Magnolol, a natural compound, induces apoptosis of SGC-7901 human gastric adenocarcinoma cells via the mitochondrial and PI3K/Akt signaling pathways.

Abstract

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies.

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INTERNATIONAL JOURNAL OF ONCOLOGY 40: 1153-1161, 2012
Abstract. Gastric cancer is the fourth most commonly diag-
nosed cancer with the second highest mortality rate worldwide.
Surgery, chemotherapy and radiation therapy are generally used
for the treatment of stomach cancer but only limited clinical
response is shown by these therapies and still no effectual
therapy for advanced gastric adenocarcinoma patients is avail-
able. Therefore, there is a need to identify other therapeutic
agents against this life-threatening disease. Plants are considered
as one of the most important sources for the development of
anticancer drugs. Magnolol, a natural compound possesses
anticancer properties. However, effects of Magnolol on human
gastric cancer remain unexplored. The effects of Magnolol on
the viability of SGC-7901 cells were determined by the MTT
assay. Apoptosis, mitochondrial membrane potential and cell
cycle were evaluated by flow cytometry. Protein expression of
Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western
blotting. Magnolol induced morphological changes in SGC-7901
cells and its cytotoxic effects were linked with DNA damage,
apoptosis and S-phase arrest in a dose-dependent manner.
Magnolol triggered the mitochondrial-mediated apoptosis
pathway as shown by an increased ratio of Bax/Bcl-2, dissipation
of mitochondrial membrane potential (ΔΨm), and sequential
activation of caspase-3 and inhibition of PI3K/Akt. Additionally,
Magnolol induced autophagy in SGC-7901 cells at high concen-
tration but was not involved in cell death. Magnolol-induced
apoptosis of SGC-7901 cells involves mitochondria and PI3K/
Akt-dependent pathways. These findings provide evidence that
Magnolol is a promising natural compound for the treatment of
gastric cancer and may represent a candidate for in vivo studies
of monotherapies or combination antitumor therapies.
Introduction
Gastric cancer is the second most common cause of cancer-
related death worldwide and approximately 800,000 people
die each year of this malignancy. So far it is the fourth most
frequently diagnosed cancer as each year more than one million
patients are annually diagnosed with gastric cancer (1,2). The
incidence of stomach cancer varies geographically, with a much
higher prevalence in Eastern countries than in the Western
ones (3). In 2005, the incidence of gastric cancer (0.3 million
deaths and 0.4 million new cases) ranked third among the
most common cancers in China (4). Although surgery remains
the gold standard for the treatment of stomach cancer but the
limitations is that it is diagnosed at an advanced stage. The
5-year survival rate of patients with advanced gastric cancer for
surgical treatment is less than 40%. The effectiveness of chemo-
therapy and/or radiation therapy, in addition to surgery, has been
actively studied over the last few decades. Unfortunately, only a
little clinical response is generally shown by chemotherapy or
radiation therapy and survival rate is also very poor (5). There
is no effective therapy for patients with advanced gastric adeno-
carcinoma. Therefore, to identify new therapeutic agents against
gastric cancer is the critical requirement to improve health and
survival chances of the patients.
The purpose of this study was to find new anticancer agents to
cure gastric cancer and for this purpose we performed screening
to find novel compounds for treatment of gastric cancer. During
the screening program, to evaluate the potential chemopreven-
tive effect of natural compounds, we screened 400 single
compounds against human gastric adenocarcinoma SGC-7901
cells. Magnolol was one of them that showed antiproliferative
effects against gastric adenocarcinoma SGC-7901 cells. Several
studies have documented that many compounds, originally
isolated from plants such as the paclitaxel, camptothecin, vinca
alkaloids, and etoposide are used in cancer chemotherapy. Plants
are considered as one of the most important sources for the
development of innovative anti-cancer drugs (6-8).
Magnolol, a natural compound, induces apoptosis of
SGC-7901 human gastric adenocarcinoma cells via the
mitochondrial and PI3K/Akt signaling pathways
AZHAR RASUL1, BO YU2, MUHAMMAD KHAN1, KUN ZHANG1,
FURHAN IQBAL3, TONGHUI MA1,2 and HONG YANG2
1Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun 130041;
2School of Life Sciences, Liaoning Normal University, Dalian 116029, P.R. China;
3Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan
Received September 23, 2011; Accepted October 27, 2011
DOI: 10.3892/ijo.2011.1277
Correspondence to: Dr Tonghui Ma, Central Research Laboratory,
Jilin University Bethune Second Hospital, Changchun 130041,
P.R. China
E-mail: math108@gmail.com
Key words: Magnolol, natural compounds, apoptosis, S-phase
arrest, SGC-7901 cells

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Magnolol, a natural compound has been reported to have
wide spectrum of biological effects including antioxidant
(9-11), antithrombotic (12), antimicrobial (13), anti-allergic (14),
antifungal (15), anti-inflammatory (16), and xanthine oxidase
inhibition (17). Moreover, Magnolol induced antiproliferative
effects in wide variety of tumor cells including melanoma cells
(18-21), colon cancer cells (22-25), prostate cancer cells (26,27),
human glioblastoma cancer cells (28,29), liver cancer cells
(24,25), lung cancer cells (30,31), leukemic cells (14), cervical
cancer (32), ovarian cancer cells (32), thyroid carcinoma cells
(33), human fibrosarcoma HT-1080 (34), and human urinary
bladder cancer 5637 cells (35,36). Several researchers reported
that Magnolol-induced cell death involve apoptosis while
Li et al (30), reported that Magnolol-induced death occurs
via autophagy but not apoptosis. Accumulated data indicate
that mechanism of Magnolol-induced cell death varies with
cell type. However, effects of Magnolol and its mechanism
on human gastric adenocarcinoma cells remain unexplored.
Therefore, the present study was conducted to explore the effects
of Magnolol on the proliferation of human gastric adenocarci-
noma SGC-7901 cells and its mechanism. Moreover, to examine
whether Magnolol-induced cell death occurs via apoptosis,
autophagy, or both. Results indicated that Magnolol effectively
inhibited the proliferation of SGC-7901 cells through arresting
the cell cycle at S-phase and induction of apoptosis which is
regulated by activation of caspase-3, down-regulation of Bcl-2,
and up-regulation of Bax.
Materials and methods
Chemicals and reagents. Magnolol was purchased from
the National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). Fetal bovine serum was
purchased from Hangzhou Sijiqing Biological Engineering
Materials Co., Ltd. DMEM, MTT [3'-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide], propidium iodide
(PI), and dimethyl sulfoxide (DMSO) were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Annexin V-FITC
Apoptosis Detection Kit was purchased from Beyotime Institute
of Biotechnology (Shanghai, China). Rabbit polyclonal anti-
human Bcl-2, anti-human Bax and cleaved caspase-3 antibodies
were purchased from Wuhan Boster Biological Technology Co.,
Ltd., Phospho-Akt (Ser-473), PI3K, and Akt antibodies were
purchased from Cell Signalling Technology (Beverly, MA, USA).
Mouse anti-β-actin and anti-rabbit antibodies were purchased
from Santa Cruz Biotechnology. Ponceou and cell lysis buffer
for Western blotting and IP were purchased from Bio SS Beijing.
Rhodamine 123 was purchased from Eugene Co. (OR, USA).
Cell culture. Human gastric adenocarcinoma SGC-7901 cells
were cultured and maintained in DMEM supplemented with
10% fetal bovine serum (FBS), 100 µm/l penicillin, and 100 µg/
ml streptomycin at 37˚C in a humidified atmosphere with 5%
carbon dioxide and 95% air. Cells were cultured in a 10-cm
culture dish and were allowed to grow to ~60-80% confluence
before experimentation.
Cell proliferation assay. The effect of Magnolol on the viability
of cells was examined by the MTT assay. SGC-7901 cells were
sub-cultured in 96-well plates and were allowed to adhere over-
night. Next day, cells were treated with various concentrations of
Magnolol (0, 10, 30, 50, 100, 200 and 300 µM) for 48 h. After
incubation, 10 µl of MTT (5 mg/ml in phosphate buffered saline)
was added to each well and incubated further for 4 h. Medium
was aspirated carefully and 150 µl of DMSO was added to each
well. The absorbance was measured on the Microplate Reader
(ELX 800, Bio-Tek Instruments, Inc.) at the wavelength of
570 nm. The effects of Magnolol were determined on viability of
cells and inhibition ratio (I%) was calculated using the following
equation (37):
I% = [A570 (control) - A570 (treated)] / A570 (control) x 100
Determination of apoptosis by flow cytometry. Apoptosis was
determined through Annexin V-FITC Apoptosis Detection Kit.
SGC-7901 cells were seeded in 6-well plates and were incubated
overnight and then treated with 40, 60, and 80 µM of Magnolol,
respectively, for 48 h. Cells were harvested by trypsinization,
washed with pre-chilled PBS (4˚C) and centrifuged at 1000
rpm for 5 min. The cell pellet was resuspended in 195 µl of
binding buffer and incubated with 5 µl Annexin V-FITC in the
dark at room temperature for 10 min. Cells were centrifuged,
washed with PBS, and re-suspended in 195 µl of binding buffer
containing 10 µl PI solution in the dark and were then analyzed
by flow cytometry (Beckman FC400 MPL, USA).
Cell cycle analysis. The distribution of cells in different phases
of cell cycle after exposure of Magnolol was analyzed with flow
cytometry. Briefly, SGC-7901 cells were harvested and washed
with PBS after exposure of 40, 60 and 80 µM of Magnolol with
control group for 48 h. The cells were fixed with 70% cold ethanol
at -20˚C overnight and then stained with PI solution consisting of
1 mg/ml PI and RNase A. The fluorescence-activated cells were
sorted in the flow cytometry, and the data were analyzed using
CellQuest analysis software.
Flow cytometric analysis of mitochondrial membrane poten-
tial. Mitochondrial transmembrane potential was assessed by
Rho-123 staining as we described previously (37). Briefly, cells
were incubated without (control) and with (40, 60 and 80 µM)
Magnolol for 48 h. After the incubation, cells were collected, cell
pellets were washed twice with ice-cold PBS and then incubated
with Rho-123 (1 µM) at 37˚C for 20 min. Stained cells were
washed twice with PBS, resuspended in 0.5 ml of PBS followed
by flow cytometric analysis. The fluorescence of treated cells
was compared with control group.
Immunoblotting. To elucidate the mechanism of the apoptotic
effect of Magnolol, we analyzed the apoptosis-related proteins
in SGC-7901 cells. After incubation of cells without (control)
and with (40, 60, and 80 µM) Magnolol for 48 h, cells were
harvested, washed twice with PBS, and cell lysates were
prepared using lysis buffer. Protein estimation was done using
NanoDrop 1000 spectrophotometer (Thermo Scientific, USA).
An equal amount of protein lysates of cells were subjected to
SDS-PAGE followed by Western blotting. The membranes
were soaked in blocking buffer (5% skimmed milk) for 2 h in
TBST at room temperature. To probe for Bcl-2, Bax, cleaved
caspase-3, phospho-Akt, pPI3K, Akt, and β-actin, membranes
were incubated overnight at 4˚C with relevant antibodies,

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followed by appropriate HRP conjugated secondary antibodies
and ECL detection.
ATP measurement. To measure the intracellular ATP level,
SGC-7901 cells were incubated without (control) and with (40,
60 and 80 µM) Magnolol for 48 h. ATP level was measured
using the ATP bioluminescence assay kit HSII (Roche
Diagnostic, Indiannapolis, IN, USA) according to the manufac-
turer's instructions.
Acridine orange staining. Staining of cells with acridine orange
was performed according to published procedure (38). In brief,
cells were incubated without (control) and with Magnolol (40,
60, and 80 µM) and with rapamycin (positive control group)
for 48 h and then acridine orange at a final concentration of
1 mg/ml was added to cells for a period of 20 min in the dark
at 37˚C. Then, cells were washed twice with PBS. Images of
cells were obtained under fluorescence microscopy.
Flow cytometric quantification of acidic vesicular organelles
(AVOs). AVOs formation (autophagosomes and autolysosomes)
is a characteristic feature of autophagy (39). For quantification
of AVOs, we used flow cytometry after cells were stained by
AO (40). AO is a weak base that accumulates in acidic spaces
and gives bright red fluorescence [punctate staining (dots)]
in the cytoplasm is detected by fluorescent microscopy. The
intensity of the red fluorescence is proportional to the degree of
acidity. Thus, the formation of AVOs can be quantified. Briefly
SGC-7901 cells were harvested after treatment of 40, 60, and
80 µM of Magnolol for 48 h. Cell pellet was collected in an
Eppendorf tube and cells were resuspended in 1 ml PBS. The
staining of cells was done with AO (1 mg/ml) for 15-20 min in
the dark at 37˚C. Cells were centrifuged at 1000 rpm for 5 min;
cells pellet was rinsed twice with PBS, and then resuspended in
400 µl PBS and analyzed on a flow cytometry using CellQuest
software.
PI staining assay. Cell death was measured by PI staining as
previously described (41). Briefly SGC-7901 cells were trypsin-
ized after the treating cells with 40, 60, and 80 µM of Magnolol
for 48 h in the presence or absence of 3-MA, collected and
resuspended with 1 ml PBS. Cells were stained with 0.5 ml of
staining solution (40 mg ml-1 PI, 100 mg ml-1 RNaseA, 0.2%
Triton-100) and cells were incubated in 37˚C for 30 min in the
dark. Cell death was measured by flow cytometry.
Statistical analysis. For the statistical analysis of data,
comparison between results from different groups were anal-
ysed with SPSS for Window Version 15.0. The Student's t-test
was employed to determine the statistical significance of the
difference between different experimental groups and control
group at P<0.05 value being regarded as statistically significant.
All experiments were repeated at least three times. Data are
presented as means ± standard deviation (SD).
Results and Discussion
We started the investigation with screening of natural
compounds against human gastric adenocarcinoma SGC-7901
cells to evaluate the potential chemopreventive effects of natural
compounds. Magnolol was one of them which showed antip-
roliferative effects against gastric adenocarcinoma SGC-7901
cells. The structure of Magnolol is shown in Fig. 1A. SGC-7901
cells were treated with different concentrations of Magnolol (0,
Figure 1. Effects of Magnolol on morphological characteristics and viability of SGC-7901 cells. (A) Chemical structure of Magnolol. (B) SGC-7901 cells were
treated with various concentrations of Magnolol for 48 h. Cell death was measured by using MTT assay. Data shown are means ± SD (n=3). (C) Morphological
changes of SGC-7901 and FRT cells were observed under the phase-contrast microscopy after treating without (control) and with 40 and 80 µM of Magnolol for 48 h.

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10, 30, 50, 100, 200 and 300 µM) for 48 h. Cell viability was
measured by MTT assay and it was observed that Magnolol
increased cell growth inhibition in a dose-dependent manner
(Fig. 1B). Furthermore, the cytotoxicity of Magnolol was
assessed by observing morphological changes in SGC-7901
cells under phase-contrasted microscopy and MTT assay. Phase
contrast microscopic analysis of cell morphology was done
following the exposure to Magnolol. Exposure of cells to 40,
60, and 80 µM of Magnolol for 48 h resulted in the significant
decrease in cells number as compared to that of the control
group. In addition, Magnolol exposure induced changes in
shape and size of the cells and they turned round and shrunk
while cells in the control group remained polygonal. The
parallel treatment with Magnolol in normal FRT cells showed
lesser effect (Fig. 1C). These results suggest that Magnolol can
act as growth inhibitor of gastric adenocarcinoma SGC-7901
cells in a similar fashion as described in previous studies
dealing with various other types of cancer cells including
melanoma cells (21), colon cancer cells (24,25), prostrate
cancer cells (26,27), human glioblastoma cancer cells (28,29),
liver cancer cells (32), lung cancer cells (30,31), leukemic cells
(14), thyroid carcinoma cells (33), and human urinary bladder
cancer 5637 cells (35).
Cell cycle arrest and apoptosis are among several effective
mechanisms involved in the induction of cell death (42). It is
now well established that various checkpoints are involved in
the proper progression of cell cycle in normal cells while in
cancer cells regulation of cell cycle is altered due to abnormal
cell growth. There are various factors such as DNA damage,
exogenous stress signals, and defects during the DNA replica-
tion or failure of chromosomes to attach with the mitotic spindle
which may disturb the normal functioning of these checkpoints
and the loss of this regulation is the hallmark of cancer (43). To
gain further insight into the mechanism of induction of cytotoxic
effects of Magnolol on SGC-7901 cells, we then investigated the
involvement of Magnolol in cell cycle arrest.
Previous studies have documented that Magnolol can
arrest the cell cycle in different types of cancer cells (28,35).
To decipher the effect of Magnolol on cell cycle progression of
SGC-7901 cells, we treated the cells with 40, 60 and 80 µM of
Figure 2. Effect of Magnolol on cell cycle distribution. SGC-7901 cells were treated with: (a) 0 µM, (b) 40 µM, (c) 60 µM, and (d) 80 µM of Magnolol for 48 h
and then they were stained with PI for flow cytometric analysis. Histograms show number of cells/channel (y-axis) vs. DNA content (x-axis). The values indicate
the percentage of cells in the indicated phases of the cell cycle. The data shown are representative of three independent experiments with the similar results.
*p<0.05; and **p<0.01 compared with the control.

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Magnolol for 48 h. The results showed that Magnolol arrested
the cell cycle at S-phase, which supported previous results (30).
The percentage of accumulation of the cells in the S-phase was
increased from 22.27% in control group to 28.84, 37.84 and
45.99% in the cells treated with 40, 60 and 80 µM of Magnolol,
respectively for 48 h (Fig. 2). In several settings, maintenance
of proper cell cycle progression in cancer cells sets the stage for
potential and effective treatment of cancer (44-46). These data
strongly implicate S-phase cell cycle arrest by Magnolol as one
of the mechanisms that induced cytotoxicity in SGC-7901 cells.
Accumulated data indicate that many of the chemotherapeutic
and chemopreventive agents have potential anti-proliferative
effects via arresting the cell division at certain checkpoints in
the cell cycle (47,48).
Apoptosis is one of the modes of cell death and induction of
apoptosis is the key characteristic of anticancer drugs as it plays
an imperative role in the elimination of damaged cells and the
maintenance of homeostasis and many of the natural chemo-
preventive agents, including Magnolol, exert their effect via
induction of apoptosis in cancer cells (21,49,50). To elucidate
the effect of Magnolol on induction of apoptosis in SGC-7901
cells, these cells were treated with Magnolol and incubated with
PI, analysed by flow cytometry and resulted in the increase of
subG1 phase which represents the apoptotic cell population.
The percentages of apoptotic SGC-7901 cells were 11.29, 24.72
and 31.76% after treating the cells with 40, 60 and 80 µM of
Magnolol respectively for 48 h (Fig. 2).
Furthermore, in order to gain confirmation of the apoptosis
and to distinguish between early and late apoptosis, we performed
flow cytometric analysis of apoptosis using annexin V-FITC and
PI double staining. The results showed that apoptotic rates were
11.52, 22.61, and 28.03% after treating the SGC-7901 cells with
40, 60 and 80 µM of Magnolol respectively for 48 h (Fig. 3).
Previous studies revealed that Magnolol inhibited the growth
of the tumor cell proliferation by inducing apoptosis in various
kinds of cancer cells such as melanoma cells (21), colon cancer
cells (24,25), prostrate cancer cells (27), lung cancer cells (31),
thyroid carcinoma cells (33), and human urinary bladder cancer
5637 cells (35). So results of present study were consistent with
previously reported results.
Apoptosis may be triggered either through the stimula-
tion of death receptors located on the plasma membrane
(extrinsic pathway) and/or within cells (intrinsic pathway) (49).
Mitochondria are important organelles which are involved to
release of apoptotic signals during an intrinsic pathway for the
execution of apoptosis (51). Dysfunction of mitochondria leads
to the dissipation of mitochondrial transmembrane potential and
subsequently release of cytochrome c from the mitochondria into
the cytosol. It is one of the mechanisms of caspase activation in
a mainly apoptotic cell death (52). To determine the effects of
Magnolol on mitochondrial transmembrane potential, SGC-7901
cells were incubated with 40, 60 and 80 µM of Magnolol for
24 h. The mitochondrial transmembrane potential was detected
with Rho-123 staining in flow cytometry (Fig. 4A). This assay is
based on the principle that decline in the fluorescence of Rho-123
was directly proportional to the decrease in the mitochondrial
transmembrane potential (53). This is also in line with the results
reported with Magnolol-induced apoptosis in human melanoma
A375-S2 cells (21), human hepatoma (Hep G2) and colon cancer
(COLO 205) cells (24), lung squamous carcinoma CH27 cells
(31), and CGTH W-2 thyroid carcinoma cells (33).
Caspases play a central role in the apoptosis and caspase-3
is a frequently activated death protease, catalyzing the specific
cleavage of many key cellular proteins (54). We examined the
effect of Magnolol treatment on the activation of caspase-3 by
Western blotting. As shown in Fig. 4B, Magnolol treatment led to
a dose-dependent activation of caspase-3, where its cleavage was
evident upon increasing the concentrations of Magnolol treat-
Figure 3. Apoptosis induced by Magnolol in SGC-7901 cells. SGC-7901 cells were treated with (A) 0, (B) 40, (C) 60 and (D) 80 µM of Magnolol for 48 h. Then they
were stained with FITC-conjugated Annexin V and PI for flow cytometric analysis. The flow cytometry profile represents Annexin V-FITC staining in x-axis and PI
in y-axis. The number represents the percentages of apoptotic cells in each condition. As shown, the cell populations in the lower right (Annexin V+/PI-) represents
early apoptotic cells, upper right (Annexin V+/PI+) represents late apoptotic cells.

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ment for 48 h. Similar results were reported in human melanoma
A375-S2 cells (21), human hepatoma (Hep G2), colon cancer
(COLO 205) cells (24), human lung squamous carcinoma CH27
cells (31), and CGTH W-2 thyroid carcinoma cells (33) in which
the caspase-3 is activated as result of treatment with Magnolol.
Together our results and previously reported studies demonstrate
that Magnolol-induced apoptosis involved the activation of
caspase-3.
Our findings showed that Magnolol induced the apoptotic
cell death associated with activation and cleavage of caspase-3
in SGC-7901 cells. Next we demonstrated the effect of
Magnolol on Bcl-2 family proteins, including the anti-apoptotic
(e.g., Bcl-2) and pro-apoptotic proteins (e.g., Bax). The balance
between these two groups could profoundly affect cellular
response to undergo apoptosis or not (55). This interaction
ablates pro-survival function and activates the Bax and Bak,
those render the cells to undergo apoptosis by permeabilizing
the mitochondrial outer membrane (56,57). To reveal the effect
of Magnolol on expression of Bcl-2 and Bax, Western blotting
was performed. It was observed that Magnolol was involved
in the up-regulation of Bax and down-regulation of Bcl-2 in a
dose-dependent manner (Fig. 4C). These results were similar
with previously reported studies in the other types of cancer
cells such as lung squamous carcinoma CH27 cells (31) and
human malignant melanoma A375-S2 cells (21).
Phosphatidylinositol 3-kinase/Akt is an important intra-
cellular pathway that is frequently overexpressed in a wide
variety of epithelial malignancies (58). Overexpression of
Akt is associated with poor prognosis, tumor progression and
resistance to systematic therapy in many types of human cancer
including gastric tumors (59,60). Phosphorylation of Akt was
able to significantly up-regulate the expression of Bcl-2, and
down-regulate the expression of Bax in gastric cancer (58).
Phosphatidylinositol 3-kinase/Akt pathway is a potential target
of most of anticancer agents. Many researchers have focused on
the PI3K/Akt pathway as potential target for therapeutic strategy
against cancer (41,60). Next we evaluated the effects of Magnolol
on the PI3K/Akt pathway by measuring the phospho-PI3K,
total Akt, and sequential level of phospho-Akt proteins. It was
observed that Magnolol markedly down-regulated the expres-
sion of phospho-Akt and PI3K in a dose-dependent manner after
48 h while total Akt protein levels remained constant during all
treatments (Fig. 4D). In agreement to our other results, Magnolol
induced a concentration-dependent down-regulation of phos-
porylated PI3K and Akt as reported previously (27,33).
It has been reported that Magnolol induced autophagy in
human lung cancer cells (30). Therefore, we hypothesized that
Magnolol may induce autophagy in gastric adenocarcinoma
SGC-7901 cells. To test our hypothesis, autophagy was analyzed
by AO staining as described in Materials and methods. The
formation of acidic vesicular organelles (AVOs) is one of the
characteristic features of cells which passed through process
of autophagy after exposure of different autophagy inducer
agents (61,62). Autophagic vacuoles (AV) or autophagosomes are
formed as result of sequestering parts of the cytoplasm or entire
organelles, respectively, during the process of autophagy (63).
We observed the effect of Magnolol treatment on the formation
of AVOs in SGC-7901 cells using fluorescence microscopy upon
Figure 4. The effects of Magnolol on mitochondrial transmembrane potential and expression levels of apoptosis-related proteins. (A) The values indicate the percentage
of Rho-123 fluorescence in the SGC-7901 cells treated without (control) and with (40, 60, and 80 µM) Magnolol for 24 h. The data shown are representative of three
independent experiments with the similar results. *p<0.05 and **p<0.01 compared with the control. (B-D) Expression levels of Caspase-3, Bcl-2, Bax, pPI3K, pAkt, and
Akt in SGC-7901 cells treated without (control) and with Magnolol (40, 60, and 80 µM) for 48 h were monitored by Western blot assay. β-actin was used as loading
control. Western blots are representative of three independent experiments.

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staining with the lysosomotropic agent, acridine orange (AO).
In fact, AO is a weak base that passes freely across the plasma
membrane in a neutral state distinguished by green fluorescence.
After entrance into acidic compartments, AO changed into
protonated form which is distinguished by bright red fluores-
cence while control cells shown green fluorescence (Fig. 5A).
For further verification of autophagy, we quantified AVOs, by
flow cytometry using AO solution as described in Materials and
methods. It was observed that there was no significant formation
of AVOs at low concentration while AVOs were formed at high
concentration of Magnolol treated cells as compared to normal
cells (30) and rapamycin was used as positive control (Fig. 5B).
Currently autophagic cell death has been studied as a potential
method for cancer therapy. To determine the role of Magnolol-
induced autophagy in cell death of SGC-7901 cells, we added the
autophagy inhibitor, 3-methyladenine (3-MA) which controlled
the autophagy pathway at various points (64). Initially effects of
3-methyladenine (3-MA) on cell growth inhibition were assessed.
The viability of cells was more than 90% when they were treated
with 3-methyladenine (3-MA) alone. Next SGC-7901 cells were
treated with 80 µM of Magnolol and autophagy inhibitor 3-meth-
yladenine (3-MA) together, cells were analyzed for cell death by
flow cytometry using PI staining assay. Cells stained with prop-
idium iodide were considered as dead cells (41). It was found that
Magnolol-induced cell death was not suppressed when the cells
were treated in combination with 3-MA (Fig. 6A). These results
Figure 5. Formation of acidic vesicular organelles (AVOs) was observed by fluorescence microscopy and quantified by flow cytometry using the AO staining. (A)
Cells were treated with Magnolol for 48 h before stained with acridine orange. Cells were examined by fluorescence microscopy. Representative images of cells from
three independent experiments are shown. (B) The number represents the percentage of AVOs formation in SGC-7901 cells in each profile after treating cells without
(control) and with Magnolol (80 µM) and rapamycin (positive control group) for 48 h. Three independent experiments were performed.
Figure 6. Effect of Magnolol-induced autophagy on cell death of SGC-7901
cells. (A) Cells were treated with Magnolol (80 µM) in the presence or absence
of 3-MA. The number represents the percentage of dead cells in each profile
after treating cells with 3-MA alone, Magnolol (80 µM) alone, Magnolol
(80 µM) together with 3-MA with negative control group for 48 h. Three
independent experiments were done. (B) Effect of Magnolol on intracellular
ATP. Cells were treated with 40, 60 and 80 µM Magnolol for 48 h before the
measurement of ATP by luminometric assay. Results shown are means ± SEM
from three independent experiments.

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RASUL et al: APOPTOSIS INDUCED BY MAGNOLOL IN SGC-7901 CELLS
1160
showed that Magnolol-induced autophagy is not involved
in the induction of SGC-7901 cell death. It has been docu-
mented that autophagy may act as enabler of apoptosis,
contributing in certain morphological and cellular events
(ATP, cells blebbing and DNA fragmentation) that take
place in apoptotic cell death, without leading to cell death
by itself (64). We assumed that Magnolol-induced apop-
tosis may be involved in the ATP alteration. To verify this
assumption, depletion of cellular ATP was observed after
exposure of various concentrations of Magnolol (Fig. 6B).
These results indicated that Magnolol-induced autophagy
may affect the ATP level in SGC-7901 cells and supported
observations which showed that autophagy may alter the
morphological and cellular events that take place in apop-
totic cell death (64).
In conclusion, Magnolol-induced cell death of SGC-7901
gastric cancer cells via induction of apoptosis as well as S-phase
cell cycle arrest. Analysis of apoptosis-related proteins in
SGC-7901 cells revealed that Magnolol triggered the mito-
chondria-mediated apoptosis pathway as shown by increased
ratio of Bax/Bcl-2, which led to dissipation of mitochon-
drial membrane potential (ΔΨm), sequential activation of
caspase-3, and inhibition of PI3K/Akt (Fig. 7). Magnolol
also induced autophagy at higher concentration but it is not
involved in cell death of SGC-7901 cells. Magnolol-induced
autophagy may alter the morphological and cellular events
that take place in apoptotic cell death. But the exact role of
Magnolol-induced autophagy in apoptosis is still unclear and
needs further research. Taken together, these results suggest
that Magnolol is a promising natural compound for the
treatment of human gastric cancer and represents a potential
candidate for in vivo studies of mono-therapies as well as
combined anti-tumor therapies.
Acknowledgements
This work has been supported by Ministry of Education of
Pakistan and China Scholarship Council of China. The authors
would like to express their deepest thanks to Xiu-Li Wang for
her excellent assistance during the flow cytometry experiments.
The authors also would like to express sincere gratitude to Syed
Manzar Abbas Shah, Vishwa Deepak and Muhammad Rauf for
language and picture editing.
References
1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM:
Estimates of worldwide burden of cancer in 2008: GLOBOCAN
2008. Int J Cancer 127: 2893-2917, 2010.
2. Hernandez BY, Green MD, Cassel KD, Pobutsky AM, Vu V and
Wilkens LR: Preview of Hawaii Cancer Facts and Figures 2010.
Hawaii Med J 69: 223-224, 2010.
3. Parkin DM, Bray F, Ferlay J and Pisani P: Global cancer statis-
tics, 2002. CA Cancer J Clin 55: 74-108, 2005.
4. Yang L, Parkin DM, Ferlay J, Li L and Chen Y: Estimates of
cancer incidence in China for 2000 and projections for 2005.
Cancer Epidemiol Biomarkers Prev 14: 243-250, 2005.
5. Roth AD: Curative treatment of gastric cancer: towards a multi-
disciplinary approach? Crit Rev Oncol Hematol 46: 59-100,
2003.
6. Amin AR, Kucuk O, Khuri FR and Shin DM: Perspectives for
cancer prevention with natural compounds. J Clin Oncol 27:
2712-2725, 2009.
7. Cragg GM and Newman DJ: Plants as a source of anti-cancer
agents. J Ethnopharmacol 100: 72-79, 2005.
8. Gordaliza M: Natural products as leads to anticancer drugs. Clin
Transl Oncol 9: 767-776, 2007.
9. Fujita S and Taira J: Biphenyl compounds are hydroxyl radical
scavengers: their effective inhibition for UV-induced mutation
in Salmonella typhimurium TA102. Free Radic Biol Med 17:
273-277, 1994.
10. Kong CW, Tsai K, Chin JH, Chan WL and Hong CY: Magnolol
attenuates peroxidative damage and improves survival of rats
with sepsis. Shock 13: 24-28, 2000.
11. Lee YM, Hsiao G, Chen HR, Chen YC, Sheu JR and Yen MH:
Magnolol reduces myocardial ischemia/reperfusion injury via
neutrophil inhibition in rats. Eur J Pharmacol 422: 159-167,
2001.
12. Teng CM, Ko FN, Wang JP, et al: Antihaemostatic and antithrom-
botic effect of some antiplatelet agents isolated from Chinese
herbs. J Pharm Pharmacol 43: 667-669, 1991.
13. Ho KY, Tsai CC, Chen CP, Huang JS and Lin CC: Antimicrobial
activity of honokiol and magnolol isolated from Magnolia offici-
nalis. Phytother Res 15: 139-141, 2001.
14. Hamasaki Y, Kobayashi I, Zaitu M, et al: Magnolol inhibits
leukotriene synthesis in rat basophilic leukemia-2H3 cells.
Planta Med 65: 222-226, 1999.
15. Bang KH, Kim YK, Min BS, et al: Antifungal activity of magnolol
and honokiol. Arch Pharm Res 23: 46-49, 2000.
16. Wang JP, Hsu MF, Raung SL, Chen CC, Kuo JS and Teng CM:
Anti-inflammatory and analgesic effects of magnolol. Naunyn
Schmiedebergs Arch Pharmacol 346: 707-712, 1992.
17. Chang WS, Chang YH, Lu FJ and Chiang HC: Inhibitory effects
of phenolics on xanthine oxidase. Anticancer Res 14: 501-506,
1994.
18. Ikeda K, Sakai Y and Nagase H: Inhibitory effect of magnolol on
tumour metastasis in mice. Phytother Res 17: 933-937, 2003.
19. Konoshima T, Kozuka M, Tokuda H, et al: Studies on inhibitors
of skin tumor promotion, IX. Neolignans from Magnolia offici-
nalis. J Nat Prod 54: 816-822, 1991.
20. Kuo DH, Lai YS, Lo CY, Cheng AC, Wu H and Pan MH: Inhibitory
effect of magnolol on TPA-induced skin inflammation and tumor
promotion in mice. J Agric Food Chem 58: 5777-5783, 2010.
21. You Q, Li M and Jiao G: Magnolol induces apoptosis via acti-
vation of both mitochondrial and death receptor pathways in
A375-S2 cells. Arch Pharm Res 32: 1789-1794, 2009.
22. Hsu YF, Lee TS, Lin SY, et al: Involvement of Ras/Raf-1/ERK
actions in the magnolol-induced upregulation of p21 and cell-
cycle arrest in colon cancer cells. Mol Carcinog 46: 275-283,
2007.
Figure 7. Hypothetical model of cytotoxic mechanism of Magnolol in human
gastric adenocarcinoma SGC-7901 cells.

Page 9

INTERNATIONAL JOURNAL OF ONCOLOGY 40: 1153-1161, 2012
1161
23. Lee SK, Kim HN, Kang YR, et al: Obovatol inhibits colorectal
cancer growth by inhibiting tumor cell proliferation and inducing
apoptosis. Bioorg Med Chem 16: 8397-8402, 2008.
24. Lin SY, Chang YT, Liu JD, et al: Molecular mechanisms of
apoptosis induced by magnolol in colon and liver cancer cells.
Mol Carcinog 32: 73-83, 2001.
25. Lin SY, Liu JD, Chang HC, Yeh SD, Lin CH and Lee WS: Magnolol
suppresses proliferation of cultured human colon and liver cancer
cells by inhibiting DNA synthesis and activating apoptosis. J Cell
Biochem 84: 532-544, 2002.
26. Hwang ES and Park KK: Magnolol suppresses metastasis
via inhibition of invasion, migration, and matrix metallopro-
teinase-2/-9 activities in PC-3 human prostate carcinoma cells.
Biosci Biotechnol Biochem 74: 961-967, 2010.
27. Lee DH, Szczepanski MJ and Lee YJ: Magnolol induces apoptosis
via inhibiting the EGFR/PI3K/Akt signaling pathway in human
prostate cancer cells. J Cell Biochem 106: 1113-1122, 2009.
28. Chen LC, Liu YC, Liang YC, Ho YS and Lee WS: Magnolol
inhibits human glioblastoma cell proliferation through upregula-
tion of p21/Cip1. J Agric Food Chem 57: 7331-7337, 2009.
29. Zhai H, Nakade K, Mitsumoto Y and Fukuyama Y: Honokiol
and magnolol induce Ca2+ mobilization in rat cortical neurons
and human neuroblastoma SH-SY5Y cells. Eur J Pharmacol 474:
199-204, 2003.
30. Li HB, Yi X, Gao JM, Ying XX, Guan HQ and Li JC: Magnolol-
induced H460 cells death via autophagy but not apoptosis. Arch
Pharm Res 30: 1566-1574, 2007.
31. Yang SE, Hsieh MT, Tsai TH and Hsu SL: Effector mechanism of
magnolol-induced apoptosis in human lung squamous carcinoma
CH27 cells. Br J Pharmacol 138: 193-201, 2003.
32. Syu WJ, Shen CC, Lu JJ, Lee GH and Sun CM: Antimicrobial
and cytotoxic activities of neolignans from Magnolia officinalis.
Chem Biodivers 1: 530-537, 2004.
33. Huang SH, Chen Y, Tung PY, et al: Mechanisms for the magnolol-
induced cell death of CGTH W-2 thyroid carcinoma cells. J Cell
Biochem 101: 1011-1022, 2007.
34. Nagase H, Ikeda K and Sakai Y: Inhibitory effect of magnolol
and honokiol from Magnolia obovata on human fibrosarcoma
HT-1080. Invasiveness in vitro. Planta Med 67: 705-708, 2001.
35. Lee SJ, Cho YH, Park K, et al: Magnolol elicits activation of
the extracellular signal-regulated kinase pathway by inducing
p27KIP1-mediated G2/M-phase cell cycle arrest in human urinary
bladder cancer 5637 cells. Biochem Pharmacol 75: 2289-2300,
2008.
36. Lee SJ, Park SS, Lee US, Kim WJ and Moon SK: Signaling
pathway for TNF-alpha-induced MMP-9 expression: mediation
through p38 MAP kinase, and inhibition by anti-cancer molecule
magnolol in human urinary bladder cancer 5637 cells. Int
Immunopharmacol 8: 1821-1826, 2008.
37. Rasul A, Khan M, Yu B, Ma T and Yang H: Xanthoxyletin, a
coumarin induces S phase arrest and apoptosis in human gastric
adenocarcinoma SGC-7901 cells. Asian Pac J Cancer Prev 12:
1219-1223, 2011.
38. Kanzawa T, Kondo Y, Ito H, Kondo S and Germano I: Induction
of autophagic cell death in malignant glioma cells by arsenic
trioxide. Cancer Res 63: 2103-2108, 2003.
39. Levine B and Yuan J: Autophagy in cell death: an innocent
convict? J Clin Invest 115: 2679-2688, 2005.
40. Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y and
Kondo S: Arsenic trioxide induces autophagic cell death in
malignant glioma cells by upregulation of mitochondrial cell
death protein BNIP3. Oncogene 24: 980-991, 2005.
41. Li DD, Guo JF, Huang JJ, et al: Rhabdastrellic acid-A induced
autophagy-associated cell death through blocking Akt pathway
in human cancer cells. PLoS One 5: e12176, 2010.
42. King KL and Cidlowski JA: Cell cycle regulation and apoptosis.
Annu Rev Physiol 60: 601-617, 1998.
43. Pan J, She M, Xu ZX, Sun L and Yeung SC: Farnesyltransferase
inhibitors induce DNA damage via reactive oxygen species in
human cancer cells. Cancer Res 65: 3671-3681, 2005.
44. Grana X and Reddy EP: Cell cycle control in mammalian cells: role
of cyclins, cyclin dependent kinases (CDKs), growth suppressor
genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene
11: 211-219, 1995.
45. Kastan MB, Canman CE and Leonard CJ: P53, cell cycle control
and apoptosis: implications for cancer. Cancer Metastasis Rev
14: 3-15, 1995.
46. Pavletich NP: Mechanisms of cyclin-dependent kinase regulation:
structures of Cdks, their cyclin activators, and Cip and INK4
inhibitors. J Mol Biol 287: 821-828, 1999.
47. Mantena SK, Sharma SD and Katiyar SK: Berberine, a natural
product, induces G1-phase cell cycle arrest and caspase-3-depen-
dent apoptosis in human prostate carcinoma cells. Mol Cancer
Ther 5: 296-308, 2006.
48. Srivastava JK and Gupta S: Tocotrienol-rich fraction of palm oil
induces cell cycle arrest and apoptosis selectively in human
prostate cancer cells. Biochem Biophys Res Commun 346:
447-453, 2006.
49. Hengartner MO: The biochemistry of apoptosis. Nature 407:
770-776, 2000.
50. Zhou HB and Zhu JR: Paclitaxel induces apoptosis in human
gastric carcinoma cells. World J Gastroenterol 9: 442-445, 2003.
51. Olson M and Kornbluth S: Mitochondria in apoptosis and human
disease. Curr Mol Med 1: 91-122, 2001.
52. Kroemer G: Mitochondrial control of apoptosis: an introduction.
Biochem Biophys Res Commun 304: 433-435, 2003.
53. Huang M, Camara AK, Stowe DF, Qi F and Beard DA: Mito-
chondrial inner membrane electrophysiology assessed by
rhodamine-123 transport and fluorescence. Ann Biomed Eng 35:
1276-1285, 2007.
54. Porter AG and Janicke RU: Emerging roles of caspase-3 in
apoptosis. Cell Death Differ 6: 99-104, 1999.
55. Mallat Z and Tedgui A: Apoptosis in the vasculature: mechanisms
and functional importance. Br J Pharmacol 130: 947-962, 2000.
56. Adams JM and Cory S: The Bcl-2 apoptotic switch in cancer
development and therapy. Oncogene 26: 1324-1337, 2007.
57. Danial NN: BCL-2 family proteins: critical checkpoints of
apoptotic cell death. Clin Cancer Res 13: 7254-7263, 2007.
58. Han Z, Hong L, Han Y, et al: Phospho Akt mediates multidrug
resistance of gastric cancer cells through regulation of P-gp, Bcl-2
and Bax. J Exp Clin Cancer Res 26: 261-268, 2007.
59. Altomare DA and Testa JR: Perturbations of the AKT signaling
pathway in human cancer. Oncogene 24: 7455-7464, 2005.
60. Lin HL, Yang MH, Wu CW, et al: 2-Methoxyestradiol attenuates
phosphatidylinositol 3-kinase/Akt pathway-mediated metastasis
of gastric cancer. Int J Cancer 121: 2547-2555, 2007.
61. Daido S, Kanzawa T, Yamamoto A, Takeuchi H, Kondo Y and
Kondo S: Pivotal role of the cell death factor BNIP3 in ceramide-
induced autophagic cell death in malignant glioma cells. Cancer
Res 64: 4286-4293, 2004.
62. Paglin S, Hollister T, Delohery T, et al: A novel response of cancer
cells to radiation involves autophagy and formation of acidic
vesicles. Cancer Res 61: 439-444, 2001.
63. Yoshimori T: Autophagy: a regulated bulk degradation process
inside cells. Biochem Biophys Res Commun 313: 453-458,
2004.
64. Eisenberg-Lerner A, Bialik S, Simon HU and Kimchi A: Life
and death partners: apoptosis, autophagy and the cross-talk
between them. Cell Death Differ 16: 966-975, 2009.