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7 authors, including:
Michael Wai Lun Chiang
Sung-Kay Chiu
SEE PROFILE
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Hon-Yeung Cheung
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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
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
752
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
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.
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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.
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.
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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.
756
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
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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
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
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.
Fig. 8. Response of HepG2 and HeLa cells at different cell cycle stages to ANDRO
treatment.
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
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