathione content (Li et al., 2007a, 2007b, 2007c; Zhang et al., 2008). Our
proteomic data suggest that oxidative stress may be the most immediate and dominant effect exerted by ANDRO on cancer cells, and the
interference in multiple signalling pathways represents the subsequent rippling of this initial impact. Intriguingly, while ANDRO induces oxidative stress in cancer cells, it is found to up-regulate the
cellular-reduced glutathione (GSH) level and antioxidant enzyme activities in terminally differentiated cells (Trivedi et al., 2007; Woo et
al., 2008; Chern et al., 2011). It will be interesting to investigate
whether these divergent responses reect the pivotal role of heme
oxygenase I up-regulation by ANDRO in different cell types.
In addition, our proteomic analysis reveals subtle differences in
the responses of HeLa and HepG2 to ANDRO. Two histones, H2A.2
and H3, were dramatically down-regulated in HepG2 but not in
HeLa. As the synthesis of histones is closely linked to cell cycle progression (Osley, 1991), the decrease of these histones in HepG2 may
be related to the ANDRO-induced cell cycle arrest. Interestingly,
chlorambucil, a chemical known to induce the down-regulation of
histone H4, also induces G2/M arrest in cancer cell lines (Dickinson
et al, 2004). Our data suggest that the oxidative stress induced by
ANDRO may accompany a suppression of H2A.2 and H3 synthesis in

759

hepatoma, which further leads cell cycle arrest. Future studies will
be needed to delineate how ANDRO deregulates histone biosynthesis.
While it is well documented that different cell lines may display
distinct fates in the wake of prolonged mitotic arrest (Shi et al.,
2008; Huang et al., 2010) , much less is known about the cell typedependent effects of toxicants during the interphase. The apparently
milder effect of ANDRO on interphase HepG2 cells is not likely due
to a poorer cellular uptake of this compound by these cells. At the
same concentration, ANDRO causes the same level of growth inhibition in HepG2 and HeLa cells in 48 h (Fig. 1A). Moreover, ANDRO appears to induce a similar extent of phospho-H2AX foci and expression
(Fig. 6B and C), which suggests that ANDRO can cause similar levels of
DNA damage in these two cell lines. The cell line-dependent phenotypes of ANDRO treatment may reect a difference in contributions
by the ATR-Chk1 and ATM-Chk2 pathways in response to this genotoxic stress. When activated by genomic instability, these two pathways lead to vastly cellular responses, in terms of apoptosis and cell
cycle arrest, in cell type-specic manner (Smith et al., 2010). It remains to be seen whether the DNA damages stimulated by ANDRO
can solicit different repair pathways in different cell types.
What we cannot eliminate, however, is the possibility that the observed differences in cellular responses to ANDRO between hepatoma
and other cell lines are caused by differences in drug metabolism and
modications within hepatoma cells. It is possible that ANDRO, or its
metabolites, can simultaneously act on multiple cellular targets and
pathways, which are differentially regulated in different cellular contexts. ANDRO is known to interfere, either directly or indirectly, with
several major signalling pathways, including the NF-kB (Xia et al.,
2004), JNK (Ji et al., 2007; Zhou et al., 2008), IL-6 (Chun et al.,
2010), and AKT and ERK pathways (Tsai et al., 2004; Lee et al.,
2010a, 2010b, 2010c). ANDRO may therefore be a useful tool in future
studies to dissect the relative importance of each of these pathways
on cellular phenotypes in different tissue types. The latest addition
to the list of ANDRO targets is serine/threonine kinase STK33. This kinase is known to promote cell viability through the suppression of
mitochondrial apoptosis only in cancer cells with k-ras mutations
(Scholl et al., 2009). ANDRO was identied in a recent high throughput screen (PubChem bioassay AID 2330) as a possible antagonist of
STK33, which suggests that ANDRO cytotoxicity may depend on the
k-ras background of the cancer. There is no obvious correlation of
ANDRO growth inhibition in the NCI60 cell lines (Suppl Fig. 1) and
their k-ras mutations (Ikediobi et al., 2006), but the possible relation
of ras transformation and the growth suppression mechanism of
ANDRO warrants future investigation.
Conclusions
Our study conrms and extends earlier observations that ANDRO exerts a distinct growth inhibitory effect on human hepatoma cells. We have
uncovered the distinct effects of ANDRO on cell cycle regulation in HepG2
cells. Our results might provide explanations to many hepatocyte-specic
actions of ANDRO, and point to new ways in which ANDRO might be used
for future therapeutic strategies in liver cancers.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.lfs.2012.04.009.
Conict of interest statement
The authors declare that there are no conicts of interest.

Acknowledgements
We thank the members of Lam and Cheung Labs for fruitful discussion. This work was funded by a research studentship (to
MTWC) and Strategic Research Grants (Project numbers 7008084
and 7002573) from the City University of Hong Kong.

760

M.T.W. Cheung et al. / Life Sciences 91 (2012) 751760

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