Alantolactone Induces Apoptosis in Glioblastoma Cells via GSH
Depletion, ROS Generation, and Mitochondrial Dysfunction
Muhammad Khan, Fei Yi, Azhar Rasul, Ting Li, Nan Wang, Hongwen Gao,
Rong Gao, and Tonghui Ma*
Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, People’s Republic of China
Glioblastoma multiforme (GBM) is the most malignant and
aggressive primary brain tumor in adults. Despite concerted
efforts to improve current therapies, the prognosis of glioblas-
toma remains very poor. Alantolactone, a sesquiterpene lactone
compound, has been reported to exhibit antifungal, antibacte-
ria, antihelminthic, and anticancer properties. In this study, we
found that alantolactone effectively inhibits growth and triggers
apoptosis in glioblastoma cells in a time- and dose-dependent
manner. The alantolactone-induced apoptosis was found to be
associated with glutathione (GSH) depletion, reactive oxygen
species (ROS) generation, mitochondrial transmembrane poten-
tial dissipation, cardiolipin oxidation, upregulation of p53 and
Bax, downregulation of Bcl-2, cytochrome c release, activation
of caspases (caspase 9 and 3), and cleavage of poly (ADP-ribose)
polymerase. This alantolactone-induced apoptosis and GSH
depletion were effectively inhibited or abrogated by a thiol anti-
oxidant, N-acetyl-L-cysteine, whereas other antioxidant (poly-
ethylene glycol (PEG)-catalase and PEG-superoxide-dismutase)
did not prevent apoptosis and GSH depletion. Alantolactone
treatment inhibited the translocation of NF-jB into nucleus;
however, NF-jB inhibitor, SN50 failed to potentiate alantolac-
tone-induced apoptosis indicating that alantolactone induces
NF-jB-independent apoptosis in glioma cells. These findings
suggest that the sensitivity of tumor cells to alantolactone
appears to results from GSH depletion and ROS production.
Furthermore, our in vivo toxicity study demonstrated that alan-
tolactone did not induce significant hepatotoxicity and nephro-
toxicity in mice. Therefore, alantolactone may become a poten-
tial lead compound for future development of antiglioma
? 2012 IUBMB
IUBMB Life, 64(9): 783–794, 2012
alantolactone; GSH; ROS; apoptosis; glioblastoma.
Glioblastoma multiforme (GBM) is the most common and
most virulent primary brain tumor in adults and accounts for
at least 80% of malignant gliomas (1, 2). Over 12,000 patients
die because of primary brain tumor in United States every
year. At present, no ideal treatment exits for GBM and the me-
dian survival rate of patients with GBM remains less than 1
year after diagnosis. Despite recent advances in surgical resec-
tion, radiation therapy, and chemotherapy, the prognosis of
glioblastoma continues to be dismal (3–5). Exploring novel
therapeutic agents and their molecular mechanism are, there-
fore, necessary for improving the outcome of glioblastoma
In recent years, a growing interest has been found to investi-
gate the role of reactive oxygen species (ROS) in different cel-
lular processes including gene expression, differentiation, and
cell proliferation. ROS are produced by eukaryotic cells during
normal oxidative metabolism and cells’ antioxidant system
scavenges ROS to maintain the redox balance. However, an
imbalance between production of ROS and cells antioxidant
system’s ability to readily detoxify ROS results in oxidative
stress (6, 7). Glutathione (GSH) is the most abundant intracellu-
lar antioxidant involved in the protection of cells against oxida-
tive damage and in various detoxification mechanisms (8–10).
Depletion of intracellular GSH results in oxidative stress which
is a known inducer of the transcription of specific genes
involved in cell death (11). On the contrary, higher level of
GSH in cells is related to apoptosis resistance (12).
Sesquiterpene lactones are plant-derived compounds used
in traditional medicines for the treatment of inflammatory dis-
eases (13). Over the past few years, a large body of pharma-
cological studies has provided convincing evidence of the
anticancer property of sesquiterpene lactones against various
human cancer cell lines (14, 15). Alantolactone is one of the
major sesquiterpene lactone compounds, isolated from the
roots of Anula helenium and possesses multiple biological
activities including antibacteria, antifungal, antihelminthic, and
Address correspondence to: Tonghui Ma, Central Research Labora-
tory, Jilin University Bethune Second Hospital, Changchun 130041,
People’s Republic of China. Tel: 186-431-8879-6667. Fax: 186-431-
8879-6667. E-mail: email@example.com
Received 26 March 2012; accepted 11 June 2012
ISSN 1521-6543 print/ISSN 1521-6551 online
IUBMBLife, 64(9): 783–794, September 2012
antiproliferative (16). To date, alantolactone has been shown
to induce growth inhibition only in a few cancer cell lines
(16–18). However, the molecular mechanism underlying alan-
tolactone-induced apoptosis remained largely unknown. This
study was, therefore, conducted to investigate the potential of
alantolactone to induce apoptosis in U87 cells and its underly-
MATERIALS AND METHODS
Chemicals and Antibodies
All the chemicals were purchased from Sigma unless other-
wise stated. Alantolactone (purity [ 99%) was purchased from
Tauto Biotech Co., LTD. (Shanghai, China). The chemical
structure of alantolactone is shown in Fig. 1. Antibodies specific
to Beta-actin, p53, Bax, Bcl-2, cytochrome c, caspase 9, caspase
3, PARP, and NF-jBp65 were purchased from Beyotime. Anti-
bodies specific to superoxide-dismutase-1 (SOD-1) was pur-
chased from BIOSS, Biosynthesis Biotechnology Co., LTD.
whereas antibody specific to catalase was purchased from
Abgent. Horseradish peroxidase-conjugated secondary antibod-
ies (goat anti-rabbit, goat anti-mouse) were purchased from
Sigma (Beijing, China).
Cell Culture and Treatments
Human glioblastoma cell lines U87, U373, and LN229 were
purchased from American Type Culture Collection (USA),
whereas the recombinant U2OS cells were obtained from
Thermo Scientific (Beijing, China). Cells were cultured in Dul-
becco’s Modified Eagle’s Medium supplemented with 10% fatal
bovine serum, 100 units/ml penicillin, and 100 lg/ml strepto-
mycin at 37 8C with 5% CO2in humidified atmosphere. Cells
were treated with various concentrations of alantolactone dis-
solved in dimethyl sulfoxide (DMSO) with a final DMSO con-
centration of 1% for various time points. DMSO-treated cells
were used as control.
Determination of Cell Viability
Cell viability was determined by 3-(4,5-Dimethylthiazol-2-
yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay as described
previously (19). Briefly, U87, U373, and LN229 cells were
seeded into 96-well culture plates in triplicates and treated with
DMSO or various concentrations of alantolactone (1–60 lM)
for 12 h. Following treatment, the MTT reagent was added (500
lg/ml) and cells were further incubated at 37 8C for 4 h. Subse-
quently, 150 ll DMSO was added to dissolve farmazan crystals,
and absorbance was measured at 570 nm in a microplate reader
(Thermo Scientific). The percentage of cell viability was calcu-
lated as follows:
Cell viability ð%Þ ¼
ðA570sample? A570blankÞ=ðA570control? A570blankÞ3100
The IC50values were calculated using GraphPad Prism 5.
Live and dead cells were quantified using live/dead assay as
describe by us previously (19). Briefly, U87 cells were incubated
with 40 lM alantolactone in the presence or absence of N-acetyl-
L-cysteine (NAC) (3 mM), catalase (200 units/ml), PEG-catalase
(200 units/ml), PEG-SOD (200 units/ml), or L-buthionine-(S, R)-
sulfoximine (BSO) (1 mM) for 0, 6, and 12 h. Subsequently,
treated (floating and adherent) and untreated cells were collected,
washed with phosphate-buffered saline (PBS), and incubated with
PBS solution containing 2 lM calcein AM and 4 lM propidium
iodide (PI) in the dark for 20 min at room temperature. After
washing, cells were resuspended in PBS and analyzed for the flu-
orescence of calcein and PI
(Olympus 1 3 71). At the end, 100 cells were counted micro-
scopically for the percentage of live and dead cells.
Flow Cytometric Analysis.
tolactone in dose- and time-dependent manner. After incubation,
floating and adherent cells were harvested, washed with PBS,
and resuspended in 500-ll binding buffer containing 5 ll annexin
V and 5 ll PI and incubated in the dark for 15 min. At the end,
samples were analyzed by flow cytometry (Beckman Coulter,
Epics XL) for the percentage of apoptotic and necrotic cells.
U87 cells were incubated with alan-
Hoechst 33258 Staining.
alantolactone for various time points (0, 6, and 12 h). The cells
U87 cells were treated with 40 lM
Figure 1. Structure of alantolactone and its effect on growth of
glioma cell lines. A: Chemical structure of alantolactone. B:
Effect of alantolactone on growth inhibition of U87, U373, and
LN229 glioma cell lines. Cells were treated with DMSO or var-
ious concentrations of alantolactone (10–60 lM) for 12 h, and
cell viability was measured by MTT assay. Data are expressed
as mean 6 SEM (n 5 3). Columns not sharing same superscript
letters differ significantly (P \ 0.05).
784KHAN ET AL.
were fixed with 4% paraformaldehyde for 30 min at room tem-
perature. After washing with PBS, cells were stained with
Hoechst 33258 (50 lg/ml) at 37 8C for 20 min in the dark. At
the end, the cells were washed and resuspended in PBS for the
observation of nuclear morphology under fluorescence micro-
scope (Olympus 1 3 71).
Determination of Intracellular Reactive Oxygen
Species, Mitochondrial Membrane Potential, and
The intracellular changes in ROS generation and mitochon-
drial membrane potential (MMP) were measured by staining the
cells with 20,70-dichlorofluorescein-diacetate (DCFH-DA) and
Rhodamine 123 as described previously (19). Cardiolipin oxida-
tion was determined by staining the cells with 10-N-nonyl acri-
dine orange (NAO), a probe specific for mitochondrial mem-
brane cardiolipin (20). Briefly, U87 cells were incubated with
40 lM alantolactone for 0, 6, and 12 h. After treatment, cells
were further incubated with DCFH-DA (10 lM), Rhodamine
123 (5 lg/ml), and NAO (5 lM) in the dark for 30 min. After
washing, the cells samples were analyzed for the fluorescence
of DCF, Rhodamine 123, and NAO.
Measurement of Intracellular GSH
using glutathione reduced assay kit (Nanjing Jiancheng, Nanj-
ing). Briefly, U87 cells were treated with 40 lM alantolactone
in the presence or absence of NAC, PEG-catalase, and PEG-
SOD for 0, 6, and 12 h. Following treatment, the intracellular
GSH was measured according to the instruction of kit. The val-
ues were expressed as nmol GSH/mg protein.
NF-jB Translocation Assay
The recombinant U2OS cells, stably expressing human NF-
jB fused to the N-terminal of enhanced green fluorescence pro-
tein were used to examine the translocation of NF-jB into nu-
cleus using NF-jB redistribution HCS assay kit (Thermo Scien-
tific, USA). Briefly, 6,000 cells were plated into 96-well plate
and incubated at 37 8C over night. After incubation, medium
was replaced with cell wash buffer and cells were further incu-
bated at 37 8C for 24 h with 5% CO2in a humidified atmos-
phere. The cells were then incubated with 10 ng/ml of tumor
necrosis factor (TNF)a or a combination of 10 ng/ml TNFa,
and 40 lM alantolactone for 30 min. Fixation, permeabilization,
and immunofluorescence staining of cells were performed
according to the manufacturer’s instructions.
Western Blot Analysis
After drug treatment, adherent and floating cells were col-
lected and proteins were isolated as described previously (19).
Nuclear and cytosolic proteins were extracted using cytosolic
and nuclear extraction kit (KeyGen, China) according to the
manufacturer’s instructions. The protein concentrations were
determined using NanoDrop 1000 (Thermo Scientific, USA). A
total of 50-lg proteins were electrophoresed on 8–12% SDS-
PAGE and transferred to polyvinylidine difluoride (PVDF)
membrane. After blocking with 5% (w/v) nonfat milk and
washing with Tris-buffered saline-Tween solution (TBST),
membranes were incubated overnight at 4 8C with p53
(1:1,000), BCL-2 (1:1,000), Bax (1:300), cytochrome c (1:200),
caspase 9 (1:1,000), caspase 3 (1:500), PARP (1:1,000), NF-jB
p65 (1:500), SOD-1 (1:300), catalase (1:2,000), and b-actin
(1:400) antibodies, respectively. After washing, the blots were
incubated with horseradish peroxidase-conjugated goat anti-rab-
bit IgG or goat anti-mouse IgG secondary antibodies (1:5,000)
for 1 h at room temperature. After washing with TBST, signals
were detected using ECL plus chemiluminescence’s kit on X-
ray film (Millipore Corporation).
In Vivo Studies
In vivo toxicity studies were conducted on 12–14-week-old
Kunming mice weighing 43–45 g. Mouse procedures were
approved by the Experimental Animal Committee of Jilin Uni-
versity (Permit No: SCXK 2007-0011). The mice were main-
tained in a specific pathogen-free grade animal facility on a 12-
h light/dark cycles at 25 6 2 8C. Mice were divided into two
groups. Group A (n 5 6) administered with 100 ll DMSO in-
traperitoneally; group B (n 5 6) administered with alantolac-
tone (100 mg/kg body weight) in 100 ll DMSO intraperitone-
ally. The experiment was conducted over a period of 5 weeks.
DMSO or drug was administered daily for 5 weeks, once a day.
At the first and last day of the experiment, the body weight of
each mouse was measured. At the end of experiment, mice
were anesthetized using Pentobarbital sodium (50 mg/kg ip),
blood was collected via cardiac puncture, allowed to clot for 10
min, and centrifuged at 1,0003g for 10 min at room tempera-
ture. Serum was separated and stored at 220 8C until analysis.
The liver and kidneys were excised and processed for hematox-
ylin and eosin staining followed standard procedures.
Blood Biochemistry Analysis.
tolactone treatment on the biochemical parameters of the experi-
mental mice were evaluated by the estimation of the serum bio-
chemical enzymes of liver function such as aspartate amino-
transferase, alanine aminotransferase, total bilirubin, alkaline
phosphatase, albumin, gamma glutamyl transpeptidase (GGT),
lactate dehydrogenase, blood glucose, and cholesterol. Nephro-
toxicity was determined by measuring the serum levels of blood
urea nitrogen and creatinine (Cr). In addition, the level of K1,
Na1, Mg11, and Ca11were also determined. These biochemi-
cal parameters were determined by an automated biochemical
analyzer (Hitechi 7170, Japan).
The toxicological effect of alan-
Evaluation of Alantolactone Transport Across the Blood–Brain
To determine the transport of alantolactone across the
blood–brain barrier, mice were injected with (200 mg/kg, ip)
785ALANTOLACTONE INDUCES APOPTOSIS IN GLIOBLASTOMA CELLS
alantolactone or with DMSO. After 6 h, brains were collected
from control and treatment groups, homogenized in PBS, and
were extracted with acetonitrile for the presence of alantolac-
tone. After centrifugation at 5,0003g for 15 min, the superna-
tants were analyzed by high performance liquid chromatography
(HPLC) (Waters) using XTerra MS C18 (5 lm, 4.6 mm 3 150
mm) column. The mobile phase was composed of acetonitrile
(A) and water (B). The gradient program was as follows: 0–30
min, A 5 65%, B 5 35%.
RNA Isolation and RT-PCR
Total RNA was isolated from treated and untreated U87 cells
using AxyPrep Multisource Total RNA Miniprep kit. Total
RNA (0.5 lg) was reverse transcribed into cDNA at 37 8C for
15 min followed by enzyme inactivation at 85 8C for 5 sec
using RT-PCR kit (Invitrogen). This was followed by 35 cycles
of (94 8C: 1 min; 52 8C: 30 sec; 72 8C: 1 min) and a final
extension of 72 8C for 10 min. PCR product was visualized on
a 1% agarose gel containing ethidium bromide. The primers
used were as follows:
GSTp, 50-GGTGAATGACGGCGTGGAG-30(Forward) and
GGCACAGGTAAAACCAAATAGTAAC-30(Forward) and 50-
50-ATGACATCAAGAAGGTGGTG-30(Forward) and 50-CAT-
(Reverse); c-GCS, 50-
The results are expressed as mean 6 standard error mean
(SEM). and statistically compared with control group or within
the groups using one way ANOVA followed by Tukey’s Multi-
ple Comparison Test. Student t-test was used to determine sig-
nificance when only two groups were compared and P \ 0.05
was considered statistically significant.
Alantolactone Inhibits Growth of Glioma Cells In Vitro
The cytotoxic effect of alantolactone on glioma was eval-
uated using U87, U373, and LN229 cell lines. Alantolactone
treatment increased the growth inhibition of U87, U373, and
LN229 cells in a dose-dependent manner (Fig. 1B). The IC50
values of alantolactone against U87, U373, and LN229 cells
were 33, 35, and 36 lM, respectively. U87 cell line was found
more sensitive to alantolactone and selected for further mecha-
nistic study. To characterize in detail whether alantolactone
induced cell death or merely inhibited the cells growth, we
treated U87 cells with 40 lM alantolactone for various time
points (0, 6, and 12 h) and morphological changes of cells were
observed. As shown in Fig. 2A, alantolactone-treated cells dis-
played drastic morphological changes of cell death including a
reduction in total number of cells and an increase in floating
cells in culture medium in a time-dependent manner. To quan-
tify the live and dead cells, we stained the cells with calcein
AM and PI. Figure 2B shows the staining of cells with calcein
AM and PI. Live cells took up calcein AM, de-esterified, and
retained the green calcein dye, whereas dead cells were unable
to retain calcein or to exclude PI. Live (green) and dead cells
(red) were counted microscopically. The data showed that the
viability of cells treated with 40 lM alantolactone for 6 and
12 h was significantly lower (59.33% and 29.6%, respectively)
compared with untreated control group (98.3%).
Alantolactone Induces Apoptosis in U87
To study the nature of alantolactone-induced cell death, U87
cells were treated with 30 and 40 lM alantolactone for 24 h
Figure 2. Microscopic analysis of U87 glioblastoma cells. A:
U87 cells were treated with 40 lM alantolactone for various
time points (0, 6, and 12 h), and morphological changes were
observed by phase contrast microscopy. Scale bar 5 100 lm.
B: U87 Cells were treated with 40 lM alantolactone for various
time points (0, 6, and 12 h). Live and dead cells were quantified
using Live/Dead assay. C: Statistical analysis of live and dead
cells from (B). Data are expressed as mean 6 SEM (n 5 3).
Columns not sharing same superscript letters differ significantly
(P \ 0.05). [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
786KHAN ET AL.
and cells undergoing apoptosis/necrosis were determined by
flow cytometry analysis after staining with annexin V-FITC and
PI. The data demonstrated that alantolactone induced a dose-de-
pendent apoptosis. However, exposure of cells to 40 lM alanto-
lactone for 24 h resulted in significant necrosis (Fig. 3A). Next,
we asked whether alantolactone-induced necrotic cell death is
dose-dependent or time-dependent. We treated the cells with 40
lM alantolactone for 6 and 12 h and cell death was determined
by flow cytometry analysis. The data demonstrated that alanto-
lactone caused only apoptotic cell death in a time-dependent
manner (Fig. 3B). The alantolactone-mediated apoptotic cell
death was further confirmed using H33258 staining and fluores-
cence microscope. U87 cells were treated with 40 lM alantolac-
tone for 0, 6, and 12 h and nuclear morphological changes were
observed under fluorescence microscope after staining with
H33258. As depicted in Fig. 3C, the nuclei in the control group
were round and stained homogeneously, whereas alantolactone-
treated cells showed condensed and fragmented nuclei in a
time-dependent manner. Taken together, the data demonstrated
that alantolactone induced dose- and time-dependent apoptosis
in U87 glioblastoma cells. The necrotic cell death is resulted
when cells are exposed to higher concentration of alantolactone
for longer time periods. Therefore, 40 lM concentration and
short-time period (6 and 12 h) were selected to investigate the
molecular mechanism of alantolactone-induced apoptosis in
U87 glioblastoma cells.
Effect of Alantolactone on ROS Generation,
Mitochondrial Membrane Potential, and Cardiolipin
As shown in Fig. 4, alantolactone increased the level of
ROS, decreased MMP, and induced cardiolipin oxidation (CL
oxidation) in U87 cells in a time-dependent manner. The level
of ROS in cells treated with 40 lM alantolactone for 6 and 12
h was significantly higher (36.93% and 51.7% vs. 13.63% in
control group, P \ 0.05). As shown in Fig. 4B, MMP in cells
treated with 40 lM alantolactone for 6 and 12 h was signifi-
cantly lower (76.6% and 46.0% vs. 92.3% in control group, P
ROS once generated, cause massive oxidation of redox sensi-
tive proteins and lipids leading to mitochondrial damage. Next,
we asked if alantolactone can cause mitochondrial cardiolipin
oxidation (CL oxidation). To examine CL oxidation, cells were
stained with NAO, and CL oxidation was determined by flow
cytometry. The data indicated that alantolactone caused CL oxi-
dation in U87 cells in a time-dependent manner (Fig. 4C).
Alantolactone Reduces Intracellular GSH in U87 Cells
Intracellular GSH plays major roles in the maintenance of re-
dox status and defense of oxidative stress. GSH depletion is an
early hallmark observed in ROS mediated apoptosis (21). We,
therefore, investigated the status of intracellular GSH in control
and alantolactone-treated U87 cells. Time-dependent study
revealed that GSH depletion was significant from 6 h of treat-
ment and increased over time. Pretreatment of cells with NAC,
completely inhibited the depletion of intracellular GSH, whereas
PEG-catalase and PEG-SOD failed to prevent GSH depletion
indicating that GSH depletion is independent of ROS generation
Figure 3. Analysis of apoptosis of alantolactone on U87 glio-
blastoma cells. A: U87 cells were treated with DMSO or 30
and 40 lM alantolactone for 24 h, and apoptosis rates were ana-
lyzed by flow cytometry after Annexin V/PI staining. B: U87
cells were treated with DMSO or 40 lM alantolactone for indi-
cated time points, and apoptosis was analyzed by flow cytome-
try. C: U87 cells were treated with DMSO or 40 lM alantolac-
tone for indicated time points and nuclear morphological
changes were observed using Hoechst 33258 staining and fluo-
rescence microscope. Arrows indicate the fragmented nuclei. D:
Data are expressed as mean 6 SEM of three independent
experiments presented in (A) and (B). Columns not sharing
same superscript letters differ significantly (P \ 0.05). [Color
figure can be viewed in the online issue, which is available at
787 ALANTOLACTONE INDUCES APOPTOSIS IN GLIOBLASTOMA CELLS
Alantolactone-Induced Cell Death is Abrogated by Thiol
Antioxidant NAC and Enhanced by BSO
We found that alantolactone disrupted MMP by depleting
intracellular GSH and increasing ROS generation. Therefore,
we observed the effect of antioxidants on cell death induced by
alantolactone. Cells were treated with NAC, BSO, PEG-cata-
lase, and PEG-SOD 1 h before the treatment of alantolactone.
As shown in Fig. 5B, pretreatment of cells with thiol antioxi-
dant, NAC completely blocked alantolactone-mediated cell
death in U87 cells, whereas BSO, a GSH depleting agent,
enhanced it. Furthermore, pretreatment of cells with 200 units/
ml PEG-catalase or 200 units/ml PEG-SOD or a combination of
both (nonthiol antioxidants) failed to protect the cells from alan-
tolactone-proceeded cell death. PEG-catalase slightly increased
the viability of cells; however, this protective effect was not
significant (P \ 0.05). To exclude the possibility of alantolac-
tone as exogenous oxidant, we treated the cells with alantolac-
tone in the presence of 200 units/ml catalase. As expected, sup-
plementation of catalase in culture medium did not improve the
viability of cells. These results were further confirmed by
observing the expression of catalase and SOD-1 by Western
blot analysis. Our Western blot results demonstrated that alanto-
lactone treatment decrease the expression of catalase in a time-
dependent manner; however, no change in the expression of
SOD-1 was observed (Fig. 5C).
Alantolactone Induces Apoptosis in U87 Cells Without
Recently, many sesquiterpene lactone compounds have been
shown to induce apoptosis in various cancer cell lines through
inhibition of NF-jB activation (13, 14). We were interested to
know if the apoptotic effect of alantolactone in U87 glioblas-
toma cells is due to its inhibition of NF-jB activation. For
this, we tested alantolactone for its inhibitory effect against
NF-jB translocation activated by TNFa, using a model cell
line U2OS, by HCS assay. As shown in Fig. 6A, alantolactone
inhibited the TNFa-induced translocation of NF-jB into nu-
cleus. Next, we treated U87 cells with TNFa alone or in com-
bination with alantolactone for 1 h and nuclear lysates were
subjected to Western blot. As shown in Fig. 6B, alantolactone
inhibited the TNFa-induced NF-jB translocation into nucleus
in U87 cells. To further confirm the involvement of NF-jB in-
hibition in apoptosis induced by alantolactone in U87 cells, we
treated the cells with 40 lM alantolactone in the presence of
20 lM SN50 (Enzo Life Science), a specific NF-jBp65 inhibi-
tor, for 6 h and apoptosis rate was determined by flow cytom-
Figure 4. Effect of alantolactone on ROS generation, mitochondrial membrane potential, and cardiolipin oxidation in U87 glioblas-
toma cells. U87 glioblastoma cells were treated with DMSO or 40 lM alantolactone for indicated time points. ROS generation (A),
mitochondrial membrane potential (B), and cardiolipin oxidation (C) were determined after staining the cells with DCFH-DA, Rho-
damine 123, and NAO, respectively. Columns representing the flow cytometry data are presented at left. Data are expressed as
mean 6 SEM (n 5 3). Columns not sharing same superscript letters differ significantly (P \ 0.05). [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
788KHAN ET AL.
etry. The data demonstrated that treatment of cells with SN50
decreased the apoptotic effect of alantolactone in U87 cells
(Fig. 6C). However, SN50 effectively inhibited TNFa-induced
NF-jB translocation into nucleus in U2OS cells (data not
shown). Because inhibition of NF-jB activation sensitizes cells
to TNFa-induced apoptotic cell death, we treated the cells
with TNFa in combination with alantolactone or SN50 and
observed the rate of apoptosis. The data showed that treatment
with TNFa in the presence of alantolactone or SN50 did not
potentiate apoptotic cell death in U87 glioblastoma cells (Fig.
6C). These findings suggest that alantolactone is effective in
inducing apoptosis in U87 glioblastoma cells through a process
that does not involve NF-jB.
Figure 5. Alantolactone induced cell death through GSH deple-
tion. A: U87 cell were treated with 40 lM alantolactone in the
presence or absence of antioxidants for indicated time points. The
intracellular GSH contents were determined according to the
instructions of kit. The values were expressed as nmol GSH/mg
protein. Data are expressed as mean 6 SEM (n 5 3). Columns
not sharing same superscript letters differ significantly (P \ 0.05).
B: U87 cells were treated with 40 lM alantolactone for 6 h in the
presence or absence of antioxidants (NAC, Catalase, PEG-catalase,
and PEG-SOD) and BSO, and cell viability was determined by
Live/Dead assay. Data are expressed as mean 6 SEM (n 5 3).
Columns not sharing same superscript letters differ significantly
(P \ 0.05). C: U87 glioblastoma cells were treated with 40 lM
alantolactone for indicated time points. The expression of catalase
and SOD-1 were determined by Western blot analysis.
Figure 6. Involvement of NF-jB in alantolactone-induced apo-
ptosis in U87 glioblastoma cells. A: U2OS cells were incubated
with 10 ng TNFa or a combination of 10 ng TNFa and 40 lM
alantolactone for 30 min, and NF-jB translocation in the nu-
cleus was observed using NF-jB redistribution HCS assay kit.
B: U87 cells were treated with 10 ng TNFa or a combination of
10 ng TNFa and 40 lM alantolactone for 1 h. Nuclear extract
was subjected to Western blot analysis. C: U87 cells were
treated with 40 lM alantolactone in the presence or absence of
10 ng TNFa and 20 lM SN50 or a combination of 10 ng TNFa
and 20 lM SN50 for 6 h, and apoptosis was determined by
flow cytometry analysis. Data are expressed as mean 6 SEM (n
5 3). Columns not sharing same superscript letters differ signif-
icantly (P \ 0.05). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
789 ALANTOLACTONE INDUCES APOPTOSIS IN GLIOBLASTOMA CELLS
Alantolactone Induces Apoptosis in U87 Glioblastoma
Cells Through Mitochondrial Pathway
Generation of ROS, disruption of MMP, and GSH depletion
are characteristic features of mitochondrial apoptosis. To gain
better insight into alantolactone-induced apoptosis in U87 cells, we
measured the expression of major mitochondrial apoptosis regula-
tors, using Western blots. The data demonstrated that alantolactone
remarkably increase the expression of p53 and Bax and decrease
the expression of Bcl-2 with concomitant release of cytochrome c
from mitochondria into cytosol (Fig. 7A). This alantolactone-medi-
ated apoptosis was further confirmed by observing the expressions
of caspase 3 and 9 and PARP. Figure 7B shows the cleavage of
caspase 3 and 9 into active fragments and cleavage of PARP into
85 kDa protein.
Alantolactone Exerts No Toxic Effect on Liver and
Kidneys In Vivo
Hepatotoxicity and nephrotoxicity are the major side effects
of cancer chemotherapeutic agents. Therefore, we investigated
the effect of alantolactone on liver and kidneys using Kunming
mice. The cytotoxic effect of alantolactone was evaluated by
measuring the changes in body weight, blood biochemistry, and
histopathology of liver and kidneys in comparison with control
group. The drug was well tolerated by mice and no mortality or
any sign of pharmacotoxicity was found at a dose of 100 mg/kg
during all the experimental periods. Body weight gains and
food consumption were comparable for control and treated mice
during the experimental period, and there were no drug-related
changes in histopathological and blood biochemistry parameters.
The histopathological changes in liver and kidneys were exam-
ined using hematoxylin and eosin staining and correlated with
liver and renal function biomarkers. No obvious histopathologi-
cal changes were observed in liver and kidneys structures of
treatment group compared with control group (Fig. 8). These
Figure 7. Effect of alantolactone on the expression of apoptosis
regulators in U87 glioblastoma cells. U87 cells were treated
with 40 lM alantolactone for indicated time intervals. Total cell
lysates and cytosolic proteins were extracted as described in
‘‘Materials and Methods’’ section. A total of 50 lg proteins
were subjected to Western blot for the expression of (A) p53,
Bax, Bcl-2, and cytochrome c, and (B) caspase 9, caspase 3,
Figure 8. Effect of alantolactone on mice liver and kidneys.
Kunming mice were administered with vehicle or alantolactone at
a dose of 100 mg/kg body weight for 5 weeks. The liver and kid-
neys from control and alantolactone-treated mice were excised
and processed for hematoxylin and eosin staining followed estab-
lished procedures. The stained sections of liver and kidneys were
photographed at different magnifications. Upper liver section,
scale bar 5 100 lm; lower liver sections, scale bar 5 20 lm;
upper kidneys section, scale bar 5 100 lm; lower kidneys sec-
tion, scale bar 5 20 lm. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
790 KHAN ET AL.
results were further confirmed by measuring the changes in liver
function biomarkers and renal function biomarkers in the serum
of control and treatment groups. The effect of alantolactone
treatment on the biochemical parameters of the experimental
mice is shown in Table 1.
Alantolactone Can Cross Blood–Brain Barrier
High performance liquid chromatography (HPLC) method
was used to check the penetration of alantolactone into mouse
brain. Brains were collected from control and treatment groups
and homogenized in PBS. The homogenates were extracted
with acetonitrile and centrifuged. The supernatant was analyzed
by HPLC. Figure 9A shows the representative chromatograms
of alantolactone standard and brain samples, respectively. The
HPLC analysis of standard solution (alantolactone) showed a
single peak at a retention time of 14.5 min. This peak was
absent in brain samples prepared from control group; however,
the peak could be easily detected in brain samples prepared
from treatment group. The data indicates clearly that alantolac-
tone can cross blood–brain barrier.
Effect of Alantolactone on Expression of
GSTp and c-GCS
Glutathione S-transferases (GST) are known to eliminate
drugs and toxic chemicals found in the food. GSTp is the major
isoenzyme of GST found in brain and its expression has been
reported to correlate with glioma cell resistance to nitrosoureas.
The induction of c-glutamylcystein synthetase (c-GCS), which
is a rate limiting enzyme in the synthesis of GSH, has been
associated with drug resistance to cancer chemotherapy. There-
fore, we examined the expressions of GSTp and c-GCS by RT-
PCR. The data demonstrated that mRNA expression of GSTp
was slightly increased, whereas no change in the mRNA expres-
sion of c-GCS was found in U87 cells exposed to alantolactone
It is well known that the redox status inside the cell is cru-
cial to the correct functioning of many enzymes and plays a
central role in the regulation of various cell functions (8).
Recent evidence has demonstrated that ROS serve as messen-
gers for normal signal transductions at lower concentrations; at
higher concentrations, however, they become toxic and induce
cell death through various signaling pathways (8, 22). As cancer
cells contain higher level of ROS than normal cells, they can be
easily poisoned by the phytochemical targeting ROS metabo-
lism (17, 18). In recent years, a growing interest in the role of
ROS in cancer prevention has prompted the identification of
phytochemical targeting ROS metabolism. Alantolactone, a
sesquiterpene lactone compound, has been reported to possess
antifungal, antibacteria, and antihelminthic properties. Very
recently, it has been shown to induce ROS generation and apo-
ptosis in HL-60 cells (22). However, the exact role of ROS in
cell death induced by alantolactone has not been explored. This
study was, therefore, conducted to examine whether alantolac-
tone can increase the level of ROS and inhibit the growth of
glioma cells. We found that alantolactone effectively inhibited
the growth of U87, U373, and LN229 glioma cell lines in a
dose-dependent manner. To further investigate the growth inhib-
itory effect of alantolactone, we performed live/dead and apo-
Effect of alantolactone on blood biochemistry of control and treated mice
ParametersControl group Treatment group Significance (P \ 0.05)
Alanine aminotransferase (U/l)
Aspartate aminotransferase (U/l)
Total bilirubin (lmol/l)
Alkaline phosphatase (lmol/l)
Lactate dehydrogenase (U/l)
Blood glucose (U/l)
Blood urea nitrogen (mmol/l)
47.10 6 7.98
147.15 6 6.34
0.93 6 0.15
44.25 6 8.72
26.55 6 1.63
2.25 6 0.47
1026 6 99.73
9.78 6 1.15
1.94 6 0.07
9.72 6 1.35
20.36 6 2.54
5.06 6 0.4
139.8 6 1.94
0.93 6 0.02
2.21 6 0.07
43.0 6 6.71
139.70 6 10.27
0.79 6 0.34
30.75 6 3.81
22.0 6 1.33
1.50 6 0.28
856.3 6 60.18
9.49 6 1.33
1.57 6 0.25
9.02 6 0.85
20.52 6 4.94
5.43 6 0.51
147.7 6 2.71
0.92 6 0.04
2.19 6 0.02
Values are mean 6 SEM, (n 5 6). s, significant; ns, not significant (P \ 0.05).
791ALANTOLACTONE INDUCES APOPTOSIS IN GLIOBLASTOMA CELLS
ptosis assays using U87 cell line. The results demonstrated that
increased growth inhibition by alantolactone was due to induc-
tion of cell death.
Oxidative stress is caused by an imbalance between produc-
tion of ROS and antioxidant defense system’s ability to readily
detoxify the reactive intermediates. The cells’ endogenous anti-
oxidant defense systems scavenge the ROS to maintain redox
balance (10, 11). The GSH redox system is one of the important
antioxidant defense systems involved in the protection of cells
against oxidative damage and in various detoxification systems
(12–14). In addition to GSH, catalase and SOD play important
role to detoxify hydrogen peroxide and super-oxides, respec-
tively. A number of therapeutic agents have been shown to
increase intracellular ROS generation by downregulating antiox-
idant enzymes such as catalase and SOD (7, 20, 23). However,
some other studies have reported that reduction in intracellular
GSH is necessary for the formation of ROS (10, 24). Hence, we
sought to clarify the exact mechanism of ROS production and
GSH depletion in alantolactone-treated U87 cells. Our data
showed that alantolactone treatment increased GSH depletion in
U87 cells in a time-dependent manner. This alantolactone-medi-
ated GSH depletion and ROS generation was completely inhib-
ited by NAC. PEG-catalase and PEG-SOD decreased the level
of ROS but failed to prevent GSH depletion in alantolactone-
treated cells (data not shown). To further confirm, we measured
the expression of catalase and SOD-1 by Western blot analysis.
The expressions of these antioxidant enzymes further support
that GSH depletion is independent of ROS generation.
Next, we explored the functional link between GSH deple-
tion, ROS production, and apoptosis. To probe the possible role
of ROS in alantolactone-mediated apoptotic cell death in U87
cells, we performed live/dead assay in the presence of different
antioxidants. Alantolactone-induced cell death was completely
blocked by thiol antioxidant NAC, whereas BSO enhanced it.
Meanwhile, nonthiol antioxidants including catalase, PEG-cata-
lase, and PEG-SOD did not protect the cells from death. This
study revealed that alantolactone-mediated oxidative stress and
cell death is mainly resulted from the GSH depletion. Our
results are also supported by a previous report that parthenolide,
a sesquiterpene lactone compound, showed a similar effect in
SH-JI cells (25).
GSH is a major neuronal antioxidant that plays a central role
in detoxification of H2O2and prevention and repair of peroxida-
tive damage to lipids, proteins, and nucleic acid (26). It is well-
documented that p53-mediated apoptosis involves generation of
ROS and depolarization of MMP. Recently, GSH depletion has
been reported to induce p53 expression in neuronal and glial
cells. p53 is a tumor suppressor protein that responds to various
stimuli including oxidative stress. Once activated, it induces
downstream modulation of gene expressions with effect on cell
survival, apoptosis, and proliferation. Apoptotic activity of p53
is crucial for eliminating defective and potentially carcinogenic
cells (26–28). Accordingly, our study demonstrates that alanto-
lactone treatment increases the expression of p53 in U87 cells
in a time-dependent manner. Other studies have shown that
GSH depletion can directly modulate MMP, Bax translocation,
and caspases activation, resulting in activation of mitochondrial
death cascade (10, 29). In this study, alantolactone disrupted
MMP and increased the expression of Bax accompanied by a
decrease in Bcl-2 expression. These results are further supported
by a previous study by Pal et al., who demonstrated a similar
effect of alantolactone in HL-60 cells (16). Release of cyto-
chrome c from mitochondria to cytosol is a hallmark of mito-
chondrial apoptosis. Cytochrome c binds with Apaf-1 forming
an oligomeric Apaf-1-cytochrome c complex (apoptosome).
This apoptosome then binds and activates caspase 9, which in
turn activates downstream caspases including caspase 3. Cas-
pase 3 has been identified as a main executioner of apoptotic
response inside the cells. Finally, activated caspase 3 cleaves
effectors proteins including PARP and induces DNA fragmenta-
Figure 9. Transport of alantolactone across blood–brain barrier.
A: HPLC chromatograms of alantolactone standard (0.5 mg/ml)
and brain samples, respectively. Mice in control groups were
injected with DMSO, whereas mice in treatment group were
injected with 200 mg/kg for 6 h. Brains were collected from
both groups and samples were prepared as described in ‘‘Mate-
rials and Methods’’ section. B: U87 glioblastoma cells were
treated with 40 lM alantolactone for indicated time and mRNA
expressions of GSTp and c-GCS were determined by RT-PCR.
792KHAN ET AL.
tion in nucleus which eventually leads to cell death (25, 30). In
line with general understanding, release of cytochrome c into
cytosol was detected in alantolactone-treated cells accompanied
by the cleavage of caspase 9, caspase 3, and PARP in a time-
dependent manner. The data demonstrate clearly that alantolac-
tone induces apoptosis in U87 glioblastoma cells through mito-
chondrial dysfunction and caspases activation.
Recently, many sesquiterpene lactone compounds have been
shown to induce apoptosis in various cancer cell lines through
inhibition of NF-jB activation (31–33). Inhibition of TNFa-
induced NF-jB activation results in apoptosis by recruiting cas-
pase 8, indicating that TNFa can induce either prosurvival or
apoptotic pathway (34). Alantolactone has been shown to
increase the expression of TNFR1 and activation of caspase 8
in HL-60 cells (16). In this study, alantolactone inhibited
TNFa-induced NF-jB activation in U2OS cells and U87 glio-
blastoma cells. To further explore that whether inhibition of
NF-jB activation is involved in alantolactone-mediated apopto-
sis in U87 glioblastoma cells, we treated the cells with 40 lM
alantolactone in the presence of 20 lM SN50 and observed the
apoptosis rate. The data demonstrated that treatment of cells
with SN50 did not potentiate alantolactone-induced apoptosis.
Similarly, treatment of cells with TNFa in combination with
alantolactone did not show any additive effect on apoptosis.
Next, we exposed the U87 cells to 10 ng/ml TNFa and 20 lM
SN50 and observed the apoptosis. We found that this treatment
failed to induce apoptosis in U87 cells. These sets of data dem-
onstrate clearly that inhibition of NF-jB activation is not
involved in alantolactone-mediated apoptosis in U87 glioblas-
toma cells. Our results are further supported by a previous
report of Anderson and Bejcek who showed that parthenolide,
another sesquiterpene lactone compound, induced apoptosis in
U87 cells without affecting NF-jB (35).
Currently available chemotherapeutic drugs not only kill the
cancer cells but also exhibit severe organ toxicity and undesirable
side effects. Hepatotoxicity and nephrotoxicity have been recog-
nized as the most important side effects of conventional chemo-
therapy. Doxorubicin and Cisplatin are widely used anticancer
drugs for various cancers including brain cancer. A large number
of reports have documented the hepatotoxicity and nephrotoxicity
of these drugs (36–39). Therefore, alternative therapeutic agents
that kill cancer cells without or with low hepatotoxicity and
nephrotoxicity are highly desirable. In this study, we investigated
the effect of alantolactone on mouse liver and kidneys. Seven
times higher dose was used for in vivo study compared with in
vitro study. We found that alantolactone did not induce signifi-
cant toxicity in liver and kidneys, highlighting its importance for
the future development of cancer chemotherapy. However, it
remained unknown in this study that whether this dose can in-
hibit the growth of tumor in vivo. We are continuing our further
in vivo study to determine the effects of alantolactone on the
growth of tumor and on normal brain tissues.
It is well-documented that cancer cells contain higher level
of endogenous ROS than normal cells (17, 18). Other studies
have shown that tumor cells have lower level of GSH (40) and
GSTp (41) than normal cells. Higher levels of GSH and GSTp
have been associated with drug resistance to chemotherapy. In
this study, alantolactone reduced the level of GSH and showed
strong growth inhibitory effect toward U87 glioblastoma cells,
whereas it did not induce any detectable toxicity in mouse liver
and kidneys. This selective toxicity of alantolactone between
cancer cells and normal cells may be due to much lower level
of oxidative stress in normal cells than cancer cells. Our data is
further supported by another study by Wen et al., who showed
that parthenolide, a sesquiterpene lactone compound, reduced
the level of GSH in cancer cells, whereas increased the level of
GSH in normal liver cells (41). Sesquiterpene lactones have
been reported to increase the level of GSH and GSTp in normal
liver cells (41, 42). Therefore, we checked the mRNA expres-
sion of c-GCS and GSTp in U87 glioblastoma cells. The
mRNA expression level of GSTp slightly increased, whereas no
difference in mRNA expression of c-GCS was observed before
and after treatment of cells with alantolactone. Future medicinal
chemistry studies may be needed to improve the anti-glioma ac-
tivity and reduce the induction of GSTp.
In summary, our data provide evidence for the first time that
alantolactone induces apoptosis in U87 glioblastoma cells via
GSH depletion and oxidative stress resulting in disruption of
MMP, p53 activation, increase Bax/Bcl-2 ratio, release of cyto-
chrome c, and cleavage of caspases (9 and 3) and PARP. How-
ever, this induced apoptosis was not affected by inhibition of
NF-jB activation. Importantly, alantolactone did not induce
significant toxicity in mouse liver and kidneys. Therefore, alan-
tolactone may become a potential lead compound for future
development of antiglioma therapy.
The work is supported by Ministry of Education of Pakistan
and Chinese Scholarship Council of China.
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