Bostrycin inhibits proliferation of human lung carcinoma A549 cells via downregulation of the PI3K/Akt pathway
Journal of Experimental & Clinical Cancer Research
Bostrycin inhibits proliferation of human lung carcinoma A549 cells via downregulation of the PI3K/Akt pathway
Wei-Sheng Chen 0 1
Jun-Na Hou 0 1
Yu-Biao Guo 0 1
Hui-Ling Yang 0 3
Can-Mao Xie 0 1
Yong-Cheng Lin 0 2
Zhi-Gang She 0 2
0 Materials and methods Cell line and cell culture The human pulmonary adenocarcinoma cell line A549 was obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat-Sen University
1 Department of Pulmonary and Critical Care Medicine, the First Affiliated Hospital of Sun Yat-Sen University , Guangzhou 510080 , China
2 Marine Microorganism Lab, Institute of Chemistry and Chemical Engineering, Sun Yat-Sen University , Guangzhou 510080 , China
3 Department of Physiopathology, Zhongshan School of Medicine, Sun Yat-Sen University , Guangzhou 510080 , China
Background: Bostrycin is a novel compound isolated from marine fungi that inhibits proliferation of many cancer cells. However, the inhibitory effect of bostrycin on lung cancers has not been reported. This study is to investigate the inhibitory effects and mechanism of bostrycin on human lung cancer cells in vitro. Methods: We used MTT assay, flow cytometry, microarray, real time PCR, and Western blotting to detect the effect of bostrycin on A549 human pulmonary adenocarcinoma cells. Results: We showed a significant inhibition of cell proliferation and induction of apoptosis in bostrycin-treated lung adenocarcinoma cells. Bostrycin treatment caused cell cycle arrest in the G0/G1 phase. We also found the upregulation of microRNA-638 and microRNA-923 in bostrycin-treated cells. further, we found the downregulation of p110a and p-Akt/PKB proteins and increased activity of p27 protein after bostrycin treatment in A549 cells. Conclusions: Our study indicated that bostrycin had a significant inhibitory effect on proliferation of A549 cells. It is possible that upregulation of microRNA-638 and microRNA-923 and downregulaton of the PI3K/AKT pathway proteins played a role in induction of cell cycle arrest and apoptosis in bostrycin-treated cells.
Lung cancer is the most common type of cancer
worldwide. Despite recent advances in surgical techniques and
chemotherapy/radiotherapy strategies, the long-term
survival rates remain poor. There is therefore an urgent
need to develop new therapeutic strategies in order to
significantly improve the prognosis in lung cancer
patients. Growth factor signaling pathways have been
shown to be important targets in lung cancer therapy.
Targeting such intracellular pathways that regulate
proliferation, apoptosis, metastasis and resistance to
chemotherapy represents an important therapeutic strategy
for lung cancer .
Marine microorganisms can grow under adverse
conditions such as low temperatures, high pressures, and
poor nutrition. The diversity of biological activities in
these environments exceeds those of land organisms.
Some metabolites from these marine microorganisms
have novel structures and biological activities including
anticancer, antiviral and immune enhancement
properties. A recent study on marine pharmacology
coordinated by multiple countries demonstrated antitumor
activity in a number of natural products derived from
marine invertebrates, algae, fungi, and bacteria, although
the mechanisms of action are still unknown .
Bostrycin, a novel compound isolated from marine
fungi in South China Sea, has been shown to inhibit cell
growth in in prostate cancer and gastric cancer [3,4].
However, since the antitumor effect of bostrycin in lung
cancer is not known, we explored the effect of bostrycin
treatment in lung cancer cells and investigated the
mechanisms underlying the inhibitory effect of bostrycin
in lung cancers.
Cells were cultured in DMEM medium (low glucose)
supplemented with 10% newborn calf serum at 37C with 5%
CO2. Cells were digested with 0.25% trypsin and
subcultured at 70% to 80% confluence Exponentially growing
A549 cells were used for all assays.
anthracene dione), a novel compound isolated from
marine fungi in P.R. China, was supplied by Marine
Microorganism Laboratory, Institute of Chemistry and
Chemical Engineering, Sun Yat-Sen University. The
chemical structure of bostrycin is shown inAdditional file 1,
Newborn calf serum, DMEM (low glucose), 0.25% trypsin
digest, and Trizol reagent were purchased from GIBCO
(Invitrogen Corporation, Carlsbad, CA, USA). MTT and
DMSO were obtained from Sigma Corporation. Mouse
anti-human phospho-Akt monoclonal antibody (mAb),
rabbit anti-human p110a mAb, rabbit anti-human p27
mAb, horseradish peroxidase (HRP)-conjugated goat
anti-mouse IgG (secondary antibody), HRP-conjugated
goat anti-rabbit IgG (secondary antibody), and prestained
protein molecular weight marker were purchased from
Cell Signaling Technology (USA).
Measurement of cell growth inhibition by MTT assay
A549 cells were seeded in 96-well plates (5 103 cells
per well) and treated with bostrycin (10, 20, and
30 mol/L). Negative control wells (containing cells but
not bostrycin), and the blank control (only medium)
were plated with 6 replicates each. Untreated and treated
cells were cultured at 37C with 5% CO2 for 12 hours.
MTT solution (20 L) was added to each well and mixed;
the wells were then incubated for an additional 4 hours.
Culture supernatant was removed, DMSO (150 L) was
added to each well and vortexed at low speed for 10
minutes to fully dissolve the blue crystals. Absorbance was
measured at 570 nm (A570) and the percentage of growth
inhibition of A549 cells was calculated at each time point
and for each concentration of bostrycin according to the
following formulae: % cell survival = (A570bostrycin
group - A570blank)/(A570negative - A570blank) 100%
and % cell growth inhibition = 1 - % cell survival. Half
maximal inhibitory concentration (IC50) values at
respective times were then calculated using linear
Cell cycle and apoptosis rate assayed by flow cytometry
A549 cells were cultured in 6-well plates (1.5 105 cells
per well) and treated with different concentrations (5,
10, and 20 mol/L) of bostrycin or complete DMEM
medium (for the control group) and incubated for 24,
48 or 72 hours. Culture supernatant from each group
was pooled and the cells were fixed for 12 h with 1 ml
of 75% ethanol (106 cells/ml) and transferred to 2 mL
Eppendorf tubes for flow cytometry and propidium
iodide (PI) staining. For PI staining, the cells were
washed twice with cold PBS and centrifuged at 1000 g
for 5 min. The pellet was washed twice in cold 0.1% Triton
X-100 PBS and incubated at room temperature for 30
minutes with 300 L DNA dye (containing 100 g/mL
propidium iodide and 20 U/mL RNase; Sigma
Corporation). Flow cytometry analysis (BECKMAN-COULTER
Co., USA) was performed. The cells were collected for the
calculation of DNA amount for cell cycling analysis using
a MULTYCYCLE software (PHEONIX, Co. USA). The
extent of apoptosis was analyzed and quantified using
WinMDI version 2.9 (Scripps Research Institute, La Jolla,
Differential expression of microRNAs
Preparation of total RNA sample
A549 cells were cultured in 6-well plates (1.5 105 cells
per well) and treated for 72 h with 10 mol/L bostrycin
for the bostrycin group or with complete medium for
the control group. The cells were lysed in 1.5 mL of
Trizol reagent and total RNA was prepared according to
the manufacturers instructions.
Microarray analysis was performed using a service
provider (LC Sciences, USA). The assay used 2-5 g total
RNA, which was size-fractionated using a YM-100
Microcon centrifugal filter (SIGMA). The small RNAs
(<300 nucleotides) isolated were 3 extended using poly
(A) polymerase. An oligonucleotide tag was then ligated
to the poly(A) tail for fluorescent dye staining. Two
different tags were used for the two RNA samples in
dualsample experiments. Hybridizations were performed
overnight on a Paraflo microfluidic chip using a
microcirculation pump (Atactic Technologies, Houston, TX,
USA). Each detection probe on the microfluidic chip
consisted of a chemically modified nucleotide-coding
segment complementary to a target microRNA
(miRBase; http://microrna.sanger.ac.uk/sequences/) or other
RNA (control or customer-defined sequences). The
probe also contained a spacer segment of polyethylene
glycol to separate the coding segment from the
substrate. The detection probes were made by in situ
synthesis using PGR (photogenerated reagent chemistry). The
hybridization melting temperatures were balanced by
chemical modifications of the detection probes.
Hybridization was done in 100 L 6 saline-sodium
phosphate-EDTA buffer (0.90 M NaCl, 60 mMNa2HPO4,
and 6 mM EDTA, pH 6.8) containing 25% formamide at
34C and fluorescence labeling with tag-specific Cy3 and
Cy5 dyes was used for detection. Hybridization images
were collected using a laser scanner (GenePix 4000B,
Molecular Device) and digitized using Array-Pro image
analysis software (Media Cybernetics). Data were analyzed
by first subtracting the background and then normalizing
the signals using a LOWESS filter (locally weighted
regression). For two-color experiments, the ratio of the two sets
of detected signals (log 2 transformed; balanced) and
P values of the t test were calculated. Differentially
detected signals were those with P < 0.01.
RT-PCR was performed using the TaqMan MicroRNA
Reverse Transcription Kit (LC Sciences, USA) and the
ABI PRISM 7000 Sequence Detection System (Life
Technologies Corporation, Carlsbad, CA, USA). 2 g
RNA was used to synthesize single stranded cDNA
according to the manufacturers instructions. Real time
PCR was performed to amplify the cDNA with the
TaqMan Universal PCR Master Mix (LC Sciences, USA) as
follows: amplification for 30 cycles at 94C for 0.5 min,
annealing at 55C for 0.5 min, and extension at 72C for
0.5 min; and then terminal elongation step at 72C for
10 min and a final holding stage at 4C. The
amplification plots were viewed and the baseline and threshold
values (as indicated in the instrument user guide) were
set to analyze the results. The relative miRNA
expression was calculated using 2-Ct where Ct is the
difference between target miRNA or reference miRNA Ct
values in the treated and control samples. Ct is the
difference between the above two Ct from target
miRNA and reference miRNA.
A549 cells (cultured in 6-well plate at 1.5 105 cells per
well) were treated with 10 mol/L bostrycin for 12, 24,
48, and 72 hours, and total proteins were extracted.
Protein samples were separated by SDS-PAGE and
electrophoretically transferred onto a polyvinylidene difluoride
membrane (Millipore, USA). The membrane was blocked
overnight at 4 degree in TBS-Tween 20 (TBST) buffer
containing 5% skimmed milk powder. The membrane
was washed with TBST (3 8 minutes). Membranes
were then incubated overnight at 4C in primary antibody
(125 L/cm3; diluted 1:1,000) with gentle shaking. The
membranes were washed with TBST (3 8 minutes) and
incubated for 1 h at room temperature in HRP-conjugated
secondary antibody (125 L/cm3; diluted 1:2,500). The
membranes were washed with TBST (3 8 minutes) and
protein signals were detected by chemiluminescence kit
(Cell signaling Technology, USA).
Normally distributed continuous variables were
compared by one-way analysis of variance (ANOVA). When
a significant difference between groups was apparent,
multiple comparisons of means were performed using the
Bonferroni procedure with type-I error adjustment. Data
are presented as means SD. All statistical assessments
were two-sided and evaluated at the 0.05 level of
significant difference. Statistical analyses were performed using
SPSS 13.0 statistics software (SPSS Inc, Chicago, IL)
Bostrycin inhibited the proliferation of A549 cells
First, we used the MTT assay to detect effect of bostrycin
on A549 cell proliferation. There was a dose-dependent
and time-dependent inhibition of A549 cell proliferation
by bostrycin (Figure 1) with an optimal linear
relationship seen between 10-30 of bostrycin. This indicated
that bostrycin could significantly inhibit A549 cell
proliferation in vitro.
Bostrycin induced cell cycle arrest and apoptosis in A549
Then, we used flow cytometry to determine cell cycle
distribution and apoptosis in A549 cells exposed to
different concentrations of bostrycin for 24, 48, and
72 hours. We showed a significant increase in the
number of G0/G1 phase cells and a decrease in the number
of S and G2/M phase cells after 72 hours of bostrycin
treatment (Figure 2A). We also used propidium iodide
staining to show that bostrycin induced apoptosis of
Figure 1 Effect of Bostrycin on the proliferation of A549 cells
by MTT assay. A549 cells were treated with 10, 20, or 30 M of
bostrycin for 24 h, 48 h or 72 h. Negative control consisted of
untreated cells, while the blank control was set up with only
medium. Statistically significant differences were observed between
groups treated with different bostrycin concentrations at each time
point and between different time points at each concentration (all
P < 0.05).
Figure 2 Effect of Bostrycin on cell cycle and apoptosis
detected by flow cytometry. A549 cells were treated with 0, 5, 10
or 20 M of bostrycin for 24 h, 48 h or 72 h. A) represents the
percentage of A549 cells at different phases of the cell cycle at
different time points and at different concentrations of bostrycin;
B) represents the percentage of apoptotic A549 cells at different
time points and at different concentrations of bostrycin; C) shows
representative flow cytometry plots. *Indicates a statistically
significant difference between the given group and its
corresponding control group. Pair-wise multiple comparisons
between groups were determined using Bonferronis test with
a = 0.017 adjustment.
A549 cells in a dose-dependent and time-dependent
manner (Figure 2B). Figure 2C shows the flow
cytometry data of cells treated with different concentrations of
bostrycin for 24 h, 48 h and 72 h.
Analysis of microRNA expression in A549 cells by
microarrays and real-time RT-PCR
We used a gene chip probe techniques to detect
changes in microRNA expression in bostrycin-treated
A549 cells when compared with untreated cells. We
found a statistically significant difference in the
expression of fifty-four microRNAs (data not shown). We
selected microRNA-638 and microRNA-923 for further
validation with real-time RT-PCR since these two
microRNAs showed the most significant difference. We
used RT-PCR to demonstrate a significant upregulation
in the levels of microRNA-638 and microRNA-923 in
bostrycin-treated A549 cells. These data were consistent
with our microarray analysis (Figure 3).
Detection of p110a, p-Akt, and p27 levels in
Finally, we detected the possible signal pathway involved
in the effects of bostrycin on A549 cells. We showed by
western blots that there was a decrease in the expression
of p110a protein over time in bostrycin-treated A549
cells. Although there was an increase in the expression
of p-Akt protein in cells treated with bostrycin for
12 hours, when compared with cells at the 0 hour time
point, we showed a gradual decrease in p-Akt levels
over time, with the most obvious reduction at 48 hours.
We also showed a time-dependent increase in the levels
of p27 protein in bostrycin-treated cells (Figure 4).
Figure 3 Relative change in expression of microRNA-638 and
microRNA-923 in A549 cells treated with bostrycin detected by
microRNA real time PCR. A549 cells were treated with 10 M
Bostrycin for 72 h and total RNA was isolated for microRNA real
time PCR assay. Expression levels of microRNA-638 and
microRNA923 were determined as described. Untreated A549 cells were used
as control. Each condition was repeated 4 times.
Figure 4 Effects of Bostrycin on intracellular expression of
p110a, p-Akt and p27 in A549 cells. A549 cells were treated with
10 mol/L bostrycin for 12, 24, 48, or 72 hours. Cells were harvested,
total proteins were extracted and immunoblotted for p110a, p-Akt
and p27. Untreated A549 cells were used as a control. Beta-actin
was used as loading control.
In this study, we demonstrated that bostrycin, a novel
compound isolated from marine fungi in the South
China Sea, inhibited cell proliferation, blocked cell cycle
progression, and promoted apoptosis of lung cancer
A549 cells. Our data also suggested that the PI3K/AKT
signaling pathway may play a role in bostrycin-mediated
inhibition of cell proliferation. Although bostrycin was
previously shown to effectively inhibit cell growth and
promote apoptosis in prostate cancer and gastric cancer
[3,4], it has not been used in lung cancer cells. To our
knowledge, ours is the first study demonstrating that
bostrycin significantly inhibited the growth of A549 cells
in a concentration- and time-dependent manner.
Regulation of the cell cycle and apoptosis is a major
determinant dictating the development and progression
of a number of cancers. PI3K/AKT inhibitors such as
Tipifarnib, cause cell cycle arrest at the G1 or G2/M
phase and induce apoptosis of human lung cancer cells
[5,6] Our data were consistent with this study and
showed that bostrycin treatment induced downregulation
of PI3K/AKT signal pathway proteins, caused G0/G1 cell
cycle arrest and promoted apoptosis in A549 cells.
PI3K is composed of a p110asubunit and p85 subunit
and the PI3K/AKT signaling pathway has been shown
to play a role in the development and progression of
lung cancer . Increased Akt activity has been reported
in the bronchial endothelial cells of long-term smokers
[8,9] and persistently high levels of activated Akt was
shown in bronchial endothelial cells from malignant
tumors or precancerous lesions. Akt activation is
thought to be related to poor prognosis of patients with
lung cancer [10-12] and may be an important molecular
target for treatment of lung cancer.
The PI3K/AKT signaling pathway inhibits apoptosis
by inactivating important members of the apoptotic
cascade, including caspase-9, forkhead, and proapoptotic
Bad [13-15] and by upregulating the transcription and
translation of antiapoptotic genes via NF B  and cell
cycle genes like cyclin D1 and p27 . The p27 gene, a
tumor suppressor, encodes a late G1 cyclin-dependent
kinase inhibitor, whose activity is dependent on
phosphorylation-dependent cytoplasmic translocation .
The PI3K/AKT pathway regulates p27 activity by 1)
directly phosphorylating it at Thr159, resulting in
cytoplasmic translocation and inactivation of p27 or 2)
phosphorylation and cytoplasmic translocation of AFX
(a forkhead transcription factor), which downregulates
p27 levels . We used p110a expression levels as a
marker of PI3K expression and showed a significant
downregulation of p110a and p-Akt levels and an
upregulation of p27 levels in bostrycin-treated A549 cells.
These data suggest that p-Akt downregulation could
inhibit cytoplasmic translocation of p27, causing a G1
cell cycle arrest of A549 cells. However, further studies
are necessary to elucidate the mechanisms underlying
bostrycin-mediated induction of apoptosis and
attenuation of the PI3K/AKT signaling pathway in A549 cells.
While we evaluated overall levels of phosphorylated Akt
and p27 in this study, we would also like to detect
changes in specific phosphorylation sites of these
proteins, in order to more completely understand the
mechanism of bostrycin action.
MicroRNAs are thought to play an important role in
the development and progression of tumors .
Microarray analysis on 104 primary non-small cell lung
carcinomas showed changes in the expression levels of
43 microRNAs in lung cancer tissue when compared
with normal lung tissue . Members of the let-7 family
of microRNAs are known to inhibit growth of non-small
cell lung carcinoma by inducing cell cycle arrest and
apoptosis , while microRNA-126 inhibits the invasion
of non-small cell lung carcinoma . microRNA-25 and
microRNA-205 have been used to predict survival and
recurrence in lung cancer patients [24,25]. Exploring
microRNA regulation may therefore provide useful
information in developing new drug targets or identifying
early disease markers . MicroRNAs 638 and
microRNA 923 were significantly upregulated in
bostrycintreated A549 cells. Both microRNAs might be related
with tumor inhibition.
Interestingly, microRNAs have also been reported to
play a regulatory role in the PI3K signaling pathway.
Recombinant microRNA-126 was shown to downregulate
the expression of p85b (a regulatory subunit of PI3K
related to the stabilization and transmission of the PI3K
signal) and p-Akt proteins in rectal cancer cells , and
microRNA-7 inhibited the Akt pathway and reduced
survival rates in spongiocytoma . It is tempting to
speculate that upregulation of microRNA-638 and
microRNA-923 in bostrycin-treated A549 cells, accompanied
by downregulation of the PI3K/AKT signaling
pathwayassociated proteins, p110a and p-Akt, are significantly
related. We would like to dissect these pathways in
greater detail in our upcoming studies, using luciferase
assays to demonstrate direct targets of microRNA-638
and microRNA-923 in bostrycin-treated cells.
In conclusion, we demonstrated that bostrycin, a novel
metabolite isolated from marine fungi, inhibited
proliferation, blocked cell cycle progression and promoted
apoptosis in pulmonary adenocarcinoma A549 cells. We
also demonstrated 1) upregulation of tumor-suppressing
transcriptional factors, the noncoding microRNA-638
and microRNA-923, and 2) downregulation of proteins
associated with the PI3K/PI3K/AKT signaling pathway
in bostrycin-treated cells, suggesting that bostrycin may
be a new PI3K/AKT signal pathway-targeting drug for
the treatment of pulmonary adenocarcinoma.
Additional file 1: Figure S1, Bostrycin
(hydroxy-methoxy-tetrahydro5-methyl anthracene dione). The file contains the molecular chemical
structure of bostrycin.
PI3K: Phosphoinositide 3-kinase; AKt/PKB: Protein Kinase B; mAb:
monoclonal antibody; IC50: the half maximal inhibitory concentration.
This work was supported by grants from The Natural Science Funds of
Guangdong Province (7001646), and the Science and Technology Project of
Guangdong Province (2008B080703022).
We thank the Marine Microorganism Laboratory, Institute of Chemistry and
Chemical Engineering, Sun Yat-Sen University, for kindly providing the test
compound, bostrycin; the Electron Microscope Center, North School Region,
Sun Yat-Sen University, for the technical support with the electron
microscope; Hangzhou Lianchuan Biological Message Ltd. Company for the
technical support in gene chip and real-time RT-PCR techniques; and Dr. Tan
Li (The Affiliated Tumor Research Centre of Sun Yat-Sen University) for the
advice on western blotting.
YBG: Conceived and designed the experiments;
WSC, JNH: Performed the experiments and analysed the data;
HLY, CMX, YCL, ZGS: Contributed reagents/material/analysis tools/.
All authors read an approved the final draft.
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