Isoorientin induces apoptosis, decreases invasiveness, and downregulates VEGF secretion by activating AMPK signaling in pancreatic cancer cells
OncoTargets and Therapy
isoorientin induces apoptosis, decreases invasiveness, and downregulates Veg F secretion by activating aMPK signaling in pancreatic cancer cells
Tingting Ye 1 2
Dinglai Yu 1 2
Mengtao Zhou 1 2
0 Zhejiang Provincial Top Key Discipline in surgery, Wenzhou Key laboratory of surgery , Wenzhou, Zhejiang Province, People's r epublic of china
1 Department of s urgery, The First a ffiliated h ospital, Wenzhou Medical University
2 Bicheng chen
Isoorientin (or homoorientin) is a flavone, which is a chemical flavonoid-like compound, and a 6-C-glucoside of luteolin. Isoorientin has been demonstrated to have anti-cancer activities against various tumors, but its effects on pancreatic cancer (PC) have not been studied in detail. In this study, we aim to investigate whether isoorientin has potential anti-PC effects and its underlying mechanism. In PC, isoorientin strongly inhibited the survival of the cells, induced cell apoptosis, and decreased its malignancy by reversing the expression of epithelialmesenchymal transition and matrix metalloproteinase and decreased vascular endothelial growth factor expression. Meanwhile, we investigated the activity of the AMP-activated protein kinase (AMPK) signaling pathway after isoorientin treatment, which was forcefully activated by isoorientin, as expected. In addition, in the PC cells that were transfected with lentivirus to interfere with the expression of the gene PRKAA1, there were no differences in the apoptosis rate and the expression of malignancy biomarkers in the tumors of the isoorientin-treated and untreated groups. Thus, we demonstrated that isoorientin has potential antitumor effects via the AMPK signaling pathway, and isoorientin merits further investigation.
pancreatic cancer; AMPK; isoorientin; apoptosis; invasiveness; VEGF
open access to scientific and medical research
O r i g i n a l r e s e a r c h
Pancreatic cancer (PC) is one of the most common malignancies of the digestive system
worldwide, and its incidence has increased over the last several decades. It is the
fourth leading cause of death by cancer in the world, and its 5-year relative survival is
currently 8%.1,2 The high mortality rate is caused by its aggressive biological
properties, late symptom onset, and lack of specific treatments.3,4 Therefore, it is necessary
to learn more about PC at the molecular level and identify a new potential therapeutic
target for anticancer drugs.
In recent years, with the universal upsurge in the study of anticancer drugs, herbal
medicine has generated much attention. A significant number of studies have indicated
that many herbal medicines that have antitumor effects can be separated into several
compounds, including isoorientin.5–7 Isoorientin (or homoorientin) is a flavone, which is
a chemical flavonoid-like compound, and a 6-C-glucoside of luteolin. Bioassay-directed
fractionation techniques led to the isolation of isoorientin as the main hypoglycemic
component in Gentiana olivieri.8 Our team confirmed that luteolin has a strong
anticancer effect in PC.9 The effects of isoorientin, as a 6-C-glucoside of luteolin, on cancer
need to be identified. With many biological activities and therapeutic effects, including
anti-inflammatory, antidiabetic, antioxidant, proapoptotic, and autophagy-inducing
effects, we believe that isoorientin has the potential to treat
inflammatory and neoplastic diseases.10–14 Thus, we
investigated whether isoorientin has potential antitumor effects on
PC and its underlying mechanism.
AMP-activated protein kinase (AMPK) is a sensor of the
cellular energy status and can be found in all types of
eukaryotes, even in very primitive ones, such as Giardia lamblia.15
8 AMPK is a heterotrimeric complex, including a catalytic α
-021 subunit and regulatory β and γ subunits. Every subunit has
l-Ju 2–3 types (α1, α2, β1, β2, γ1, γ2, and γ3); hence, there are
n21 at least 12 types of AMPKs.16 Under specific circumstances,
2o1 AMPK appears to have different capacities as a tumor
suppres.591 sor or a tumor promoter.17–24 AMPK activators, such as
met..32 formin, phenformin, and 5-aminoimidazole-4-carboxamide
213 ribonucleotide (AICAR), exhibit antineoplastic effects on
/yb many cancers (acute myelocytic leukemia, renal cell
car.com cinoma, breast cancer, malignant melanoma, PC, thyroid
ss cancer, glioblastoma, colon cancer, etc.) in vivo or in
rvpee l.yno vitro.25–29 It has been noted that activated AMPK inhibits
.dow lsue cell proliferation and kills cancer cells via the induction of
/ww ano apoptosis. Furthermore, AMPK is required for the induction
ttp pe of the epithelial–mesenchymal transition (EMT), which is
h ro an important process that contributes to cancer metastasis.30
from F Moreover, metformin can reverse multidrug resistance in
eadd human breast cancer cells by activating AMPK.31
lon Although several aspects of the mechanisms of AMPK
odw in cancer have been studied, the pathophysiological role of
ryap AMPK in PC has not been fully elucidated. In this study,
heT our aim is to explore the medicinal benefits of isoorientin on
adn PC and its relationship with AMPK, and our study focused
tseg on its effects on the cell proliferation, apoptosis, cell
migraraT tion, and invasion of the human PC cell lines PANC-1 and
Materials and methods
Fetal bovine serum (FBS) was purchased from Sigma
Chemical (St Louis, MO, USA). Roswell Park Memorial
Institute (RPMI)-1640 (11875093), Dulbecco’s Modified
Eagle’s Medium (DMEM), and trypsin were purchased from
Gibco (Grand Island, NY, USA). The anti-AMPK (64 kDa,
ab80039), anti-phospho-AMPK (64 kDa, ab133448),
antiE-cadherin (97 kDa, ab133597), anti-N-cadherin (125 kDa,
ab18203), anti-VEGF (42 kDa, ab46154), anti-matrix
metalloproteinase (MMP) 2 (75 kDa, ab86607), and anti-MMP9
(92 kDa, ab76003) antibodies were purchased from Abcam
(Cambridge, UK). The anti-Bax (20 kDa, 5023s) and
antiBcl-2 (26 kDa, 4223s) antibodies were purchased from Cell
Signaling Technology (Danvers, MA, USA). The anti-GAPDH
antibody (36 kDa, MB001) was purchased from Bioworld
Technology (St Louis Park, MN, USA). Polyvinylidene
difluoride (PVDF) membranes were purchased from Millipore
(Billerica, MA, USA). Power SYBR Green PCR Master Mix
was purchased from Applied Biosystems (Foster City, CA,
USA), and a RevertAid First Strand cDNA Synthesis Kit was
purchased from Thermo Fisher Scientific (Manassas, VA,
USA). PCR primers were purchased from Generay Biotech
(Shanghai, People’s Republic of China). Human VEGF
enzyme-linked immunosorbent assay (ELISA) kit was
purchased from NeoBioscience (Shenzhen, People’s Republic of
China), and isoorientin (E-1060) was purchased from Tauto
Biotech (Shanghai, People’s Republic of China).
The human PC cell line PANC-1 cell was obtained from the
Cell Bank of the Chinese Academy of Sciences (Shanghai,
People’s Republic of China), and the human PC cell line
PATU-8988 was provided by the American Type Culture
Collection (Manassas, VA, USA). PANC-1 was cultured with
DMEM and PATU-8988 was cultured with RPMI-1640 at
37°C with 5% CO2. Both media contained 100 U/mL
penicillin, 10% FBS, and 100 μg/mL streptomycin, and it was
changed every 2 days. When cells were ~80% confluent,
we detached the cells with 0.25% trypsin–0.02%
ethylenediaminetetraacetic acid for subculture or for the following
PANC-1 and PATU-8988 cells were plated into 6-cm
culture dishes. When the cells reached 70%–90% confluence,
isoorientin was added to the FBS-free medium for 24 hours.
Then, the cells were prepared for subsequent experiments,
such as extracting protein, isolating RNA, and transwell
silencing the expression of the gene
Lentivirus vectors containing the short hairpin RNA
(shRNA) of PRKAA1 (protein kinase AMP-activated
catalytic subunit alpha 1, sequence:
GCTTGATGCACACATGAAT) and their negative control were designed and
produced by GeneChem (Shanghai, People’s Republic of
China). First, PANC-1 and PATU-8988 cells were seeded
in six-well plates (5×104 cells) and allowed to grow until
nearly 30%–40% confluent. After the culture medium was
discarded, the six-well plates were washed with
phosphatebuffered saline (PBS) three times. Then, each of the plates was
filled with a transfection-enhancing solution with lentivirus
(2 multiplicity of infection for PANC-1 and 10 multiplicity
of infection for PATU-8988) and 10 μg/mL polybrene. After
8–12 hours of incubation and transfection at 37°C with 5%
CO2, we replaced the transfection medium with the common
culture medium. When the cells reached 80% of the plate,
the cells were harvested for passage or experiments. In all of
the operations we mentioned above, the cells were processed
using the proper biohazard safety equipment.
cell viability detection using a cell
counting Kit 8 assay
PANC-1 and PATU-8988 cells were plated onto 96-well
plates. Each well contained ~5,000 cells and 200 μL of the
medium with 10% FBS. When the cells of each well reached
70% confluency, the medium was changed, and FBS-free
medium with different concentrations of isoorientin was
added. After 24 hours, the cells were washed with PBS once,
the medium containing isoorientin was discarded, and 100 μL
of FBS-free medium with 10 μL of the Cell Counting Kit 8
(CCK8; Dojindo, Kumamoto, Japan) reagent were added.
The cells were incubated for another 1–2 hours at 37°C, and
the absorbance of each well was detected using an ELISA
reader (BioTek, Winooski, VT, USA) at 490 nm based on
the manufacturer’s instructions. Cell viability was expressed
as the fold change of absorbance.
Cells were plated in 6-cm culture dishes (5×105 cells per dish)
and treated with isoorientin (0, 20, 40, 80, and 160 μM) for
24 hours. The cell apoptosis rate was measured using the
Annexin V-FITC/PI Apoptosis Detection Kit (BD, Franklin
Lakes, NJ, USA), according to the manufacturer’s
instructions. After the treatments, the fluorescence intensity was
measured using a BD Accuri C6 (BD).
rna extraction and quantitative
realtime Pcr analysis
After the cells were treated as described earlier, the total RNA
was isolated by adding TRIzol (Ambion, Carlsbad, CA, USA)
in accordance with the manufacturer’s instructions. Then,
the RevertAid First Strand cDNA Synthesis Kit was used to
obtain cDNA from the RNA. Next, the quantitative real-time
(qRT)-PCR was performed using the SYBR Green Master
Mix in a 7500 Real-Time PCR System (Applied Biosystems).
β-Actin was amplified as an internal standard. An analysis
of the qRT-PCR data was performed using the ∆Ct values.
All the primer sequences are listed in Table 1.
Western blot analysis
After treatments, cells were lysed in
radioimmunoprecipitation assay buffer (Beyotime, Shanghai, People’s Republic
of China) containing 10% phosphatase inhibitor (Roche
Diagnostics GmbH, Mannheim, Germany) and 1%
phenylmethylsulfonyl fluoride (Beyotime) for 30 minutes. Then, the
cell lysate was centrifuged at 12,000×g , and the supernatant
was collected. The protein concentration of each group
was measured using a BCA Protein Assay Kit (Beyotime).
After denaturation, 50 μg of protein for each group was
then subjected to sodium dodecyl sulfate–polyacrylamide
gel electrophoresis, transferred to PVDF membranes and
incubated with specific antibodies overnight at 4°C. Protein
detection was performed using the enhanced
chemiluminescence reagent obtained from Thermo Fisher Scientific, and
the immunocomplexes were visualized using the
enzyme-linked immunosorbent assay
After the cells were treated properly, the culture medium
was collected. VEGF protein involved in the culture media
was directly measured by ELISA kit according to the
manufacturer’s instructions (NeoBioscience). The absorbance
was measured at 450 nm. The concentration of VEGF was
calibrated with the VEGF standard curve.
Transwell migration and invasion assay
According to the manufacturer’s protocol, transwell inserts
(Corning, Franklin Lakes, NJ, USA) with 8-μm pore
membrane filters were used for the migration and invasion assay.
Briefly, Matrigel Basement Membrane Matrix (Corning)
was laid in the upper chambers to test for invasion, but not
for migration. A volume of 500–600 μL RPMI-1640 (for
PATU-8988) or DMEM (for PANC-1) with 10% FBS as a
chemoattractant was added to the lower compartment. After
the cells were starved in FBS-free medium overnight, the cells
(migration: 5×103, invasion: 5×104) were placed in the upper
compartment (FBS-free medium) and incubated for 24 hours.
Then, we gently scrubbed off the cells on the upper surface with
a cotton swab, washed the filters with PBS three times, and
then fixed the cells with 4% paraformaldehyde for 20 minutes.
Subsequently, the cells were stained with a 0.05% crystal violet
solution, followed by three washes with PBS. Then, the inserts
were allowed to dry at room temperature, and the membranes
were photographed under an inverted microscope.
isoorientin inhibits cell proliferation
At the beginning of our study, we performed a CCK8 assay
to assess whether isoorientin affects the cell viability of
PANC-1 and PATU-8988 cells. Both cell lines were grown
for 24 hours in the presence of isoorientin (0, 20, 40, 80, and
160 μM), and a CCK8 solution was added. We found that the
cell viability decreased significantly at the concentrations of
20, 40, 80, and 160 μM (Figure 1A). Then, we performed a
CCK8 assay in the shRNA-interfering group. The cell
proliferation in the isoorientin-treated group was not significantly
different from that in the untreated group in both the PANC-1
and PATU-8988 cell lines (Figure 1B).
aMPK is constitutively activated by
After the cells were cultured with isoorientin (0, 20, 40, 80,
and 160 μM for PANC-1; 0, 20, 40, 80, 160, and 320 μM
for PATU-8988) for 24 hours, we assessed the expression
of p-AMPK and AMPK by Western blotting. After the
isoorientin treatment, the p-AMPK expression was increased
(Figure 2A). Then, in the shRNA group, we choose the
NC shRNA shRNA
– – +
Isoorientin (µM) WT –
NC shRNA shRNA
– – + Isoorientin
concentration of 80 μM to detect the effects of isoorientin.
As shown in Figure 2B, the expression levels of AMPK and
p-AMPK were much lower in the shRNA group than in the
wild-type PC cells (WT) and the group that was transfected
with a negative control lentivirus (NC). In the shRNA group,
with or without isoorientin treatment, the expression of
AMPK and p-AMPK was not different (Figure 2B).
Pc cells are susceptible to
Because AMPK is interrelated with the regulation of cell
apoptosis in a large variety of cancer cells, the effect of
isoorientin treatment was analyzed for up to 24 hours on the
cell apoptosis rate of PANC-1 and PATU-8988 cells. Except
for the cells that were cultured with or without isoorientin,
all cells were treated the same way. The isoorientin-induced
apoptotic response was increased in the normal PC group
(Figure 3A–C). In contrast, apoptosis was not significantly
different in the shRNA groups with or without isoorientin
treatment (Figure 3D–F). Thus, we can conclude that the
proapoptotic response of isoorientin was increased via the
activation of the AMPK signaling pathway.
isoorientin downregulates the expression
of VegF, MMPs, and the proteins involved
in the eMT
Since VEGF, MMPs, and EMT are associated with changes
in the survival, motility, migration, and invasion of tumors
and, thus their malignancy, we evaluated whether isoorientin
is able to regulate the expression of VEGF, MMPs, and EMT.
The cells were treated with isoorientin (0, 20, 40, 80, and
160 μM) for 24 hours, and the expression of VEGF, EMT
hallmarks (N-cadherin and E-cadherin), and MMP hallmarks
(MMP2 and MMP9) was evaluated by real-time PCR assay
and Western blotting. We also test the releasing VEGF
level in medium by ELISA. We found that PC cells treated
with isoorientin (20, 40, 80, and 160 μM) had significantly
reduced expression levels of VEGF, N-cadherin, MMP2, and
MMP9 and increased expression levels of E-cadherin both
at the mRNA and the protein levels (Figure 4). Meanwhile,
in the shRNA group, these changes were not observed
isoorientin inhibits the migration and
invasion of Pc cells
To further evaluate the effects of isoorientin on PC cell
migration and invasion, we utilized transwell migration/invasion
assays. The cells were plated onto the upper chamber of the
transwell system in the presence or absence of isoorientin
(20, 40, 80, and 160 μM) for 24 hours. After the cells were
stained with crystal violet nuclear dye, micrographs of the
cells migrating onto the lower surface of the transwell
membrane were obtained. Compared with the respective untreated
control, the density of the migrated cells, which were stained
purple, was clearly decreased in the groups treated with
isoorientin (20, 40, 80, and 160 μM; Figure 5A). In the
shRNA group, no significant difference was found between
the isoorientin-treated and untreated groups (Figure 5B).
PC is considered one of the most invasive tumor diseases, and it
has a very poor prognosis. The mortality rate will not decrease
significantly unless new chemotherapy, radiotherapy, and
biotherapy treatments emerge. It has been reported that AMPK
is a potential therapeutic target for tumor treatment. AMPK
is a ubiquitous and highly conserved sensor of the cellular
energy status, and it regulates cellular energy homeostasis
by phosphorylating various enzymes that are involved in
glucose, protein, and lipid metabolism.32 It has been reported
that increasing the activation of AMPK in papillary thyroid
cancer cell lines leads to a conspicuous antitumor response,
as measured by the inhibition of cell proliferation and
migration, the induction of cell death, and the reversal of the EMT.33
Figure 4 (Continued)
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Furthermore, the exposure of cancer cells to OSU-53, a novel
the AMPK signaling pathway. In this study, we choose the
AMPK activator, was sufficient to reverse the EMT in those
PANC-1 and PATU-8988 cell lines to test the antitumor
cells.34 Several other AMPK activators, such as metformin,
capacity of isoorientin. We found that isoorientin can lower
phenformin, and AICAR, have been shown to regulate cell
the malignancy of PC cells, at least partly, by activating the
survival and metastasis in a variety of cancer types.25–31,35
AMPK signaling pathway, further demonstrating isoorientin’s
Herbal medicine has become a hot topic in the search of
potential as a new therapeutic agent for PC.
antitumor agents. Many studies have noted that various herbal
Apoptosis plays a vital role in the progress of cell
prolifermedicines that have antitumor effects can be separated into
ation, differentiation, senescence, and death. Previous studies
several compounds, including isoorientin.5–7 Flavonoids, such
have concluded that BCL-2 and BAX control programmed
as baicalin, quercetin, luteolin, and epigallocatechin, have
cell death (apoptosis).38–41 Therefore, we determined the levels
been shown to activate the AMPK signaling pathway.36,37
of BCL-2 and BAX after isoorientin treatment and found that
Here, we hypothesized that, as a flavone, isoorientin could
isoorientin effectively inhibited the expression of BCL-2,
induce cancer cell death and lower tumor malignancy via
whereas a contrary trend was observed in the expression
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of BAX. To further examine the apoptotic effect of isoorientin,
we examined the apoptosis rates by flow cytometry.
To some extent, the biological behaviors of cancer cell
malignancy are invasiveness and high vascularization. EMT
is a biological process in which epithelial cells transform into
special cells with mesenchymal phenotypes, and these cells
can be isolated during cancer metastasis. Previous studies
have found that changes in the EMT are often followed with
increased expression of MMPs.42,43 Thus, we determined the
levels of E-cadherin, N-cadherin, MMP2, and MMP9 after
isoorientin treatment and found that isoorientin efficaciously
inhibited the expression of N-cadherin, MMP2, and MMP9,
while the level of E-cadherin was increased. The EMT and
the degradation of the extracellular matrix make cancer cell
migration possible.44,45 We performed a transwell invasion
and migration assay to further validate the inhibitory effect
of isoorientin on cancer cell invasiveness in vitro. VEGF is
one of the most effective proangiogenic growth factors. The
inhibition of VEGF can suppress tumor growth in vivo.46
Moreover, VEGF can promote the migration and
invasion capacity of tumor cells, resulting in an increase in the
degree of the tumor cell malignancy.47,48 In our experiments,
VEGF was significantly reduced by isoorientin treatment in
PC cell lines, both at the mRNA and protein levels. All the
data demonstrated that isoorientin can decrease the
malignancy of PC. More importantly, in this study, we observed
that the phosphorylation of AMPK was accompanied by
the upregulation of E-cadherin and the downregulation of
Figure 5 (Continued)
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VEGF, N-cadherin, and MMPs, similar to apoptosis. This
suggested that isoorientin lowers malignancy and induces
the apoptosis of PC cell through the activation of the AMPK
Although the antitumor effects of AMPK were tested in
our experiments, the specific mechanism by which AMPK
regulates cell apoptosis, migration, and invasion was not
fully demonstrated in this article. In many reports, it has
been confirmed that mTOR, which is negatively regulated
by AMPK, is involved in cancer cell death.49,50 Similarly, the
tumor suppressors TSC251–54 and p5354–57 are downstream
targets of AMPK activity. Intriguingly, a survey of the literature
available shows that the activation of AMPK is a necessary
but not sufficient condition for the induction of the EMT.30
Altogether, it is certain that the relationship of isoorientin
with the antitumor effect of AMPK and their relationship
with other signaling pathways are very complex and need
to be further investigated.
In conclusion, our study shows that isoorientin can inhibit
the expression of VEGF, VEGF, N-cadherin, MMP2, MMP9,
This research was supported by grants from the National
Natural Science Foundation of China (Nos 81370563 and
81570583), the Outstanding Youth Fund of Zhejiang Province
(No LR14H30001), and the Health and Family Planning
Commission of Zhejiang Province (2013ZDA014).
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
targets for therapy and treatment protocols employed to improve the
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of management programs and new therapeutic agents and protocols on
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