Targeted inhibition of the phosphoinositide 3-kinase impairs cell proliferation, survival, and invasion in colon cancer
OncoTargets and Therapy
Targeted inhibition of the phosphoinositide 3-kinase impairs cell proliferation, survival, and invasion in colon cancer
Fei Yang 2
Jun-Yi gao 1
Zhen-hua Du 2
Xue-Qun Zhang 0
0 g raduate s chool, Taishan Medical University , Xintai
1 Department of clinical Medicine, Weifang Medical college , Weifang
2 Department of Pathology, Jinan central hospital affiliated to shandong University , Jinan
3 Department of Oncology, Jinan c entral h ospital affiliated to shandong University , Jinan, People's republic of china
8 1 0 2 - l u J - 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s re .
human colon cancer; PI3K/Akt/mTOR pathway; BEZ235; PI3KCA knockdown
open access to scientific and medical research
O r i g i n a l r e s e a r c h
*These authors contributed equally
to this work
Colon cancer is one of the most common cancers in the world, and the mortality rate
is rising every year. In the case of metastasis, the 5-year survival rate for colon cancer
patients is only 10%.1 With the continuous study of colon cancer, its occurrence and
development are found to be multi-stage, multi-step processes involving numerous
The targets of the PI3K/protein kinase B (Akt)/mammalian rapamycin (mTOR)
are usually associated with the development of cancer, including colon cancer.2–5
This pathway is upregulated in cancers and is associated with increased proliferation,
decreased apoptosis, and promoted cancer pathogenesis.6 Moreover, the activation of
PI3K/Akt/mTOR pathway is closely related to the drug resistance of colon cancer,
thus reducing the success rate of treatment.7,8 In addition, the activation of PI3K
regulates various downstream proteins involved in tumor progression, such as p70S6K,
cyclin D1, β-catenin, E-cad, Bcl-2/Bax, and so on. Among them, p70S6K is a substrate
for mTOR that is regulated by PI3K/Akt/mTOR and related to cell proliferation,
survival, and epithelial–mesenchymal transition (EMT).9,10
Therefore, inhibiting the PI3K/Akt/mTOR pathway may
have the potential for cancer treatment and may enhance the
sensitivity of chemotherapy and radiotherapy.
BEZ235 has dual inhibitory effects on PI3K and m-TOR,
blocking the PI3K activity by simultaneously inhibiting
mTOR. In tumor research and clinical trials, BEZ235
has been used because of its effectiveness and low side
1820 effects.11–14 A previous study showed that, even in PIK3CA
l--Ju mutation status, the cell proliferation of colorectal cancer cell
12 lines (HCT116, DLD-1, and SW480) was effectively
inhibon7 ited by BEZ235.15 Chen et al found that BEZ235 suppressed
.062 colon cancer cell HCT-116 proliferation, leading to cell
.495 apoptosis.16 Therefore, BEZ235 was selected as an inhibitor
.37 to investigate the effects of targeting the PI3K/Akt/mTOR
/yb pathway in colon cancer cells. shRNA transfection is a
.com simple and effective way to knock down PI3K and PI3KCA,
rsse . which is used in various cancer studies including HT-29 and
.vdoep lsyeon explore the effects of targeted inhibition of PI3K on proliferation,
HCT-116 cells.17–19 In the present study, we determined to
/:/ww loan migration, invasion, and apoptosis of colon cancer cells.
Methods and materials
The colon cancer HT-29 and HCT-116 cell lines were
purchased from China Cell Bank (Shanghai, China) and
cultured at 37°C with 5% CO2 in DMEM medium (Thermo
Fisher Scientific, Waltham, MA, USA) containing 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific), 100 U/mL
penicillin, and 100 mg/mL streptomycin (Thermo Fisher
Scientific). The cells in the logarithmic phase with the sound
condition were used for further detection. When the cells
were in the total growth phase, they were divided into three
groups and treated under three culture conditions: BEZ235,
shRNA transfection, and dimethyl sulfoxide (DMSO) as
inhibition of Pi3K/akt/mTOr by BeZ235
Different concentrations of BEZ235 (Cell Signaling
Technology, Beverly, MA, USA; dissolved in DMSO), the
specific inhibitor of PI3K/Akt pathway, were used to inhibit
the PI3K/Akt/mTOR pathway based on the manufacturer’s
instruction. Approximately 5×106 HT-29/HCT-116 cells were
seeded in a 6-cm diameter dish with DMEM and cultured
at 37°C with 5% CO2 for 24 hours. Then, the cells treated
with BEZ235 at 0, 1 nM, 10 nM, 100 nM, and 1 µM were
incubated shortly thereafter for 24 hours. The appropriate
concentrations of BEZ235 which achieved the maximum
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inhibition effect were selected for further experiments.
DMSO was used as a control reagent. This experiment was
repeated independently at least two times.
Knockdown of Pi3Kca by shrna
We designed and synthetized the PI3KCA-shRNA as
follows: shRNA (GCATTAGAATTTACAGCAAGA). The
target vector pLVX-shRNA2 (Clontech Co. CA, US) was
digested by BamHI and EcoRI. According to the study of
Dang et al,20 the shRNA vector was constructed and
transfected into HT-29 and HCT-116 cells by Liposomes 2000
(Thermo Fisher Scientific) according to the manufacturer’s
instructions. The transfection efficiency for HT-29 and
HCT-116 cells was 81% and 85%, respectively. Forty-eight
hours after transfection, the cells were collected for RT-PCR,
Western blotting analysis, and other assays.
real-time quantitative r T-Pcr analysis
The mRNA level of PI3KCA was quantitatively estimated
by real-time quantitative RT-PCR, to determine a definitive
measurement of PI3KCA knockdown efficiency. Total RNA
was isolated by RNeasy kit (Sigma, St Louis, MO, USA).
Reverse transcription reactions were performed by using the
PrimeScript® 1st strand cDNA synthesis kit (TaKaRa, Beijing,
China). SYBR Green qPCR Master Mix (2×) kit (Thermo
Fisher Scientific) was performed to analyze real-time RT-PCR
with the following program: 95°C 10 minutes, 95°C 15
seconds, 60°C 60 seconds for annealing and extension, with a
repetition of 40 cycles. Real-time RT-PCR data were analyzed
by comparative Ct (ΔΔct) method. The expression level of
PI3KCA mRNA was normalized to an endogenous control,
GAPDH. Primer sequences were as follows: PI3KCA:
forward primer 5′-CAATCGGTGACTGTGTGGGA-3′, reverse
primer 5′-ACAGGTCAATGGCTGCATCA-3′; GAPDH:
forward primer 5′-CATGAGAAGTATGACAACAGCCT-3′,
reverse primer 5′-AGTCCTTCCACGATA CCAAA GT-3′.
The expression of PI3K/Akt/mTOR-related proteins and
proteins associated with apoptosis was determined by Western
blotting method. HT-29 and HCT-116 cells were treated with
BEZ235 at different concentrations (0, 1 nM, 10 nM, 100 nM,
and 1 µM) for 24 hours to detect inhibition efficiency. HT-29
and HCT-116 cells were then treated with 1 µM BEZ235 or
transfected with PI3KCA shRNA transfection, respectively.
Both the treatments lasted for 24 hours. The cells were then
collected to extract proteins, which were quantified using a
BCA Protein Assay Kit. Proteins were then separated with
10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.
After blocking with 5% nonfat dry milk in Tris-buffered saline,
0.1% Tween 20 (TBST) for 1 hour, membranes were
incubated with the primary antibody (1:1,000) at 4°C overnight.
After washing, secondary antibodies were applied to
membranes and incubated at room temperature for 1 hour. Labeled
protein bands were exposed using electro-chemiluminescence
(ECL) solution. For quantification analyses, Western blotting
was repeated for three times. The band intensity was measured
by using ImageJ. The relative expression levels of proteins
were calculated by standardized the band intensity with the
amount of β-tubulin from the same sample.
cell proliferation assay
According to the manufacturer’s instructions, Cell Counting
Kit-8 (CCK8; Dojindo, Japan) was adopted to quantify the
cell proliferation after treatment with BEZ235 and PI3KCA
knockdown. HT-29 and HCT-116 cells were treated with 1 µM
BEZ235 and shRNA transfection for 24 hours, respectively.
Then, the cells were seeded in 96-well plates at a density of
1,000 cells/well and were constantly cultured with BEZ235 in
normal medium. The supernatant was replaced with 100 mL
of medium containing 10 mL of CCK8, enabling detection of
optical density (OD) values at 450 nm every 24 hours.
For colony formation assays, 200 HT-29 and HCT-116
cells of three groups with 24-hour treatment of BEZ235,
PI3KCA shRNA transfection, and respective NC were seeded
in a disposable dish and cultured in a medium containing
10% FBS for 1 week. Colonies were fixed with methanol,
stained with 0.1% crystal violet, and photographed and
counted with an inverted microscope. Each treatment was
performed three times.
cell migration and invasion assay
Transwell chamber model was performed to measure cell
migration and invasion after treatment with BEZ235 and
PI3KCA shRNA transfection.
HT-29 and HCT-116 cells with 24-hour treatment of 1 µM
BEZ235 and PI3KCA shRNA transfection were suspended at
a concentration of 3×104 cells/mL. 100 µL of the suspension
was added into the upper chamber of Transwell
apparatus (EMD Millipore, Billerica, MA, USA; 0.8 µm).
After incubation for 24 hours, we removed the cells that
remained in the top chamber.
Matrigel (BD Biosciences, San Jose, CA, USA) was used to
coat the upper chambers, and the cells with the BEZ235 or
PI3KCA shRNA transfection treatment were added to the
upper chamber at a density of 5×104 cells/chamber (100 µL).
100 µL of medium with 10% FBS was added into the lower
chamber. After incubation for 24 hours, cells that attached to
the lower chamber were fixed in 4% paraformaldehyde and
stained with 0.1% crystal violet to quantify cell migration.
The Transwell assays were repeated three times.
cell apoptosis assay
HT-29 and HCT-116 cells were seeded in six well plates
and treated with BEZ235 and PI3KCA shRNA transfection
at the logarithmic growth phase. After 48 hours of
incubation, cells were collected at the concentration of 105/mL in
binding buffer and stained with 5 µL Annexin V fluorescein
isothiocyanate (FITC) and 10 µL propidium iodide (PI) (BD
Biosciences). Flow cytometry was then performed to assess
apoptosis. The experiment was repeated three times.
The quantitative experimental data were presented as
mean ± SD and analyzed by SPSS 16.0 statistical software
with one-way analysis of variance and unpaired t-test to
analyze the differences between groups. P,0.05 for the
difference was statistically significant.
BeZ235 effectively inhibited Pi3K/akt/
mTOr activation in hT-29 and
To establish a suitable dose of BEZ235 on PI3K/Akt/
mTOR pathway, HT-29 and HCT-116 cells were treated
with BEZ235 at 0, 1 nM, 10 nM, 100 nM, or 1 µM. Indeed,
we found that BEZ235 efficiently decreased p-Akt levels
in a dose-dependent manner. As 1 µM of BEZ235 had the
greatest inhibitory effect, this concentration was used in the
subsequent experiments (Figure 1A). The levels of p-Akt
and p-mTOR in HT-29 and HCT-116 cells decreased when
compared with the control groups (Figure 1B).
inhibition of Pi3K/akt/mTOr by BeZ235
resulted in a decline in hT-29 and
hcT116 cell proliferation
In order to describe the BEZ235 effect on proliferative activity
of HT-29 and HCT-116, after treating with 1 µM of BEZ235,
the cell viability was detected with CCK8 and colony formation
assay. The proliferation of HT-29 and HCT-116 cells was
significantly inhibited by BEZ235 in 24 hours (Figure 1C). Compared
with the control group, the proliferation ability of cells increased
significantly during the treatment period. Similar results were
observed in the analysis of colony formation. BEZ235 inhibition
of the PI3K/Akt/mTOR pathway led to a significant reduction
in the number of cell colonies of HT-29 and HCT-116 cells,
compared with the control group (Figure 1D and E).
Pi3Kca knockdown reduced hT-29 and
hcT-116 cell proliferation
To confirm the inhibitory effect of the PI3K/Akt/mTOR
pathway on colon cancer cells, PI3KCA shRNA
transfection was used to inhibit PI3KCA expression. In knockdown
HT-29 cells, the expression of PI3KCA mRNA was reduced
by 70%, while the knockdown of HCT-116 cells decreased
by 68%. The cells were detected by RT-PCR and compared
with control cells of 48 hours past-transfection (Figure 2A).
In knockdown HT-29 and HCT-116 cells, the PI3K/Akt/
mTOR pathway activation was also significantly suppressed
(Figure 2B and C). PI3KCA knockdown resulted in a
significant decrease in the proliferation of HT-29 and HCT-116 cells
at the time of 48 and 72 hours, respectively (Figure 2D),
compared to the control group. These results were also confirmed
by colony formation assays. Compared with the control group,
the number of cells in the PI3KCA-knockdown HT-29 cell
group decreased by .93% (Figure 2E), while the
PI3KCAknockdown HCT-116 cells (Figure 2F) decreased by 92%.
inhibition of Pi3K/akt/mTOr suppressed
migration and invasion of hT-29 and
In order to investigate the effect of inhibiting PI3K/Akt/
mTOR pathway on HT-29 and HCT-116 cell migration
and invasion, a Transwell assay was carried out. PI3K/Akt/
mTOR inhibition by BEZ235 and PI3KCA knockdown both
resulted in a migration rate through an 8 µm polycarbonate
membrane filter dropped by 73% and 58% on HT-29 cell
and HCT-116 cell migration rates were reduced to 68%
and 55%, respectively (Figure 3A). The Transwell invasion
assay showed that BEZ235 and PI3KCA knockdown both
significantly decreased cell invasion in HT-29 (75%, 63%)
and HCT-116 (72%, 60%) cells (Figure 3B).
inhibition of Pi3K/akt/mTOr induced
hT-29 and hcT-116 cell apoptosis in vitro
To further evaluate the inhibitory effects of PI3K/Akt/mTOR
by BEZ235 and PI3KCA knockdown on cell apoptosis, flow
cytometry assay and Western blotting were performed. As
shown in Figure 4A, 16% of apoptotic cells were found in
the negative control group, but the proportions of apoptotic
cells in HT-29 cells were significantly increased to 35% and
26% with treatment of BEZ235 and PI3KCA knockdown,
respectively. In HCT-116 cells, BEZ235 treatment and
PI3K knockdown also promoted apoptosis (Figure 4A).
The expression levels of proteins associated with apoptosis
changed, with the expression of cleavage caspase 3 and Bax
increasing while Bcl-2 and Bim decreasing significantly
Colon cancer is the third most common cancer in the world
and has not yet been adequately treated.21 Current clinical
routine treatment for colon cancer includes surgical resection
of primary tumor, chemotherapy, radiotherapy, and
immunotherapy, with limitations and side effects. Drug resistance
also affects the treatment efficacy.22,23 The PI3K/Akt/mTOR
pathway is closely related to the pathogenesis and process
of cancer and is involved in drug resistance.3–8 Therefore,
inhibition of PI3K/Akt/mTOR signaling pathway may be of
great significance to the treatment of cancer.
Activated Akt will promote cell growth and survival by
inhibiting the pro-apoptotic proteins of Bcl-2 family.24 mTOR
is a serine/threonine kinase that can be induced by the activity
of activated Akt.25 Activated mTOR is involved in gene
transcription and protein translation. It has been proven that
Akt and mTOR have strong expression in colon cancer tissue
and HT-29 cells.26 The PI3K/Akt/mTOR pathway is a crucial
regulator in cell growth, including proliferation, growth,
differentiation, and survival. Therefore, abnormal activation of
PI3K/Akt/mTOR signaling pathway is commonly involved
in the development and progression of various tumors.27–31
PI3K/Akt/mTOR signaling pathway is upregulated and
plays an important role in colon cancer.2–4,32,33 In addition, a
large number of studies have shown that this pathway is also
related to drug resistance.2,5–7 Thus, targeting the PI3K/Akt/
mTOR pathway may be a promising technique for treating
BEZ235 is a novel dual inhibitor of both PI3K and
mTOR and has been used in many research studies.29
Several previous reports in these studies suggested that,
in the form of apoptosis or autophagy in tumors, the
treatment of BEZ235 can inhibit cell proliferation and induce
cell death autophagy.11–14,34 It has been proven that BEZ235
suppresses HCT-116 cell proliferation.16 Hong confirmed
that by inducing apoptosis, BEZ235 has inhibitory effects
on HT-29 and HCT-116.35 In our studies, we found that
BEZ235 can effectively inhibit the activation of PI3K/Akt/
mTOR pathway, cell proliferation, migration, invasion, and
induced apoptosis in HT-29 and HCT-116 cells (Figures 1, 3,
and 4). Inhibition of PI3K/Akt/mTOR pathway by PI3KCA
knockdown can also reduce cell proliferation, migration,
invasion, and induced apoptosis (Figures 2–4). Therefore,
inhibition of PI3K/Akt/mTOR pathway may significantly
inhibit cell progression in colon cancer.
Caspase 3 is a key member of the cysteine-aspartic acid
protease family, an important protease that causes
apoptosis.36 Bcl-2 and Bax are important regulators of apoptosis,
involved in the regulation of cell death.37 Bcl-2 is an
antiapoptosis protein that is localized in mitochondria, while
Bax is a pro-apoptotic protein that mainly resides in the
cytoplasm. The ratio of Bcl-2/Bax determines cell survival,
as a reduction in the ratio promotes cell apoptosis.38,39 Bim
(cell death mediated by Bcl-2) is another important member
of the Bcl-2 family and has a similar role to Bax in
promoting cell apoptosis.40 The present study shows that through a
downregulation process of Bcl-2 and Bim expressions,
inhibition of PI3K/Akt/mTOR pathway by BEZ235 and PI3KCA
knockdown induced HT-29 and HCT-116 cells apoptosis, at
the same time keeping an upregulation in cleavage caspase 3
and Bax (Figure 4B).
In summary, this study reveals that targeted inhibiting
PI3K/Akt/mTOR pathway by BEZ235 and PI3KCA
knockdown could inhibit cell proliferation, survival, and invasion
in human colon cancer. Therefore, in addition to surgery,
chemotherapy, and radiotherapy, targeted inhibiting PI3K/
Akt/mTOR pathway may be a valuable technique for human
colon cancer therapy.
This work was supported by the Science and Technology
Plan of Shandong (Grant No 2014GSF118008).
The authors report no conflicts of interest in this work.
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OncoTargets and Therapy is an international, peer-reviewed, open
access journal focusing on the pathological basis of all cancers, potential
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