J. Pineal Res.

2012; 53:180187

 2012 John Wiley & Sons A/S

Journal of Pineal Research

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/j.1600-079X.2012.00985.x

Melatonin suppresses migration and invasion via inhibition of

oxidative stress pathway in glioma cells
Abstract: Melatonin, an indolamine produced and secreted predominately by
the pineal gland, exhibits a variety of physiological functions, possesses
antioxidant and antitumor properties. In this study, we have shown that
pharmacologic concentration (1 mm) of melatonin signicantly reduced cell
migration and invasion of T98G and U251 glioma cells after 24-hr treatment
and inhibited expression of matrix metalloproteinase 2 (MMP 2) and MMP 9.
The melatonin inhibition of cell migration and invasion was associated with
its reduction of intracellular basal free radical generation. Melatonin at
pharmacologic concentration also inhibited the constitutive activation of the
reactive oxygen species downstream transcription factor nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-jB). Furthermore,
pyrrolidine dithiocarbamate, a NF-jB-specic inhibitor, at 10 lm displayed
anti-migration and invasion eects and inhibition of MMP 2 and MMP 9
expression resembling that of melatonin. Taken together, it is concluded that
inhibition of migration and invasion of glioma cells by melatonin is
associated with the latter in its inhibition of oxidative stress pathway. This
suggests a potential therapeutic application of melatonin in the treatment of
glioma.

Juntao Wang1, Hongbo Hao2,

Linli Yao3, Xiaodan Zhang4,
Shidou Zhao5, Eng-Ang Ling6,
Aijun Hao3 and Gang Li1
1
Department of Neurosurgery, Qi Lu Hospital,
Shandong University, Jinan, China;
2
Department of General Surgery, Provincial
Hospital affiliated to Shandong University,
Jinan, China; 3Key Laboratory of the Ministry
of Education for Experimental Teratology,
Shandong Provincial Key Laboratory of Mental
Disorders, Department of Histology and
Embryology, Shandong University School of
Medicine, Jinan, China; 4Department of
Traditional Chinese Integrated Western
Medicine, Qi Lu Hospital, Shandong
University, Jinan, China; 5Center for
Reproductive Medicine, National Research
Center for Assisted Reproductive Technology
and Reproductive Genetics, The Key
Laboratory for Reproductive Endocrinology of
Ministry of Education, Shandong Provincial
Key Laboratory of Reproductive Medicine,
Shandong University, Jinan, China;
6
Department of Anatomy, Yong Loo Lin School
of Medicine, National University of Singapore,
Singapore.

Key words: glioma, invasion, melatonin,

migration, NF-jB, ROS
Address reprint requests to Aijun Hao, PhD,
Key Laboratory of the Ministry of Education for
Experimental Teratology, Shandong Provincial
Key Laboratory of Mental Disorders, Department of Histology and Embryology, Shandong
University School of Medicine, 44#, Wenhua Xi
Road, 250012, Jinan, China.
E-mail: aijunhao@sdu.edu.cn
Gang Li, MD, PhD, Department of Neurosurgery, Qi Lu Hospital, Shandong University,
107#, Wenhua Xi Road, Jinan 250012, China.
E-mail: doctorligang@126.com
Received December 8, 2011;
Accepted February 1, 2012.

Introduction
Invasion is a characteristic feature of human glioma being
most responsible for the poor prognosis with average
survival no more than 1 yr [1]. Diused inltration into
surrounding brain tissue restricts curative resection and
limits eective delivery of radio- and chemotherapy.
Therefore, the control of metastasis and invasion represents
an important therapeutic target in the treatment of glioma.
180

However, the present therapy strategies are limited in view

of neural toxicity, hematologic toxicity, or diculty in
crossing the bloodbrain barrier (BBB) of many drugs [2].
Thus, development of new therapeutic strategies that are
eective in inhibition of tumor progression and less toxicity
as well as readily crossing the BBB is desirable.
Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that is synthesized by the pineal gland and the
suprachiasmatic nucleus as well as peripheral tissues [36].

Melatonin inhibits migration and invasion of glioma cells

It is soluble in both lipid and water; hence, it can easily
cross the BBB. Besides a variety of important physiological
functions [710], melatonin exerts a neuroprotective eect
in many pathological conditions of the central nervous
system [11], including Parkinsons disease [12], Alzheimers
disease [13], and ischemic brain injury [14]. Also, melatonin
displays antioxidant eects and acts as a free radical
scavenger [11, 1520]. Recent interest on melatonin has
been focused on its potential antitumor eects. Indeed, the
antitumor eects of melatonin have been tested in many
types of tumors, including prostate cancer, sarcomas,
colorectal cancer, hepatocarcinomas, melanoma, ovarian
cancer, breast cancer, as well as brain tumors (see review
[21]). Melatonin inhibits cell proliferation and induces
apoptosis in most cell lines of the aforementioned tumors
and decreases tumor growth in murine tumor models.
Furthermore, recent studies have reported that melatonin
can inhibit tumor invasion through increased adhesion by
elevating E-cadherin and b1-integrin expression [22] or
modulating microlament [23, 24], and decrease matrix
metalloproteinases (MMPs) production [25]. Anti-invasion
eect of melatonin has been shown in human mammary
epithelial cancer MCF-7 cells [22, 23, 25] and MDCK cells
[24]. In glioma cells, millimolar concentration of melatonin
can reduce growth and inhibit cell progression from G1 to S
phase of the cell cycle [26]. However, the eect of melatonin
on migration and invasion of glioma has remained to be
explored and elucidated.
In this study, we investigated the eect of melatonin on
migration and invasion of T98G and U251 glioma cells. We
report here that pharmacologic concentration (1 mm), but
not physiological concentration (1 nm), of melatonin
displays signicant inhibition eect on migration and
invasion of glioma cells. Additionally, we have shown that
the anti-migration and invasion eects of melatonin are
closely linked to the latter in its inhibition of reactive
oxygen species (ROS)/NF-jB/MMPs pathway. Our experimental data have therefore amplied the benet eects of
melatonin, among many others, including its potential
therapeutic application for treatment of glioma.

(PDTC) (Sigma) was rst dissolved in double-distilled

water and then diluted with DMEM/HG.
The human T98G and U251 glioma cell lines were
obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA) and conserved in our laboratory for
<2 yr.
Cell culture and treatment
Glioma cell lines T98G and U251 cells were cultured in
DMEM/HG (Hyclone) supplemented with 10% FBS
(Gibco, Invitrogen, Carlsbad, CA, USA), penicillin
(100 U/mL), and streptomycin (100 lg/mL). All cells were
maintained at 37C in a humidied incubator with 5%
CO2. All experiments were conducted using 8085%
conuent cells. Before each experiment, the plated cells
were incubated with serum-free DMEM/HG medium for
6 hr. After this, the medium was replaced with serum-free
DMEM/HG containing either physiological concentration
(1 nm), pharmacologic concentration (1 mm) of melatonin
(Sigma-Aldrich) or PDTC (10 lm, Sigma) for indicated
times. Trypan blue stain was used to determine percentages
of viable cells after each treatment.
Cell viability assay
Cell viability was tested by the MTT assay as described
previously [27, 28]. Briey, 1 105 cells with 200 lL of
culture medium per well were plated into 96-well culture
plates. When reaching 80% conuency, the cells were
incubated, respectively, in a serum-free medium containing
1 nm melatonin, 1 mm melatonin, or 10 lm PDTC for
24 hr. Then, 20 lL of MTT solution (5 mg/mL; SigmaAldrich) was added to each well and incubated at 37C for
4 hr. The culture medium was aspirated and followed by
addition of 200 lL of dimethyl sulfoxide. Cells incubated
with serum-free medium were used as control. The absorbance value was measured in a microplate reader (Bio-Rad
Labs, Hercules, CA, USA) at 490 nm. Values were expressed
as a percentage relative to those obtained in controls. At least
three independent experiments were conducted.

Materials and methods

Materials
Fetal bovine serum (FBS) was purchased from Gibco
(Invitrogen, Carlsbad, CA, USA). Cell culture media and
supplements were from HyClone Laboratories (Logan, UT,
USA). Protease and phosphatase inhibitors (trypsin, phenylmethylsulfonyl uoride), trypsinEDTA solution, 3-(4, 5,
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
(MTT) were purchased from Sigma (Sigma-Aldrich,
St Louis, MO, USA). Anti-p65 antibody and anti-p-p65
antibody were purchased from Cell Signaling Technology
(CST, Danver, MA, USA). Anti-b-actin antibody was
purchased from Abcam (Cambridge, MA, USA). Transwell
system was purchased from Costar (Corning, NY, USA).
Melatonin (Sigma) was rst dissolved in absolute ethanol
and then diluted with Dulbeccos modied essential
medium (DMEM)/high glucose (HG): the nal ethanol
concentration was <0.5%. Pyrrolidine dithiocarbamate

Migration assay
Wound-healing assay (WHA) was performed as previously
described [29]. Briey, a 10-lL pipette was used to create a
scratch in the monolayer cells, which were then incubated
with physiological concentration (1 nm) of melatonin,
pharmacologic concentration (1 mm) of melatonin (SigmaAldrich), or PDTC (10 lm, Sigma), a specic inhibitor of
NF-jB. Photographs were taken at 0 and 24 hr at the same
site, and the migration distance was measured.
Invasion assay
Invasion assay was measured in a Transwell culture
chamber system following the protocol described previously [29]. Briey, 1 104 cells were seeded into the upper
chamber and incubated with physiological concentration
(1 nm of melatonin), pharmacologic concentration (1 mm)

181

Wang et al.
of melatonin, or PDTC. Six hundred microliters of FBScontaining medium was added into the lower chamber. The
adherent cells were gently detached from the upper surface
of the membrane. Cells on the bottom side of the
membrane were stained with 0.1% crystal violet in methanol for 10 min. The cells were counted under a light
microscope (IX71; Olympus, Tokyo, Japan) and photographed.

ma-Aldrich) followed by enhanced chemiluminescence

development (Millipore, Billerica, MA, USA). Normalization of the results was carried out by running parallel
Western blots using b-actin as control. The optical density
was quantied using AlphaEase FC Version 4 analysis
software (Alphalmager HP, Alpha Innotech, and San
Leandro, CA, USA).
Data analysis

ROS analysis
Intracellular ROS levels were measured by dihydroethidium
(DHE) assays. DHE is a membrane-permeable dye that is
oxidized by intracellular ROS to the uorescent product
ethidium. The uorescence excitation and emission wavelengths were 510/595 nm for DHE. The ROS analysis was
performed following the protocol described previously [28].
Briey, glioma cells were treated with 1 nm, or 1 mm
melatonin for 24 hr, and then incubated with 2 lm DHE
(Molecular Probes, Eugene, OR, USA) for 20 min at 37C.
The uorescence was measured with a uorescence plate
reader (Fluroskan Ascent II; Labsystems, Helsinki, Finland). Results in arbitrary units were expressed as a ratio to
the uorescence signal of untreated cells (control) set at 1.0.
RNA extraction and real-time reverse transcription
polymerase chain reactions (real-time RT-PCR)
Total RNA was extracted using TRIzol reagent (Takara,
Otsu, Shiga, Japan). RNAs (1 ng) were reverse transcribed
into complement DNA (cDNA) by a commercial RT-PCR
kit (Fermentas, Vilnius, Lithuania) according to the manufacturers instructions. For real-time PCR, the LightCycler 2.0 instrument (Roche Applied Science, Mannheim,
Germany) and the LightCycler FastStart DNA Master
SYBR Green I Reagent Kit (Roche Molecular Biochemicals) were used according to the manufacturers protocol.
PCR conditions for amplication were used as described
previously [30], and the primers were as follows: b-actin:
sense: 5-AGATGTGGATCAGCAAGCAG-3 and antisense:
5-GCGCAAGTTAGGTTTTGTCA-3; MMP 2: sense: 5GGCCTCTCCTGACATTGACCTT-3 and antisense: 5GGCCTCGTATACCGCATCAATC-3; and MMP 9:
sense: 5-TTGACAGCGACAAGAAGTGG-3 and antisense: 5-CCCTCAGTGAAGCGGTACAT-3. Melting curve
analysis was used to conrm amplication specicity. The
quantication data were analyzed with LightCycler analysis
software version 4.0 (Roche Applied Science). The relative
expression was normalized on the basis of b-actin. At least
three independent experiments for each condition were
conducted.

Quantitative data were presented as the mean S.D. of at

least three independent experiments. Statistical analysis of
data was carried out by Students t-test or by one-way
ANOVA using Dunnetts test in multiple comparisons of
means. Dierences were considered statistically signicant
if the P-value was <0.05.

Results
Melatonin at both physiological concentration (1 nm) and
pharmacologic concentration (1 mm) was tested to evaluate
its inuence on cell viability using MTT assay. The results
showed that neither physiological concentration nor pharmacologic concentration of melatonin aected the mitochondrial respiration in glioma cell line T98G and U251
cells after 24-hr treatment (Fig. 1A,B). Trypan blue staining (data not shown) revealed that at concentrations 1 nm
and 1 mm, melatonin did not induce glioma cell death
under the present experimental conditions.

Western blot assay

Equal amounts of proteins were loaded on 10% SDSpolyacrylamide gel. After electrophoresis, the proteins were
transferred to polyvinylidene uoride membranes, and the
blots were subsequently probed with the following antibodies: polyclonal p-p65 (1:1000; CST) and polyclonal p65
(1:1000; CST). For detection, horseradish peroxidaseconjugated secondary antibodies were used (1:5000; Sig182

Fig. 1. Melatonin (MEL) at both physiological concentration

(1 nm) and pharmacologic concentration (1 mm) did not aect cell
viability of T98G and U251 glioma cells. T98G (A) and U251 (B)
glioma cells were incubated with 1 nm or 1 mm melatonin for
24 hr, and then cell viability was tested by MTT assay. Cells
incubated with medium free of melatonin were used as control.
Results are presented as mean S.D. of three independent
experiments. *P < 0.05, in comparison with control.

Melatonin inhibits migration and invasion of glioma cells

To study the eect of melatonin on migration of glioma
cells, WHA was employed. The results showed that a
signicantly smaller number of fewer glioma cells treated
with pharmacologic concentration of melatonin migrated
into the denuded area in comparison with the untreated
control cells; whereas, almost the same number of glioma
cells treated with physiological concentration of melatonin
migrated to the denuded area compared with the untreated
control cells (Fig. 2A). Pharmacologic concentration of
melatonin signicantly decreased the wound-healing ability
of both T98G and U251 glioma cells compared with the
untreated control T98G and U251 glioma cells (P < 0.05),
respectively; however, physiological concentration of melatonin did not exist such an eect (P > 0.05) (Fig. 2B).
To investigate the eect of melatonin on the invasion
ability of glioma cells, matrigel-coated transwell system was
employed. Glioma cells were seeded on the upper chamber
and treated with physiological or pharmacologic concentration of melatonin. Twenty-four hours later, invasion
cells on the bottom side of the membrane were enumerated.
The results showed that pharmacologic concentration of
melatonin signicantly inhibited the invasion ability of
glioma cells as demonstrated by a decrease in cells on the
bottom side of the membrane compared with the untreated
control groups (P < 0.05), while physiological concentration of melatonin did not reduce the invasion ability of
glioma cells (P > 0.05) (Fig. 3A,B).

Fig. 2. Melatonin at pharmacologic concentration (1 mm) inhibited migration ability of T98G and U251 glioma cells. (A) Eect of
melatonin on cell migration of glioma cells was tested by woundhealing assay. Cells were starved for 6 hr, and then scratches were
created using a 10-lL pipette. After that, cells were incubated with
1 nm or 1 mm of melatonin for 24 hr. Photographs were taken
under a microscope, and scale bar represents 50 lm. (B) Quantication of cell migration distances. The migration distances were
obtained by widths of the scratches at 0 hr subtracts that at 24 hr.
Cells incubated with medium free of melatonin were used as control. Results are presented as mean S.D. of three independent
experiments. *P < 0.05, in comparison with control.

It is well documented that MMPs are involved in the

invasion and metastasis of glioma cells [31]. To determine
the relevant mechanisms involved in the melatonin inhibition of glioma invasion, expression of MMP 2 and MMP 9
was evaluated by real-time RT-PCR. Expression of both
MMP 2 and MMP 9 was signicantly inhibited by
pharmacologic concentration of melatonin. MMP 2
mRNA levels were decreased by about 37% and 31% in
T98G and U251, respectively; MMP 9 mRNA levels were
decreased by about 40% and 50% in T98G and U251,
respectively, following treatment with pharmacologic concentration of melatonin (P < 0.05 or P < 0.01)
(Fig. 4A,B).
Melatonin has been shown to be an eective free radical
scavenger. ROS has been linked to tumor progression as a
constitutive elevation of ROS has been detected in cancer
cells [32]. Furthermore, oxidative stress pathway has been
found to be closely associated with cell invasion [33, 34].
Thus, to evaluate the possible involvement of antioxidant
eect of melatonin in the anti-invasion eect, intracellular
free radical levels were measured. As shown in Fig. 5,
physiological concentration of melatonin did not decrease
ethidium uorescence, whereas pharmacologic concentra-

Fig. 3. Melatonin at pharmacologic concentration (1 mm)

decreased invasion ability of T98G and U251 glioma cells. (A)
Eect of melatonin on cell invasion of glioma cells was tested by
matrigel-coated transwell system. Cells were starved for 6 hr, then
suspended in serum-free medium containing dierent concentrations (1 nm, 1 mm) of melatonin, seeded onto the upper chamber,
and incubated for 24 hr. Invasion cells on the bottom side of
the membrane were xed and stained with 0.1% crystal violet.
Photographs were taken under a microscope; scale bar represents
20 lm. (B) Quantication of invasion cells. The invasion cells were
counted in 20 dierent elds. Cells incubated with medium free of
melatonin were used as control. Results are presented as
mean S.D. of three independent experiments. *P < 0.05, in
comparison with control.

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Wang et al.

Fig. 4. Melatonin at pharmacologic concentration (1 mm) reduced

expression of matrix metalloproteinase (MMP) 2 and MMP 9 in
T98G and U251 glioma cells. T98G (A) and U251 (B) glioma cells
were treated with dierent concentrations (1 nm, 1 mm) of melatonin, and then total RNA was extracted, and the MMP 2 and
MMP 9 were amplied by real-time RT-PCR. The relative
expression was normalized on the basis of b-actin. Cells incubated
with medium free of melatonin were used as control. Results are
presented as mean S.D. of three independent experiments.
*P < 0.05, in comparison with control.

tion of melatonin decreased the intracellular basal free

radical levels by 40% and 48% in T98G and U251 (both
P < 0.05), respectively (Fig. 5A,B).
To further determine the involvement of oxidative stress
pathway in the anti-invasion eect of melatonin, activation
of NF-jB, an ROS downstream transcription factor, was
measured. Both T98G and U251 glioma cells showed basal
activation of NF-jB as demonstrated by a detectable level
of p-p65-NF-jB. The physiological concentration of melatonin did not inhibit the activation of NF-jB; however,
the pharmacologic concentration of melatonin decreased
the basal levels of p-p65-NF-jB by about 60% and 35% in
T98G and U251 glioma cells (both P < 0.05), respectively
(Fig. 6A,B). Pyrrolidine dithiocarbamate, a specic inhibitor of NF-jB [35], was employed as a positive control.
Remarkably, PDTC at 10 lm decreased the p-p65-NF-jB
expression level comparable with melatonin in glioma cells
(Fig. 6A,B).
We next determined whether inhibition of migration of
glioma cells by melatonin is mediated by the inhibition of
the oxidative stress pathway. Remarkably, 10 lm of PDTC
signicantly inhibited the migration and invasion as well as
expression of MMP 2 and MMP 9 in glioma cells in parallel
to the pharmacologic concentration of melatonin (Fig. 7BE).
184

Fig. 5. Melatonin at pharmacologic concentration (1 mm) suppressed basal generation of reactive oxygen species in T98G and
U251 glioma cells. Glioma cells were treated with dierent concentrations (1 nm, 1 mm) of melatonin for 24 hr and then incubated with 2 lm dihydroethidium for 20 min at 37C. The
immunouorescence images were taken by a uorescence microscope; scale bar represents 10 lm (A). (B) The uorescence was
measured with a uorescence plate reader. Results in arbitrary
units were expressed as a ratio to the uorescence signal of
untreated cells (control) set at 1.0. Results are presented as
mean S.D. of three independent experiments. *P < 0.05, in
comparison with control.

Additionally, 10 lm of PDTC did not induce cell viability

changes in glioma cells (P > 0.05) (Fig. 7A).

Discussion
In this study, we investigated the eect of melatonin as well
as the underlying mechanisms on cell migration and
invasion of glioma cells. We have shown that melatonin
eectively inhibits migration and invasion of glioma cells in
vitro. More importantly, melatonin did not aect the cell
viability in the present experimental conditions. Melatonin
treatment reduces expression of MMP 2 and MMP 9,
which have been reported to be closely related to cell
migration and invasion [31]. Moreover, melatonin inhibits
the persistent and elevated production of ROS and
constitutive activation of downstream transcription factor
NF-jB in glioma cells. We have also extended that PDTC,
an NF-jB-specic inhibitor, exerts an inhibitory eect on
glioma cells similar to that of melatonin. Together, our data
suggest that melatonin, a free radical scavenger [11, 1520],
at pharmacologic concentration can suppress migration
and invasion of glioma cells and that ROS/NF-jB/MMPs
pathway is involved in the anti-invasion eect of melatonin.
It has been reported that melatonin at concentration of
1 mm decreases cell proliferation in C6 glioma cells [26].

Melatonin inhibits migration and invasion of glioma cells

Fig. 6. Melatonin at pharmacologic concentration (1 mm) inhibited activation of

p65-NF-jB in T98G and U251 glioma
cells. Glioma cells were treated with different concentrations (1 nm, 1 mm) of
melatonin or pyrrolidine dithiocarbamate
(10 lm) for 24 hr, and then protein was
extracted and measured. Expression levels
of p-p65-NF-jB and p65-NF-jB were
evaluated by Western blot assay. (A) and
(C) show Western blot results. (B) and (D)
show quantication of p-p65-NF-jB and
p65-NF-jB levels by optical density. Cells
incubated with medium free of melatonin
were used as control. Results are presented as mean S.D. of three independent experiments. *P < 0.05, #P < 0.01,
in comparison with control.

Fig. 7. Pyrrolidine dithiocarbamate (PDTC)

at 10 lm displayed anti-migration and
invasion eects and matrix metalloproteinases (MMPs) inhibition similar to that
of 1 mm melatonin. (A) MTT results
showed that 10 lm of PDTC did not aect
cell viability of T98G and U251 glioma
cells. (B) PDTC at 10 lm decreased
migration ability of T98G and U251 glioma cells. (C) PDTC at 10 lm suppressed
invasion ability of T98G and U251 glioma
cells. (D, E) PDTC at 10 lm inhibited
MMP 2 and MMP 9 expression in T98G
and U251 glioma cells. Cells incubated
with medium free of melatonin were used
as control. Results are presented as
mean S.D. of three independent
experiments. *P < 0.05, #P < 0.01, in
comparison with control.

However, in this study, we have shown that neither

physiological concentration (1 nm) nor pharmacologic
concentration (1 mm) aects the cell viability of T98G
and U251 glioma cells. The discrepancy between our results
and that of Martin et al. may be attributed to dierences in
sensitivity of dierent glioma cells to melatonin or dierent
methodology used. In agreement with previous reports [26,
36], melatonin at both physiological and pharmacologic
concentration did not induce cell death in glioma cells.

In this study, we have used WHA and matrigel-coated

transwell assay to assess the migration and invasion ability,
respectively, in human glioma cells. Our data showed that
the migration and invasion potential of T98G and U251
glioma cells was inhibited by melatonin at 1 mm. Together
with previous ndings in MCF-7 [22, 23, 25], it is suggested
that melatonin displays anti-migration and anti-invasion
eects on tumor cells. In addition, we demonstrated that
MMP 2 and MMP 9 expression was signicantly
185

Wang et al.
suppressed by melatonin. MMP 2 and MMP 9 are members
of MMPs family, which can catalyze extracellular proteolysis and play an essential role in cell migration and tissue
remodeling [37]. The invasion property of glioma is
predominantly associated with MMPs, especially MMP 2
and MMP 9 [31]. Our results suggest that inhibition of
glioma invasion by melatonin is linked to its modulation of
MMP 2 and MMP 9.
Oxidative stress has been linked to cancer as elevated
levels of free radical were detected in many cancers [32, 38].
Intracellular ROS produced by exogenous stimuli as well as
exogenous administration of ROS has been shown to
enhance the proliferation of numerous cancer types,
including hepatoma, breast cancer, ovarian cancer, and
glioma (see review [21] and [39]). ROS is essential in both
extrinsic and intrinsic pathway of apoptosis [40]; however,
ROS is also required for cancer cell survival [41, 42].
Excessive levels of ROS may induce cell death, while a
modest level is required for cancer cell survival. Recently, it
has been reported that ROS is involved in metastasis and
angiogenesis in cancers [4345]. The present results have
shown that melatonin not only suppresses migration and
invasion of glioma cells but also inhibits production of
ROS, along with its downstream transcription factors and
genes indicating that melatonin can regulate these processes
or pathways involved in glioma metastasis.
NF-jB is one of the transcription factors that can be
activated by ROS [46]. Activated NF-jB induces expression
of numerous genes that are associated with tumor growth
and progression [47]. Also, its constitutive activation in
cancer cells is correlated with migration and invasion, and
inhibition of its activation leads to decrease in migration and
invasion ability in MCF-7 cells [33, 34]. In the present study,
we have shown that pharmacologic concentration of melatonin reduces intracellular ROS generation and inhibits
phosphorylation of p65-NF-jB. NF-jB-specic inhibitor
PDTC exerted anti-migration and invasion eects similar to
that of melatonin. Furthermore, consistent with previous
ndings, inhibition of NF-jB decreased expression of MMP
2 and MMP 9. Taken together, our data demonstrated that
the antioxidant eect of melatonin is involved in its antimigration and invasion eects in glioma cells.
Arising from the present results, it is concluded that the
anti-migration and invasion eect of melatonin is associated with its inhibition of ROS/NF-jB/MMPs pathway,
and this will provide evidence for the correlation of cellular
redox state with metastasis behavior in glioma cells.
Furthermore, our ndings support the potential application
of melatonin in the treatment of glioma.

Acknowledgements
This work was supported by funding from the National
Natural Science Foundation of China (No. 30872645,
81071057) and the Natural Science Foundation of Shandong Province (No. Y2008C57, JQ200823).

Authors contribution
AJH and GL were involved in study design, data interpretation and manuscript editing; JTW performed the majority
186

of the laboratory work and contributed to the analysis of

data and writing of the manuscript; HBH and SDZ
contributed to the analysis of data; LLY and XDZ were
responsible for the cell culture and part of laboratory work;
EAL was involved in manuscript editing. All authors
declared no conict of interest.

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