European Journal of Pharmacology 893 (2021) 173840

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European Journal of Pharmacology

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Effects of atorvastatin in combination with celecoxib and tipifarnib on

proliferation and apoptosis in pancreatic cancer sphere-forming cells
Xue-Tao Xu a, b, Jie Chen a, Xiang Ren a, Yu-Ran Ma a, Xiao Wang a, Yan-Yan Ma a, b,
Den-Gao Zhao a, b, Ren-Ping Zhou c, Kun Zhang a, b, Susan Goodin d, Dong-Li Li a, b, Xi Zheng c, d, *
a
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, 529020, PR China
b
International Healthcare Innovation Institute (Jiangmen), Jiangmen, 529020, PR China
c
Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA
d
Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 08903, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Cancer stem cell (CSC) plays an important role in pancreatic cancer pathogenesis and treatment failure. CSCs are
Pancreatic cancer characterized by their ability to form tumor spheres in serum-free medium and expression of CSC related
Atorvastatin markers. In the present study, we investigated the effect atorvastatin, celecoxib and tipifarnib in combination on
Celecoxib
proliferation and apoptosis in Panc-1 sphere-forming cells. The sphere-forming cells were isolated from Panc-1
Tipifarnib
Stem cells
cells by sphere-forming method. These sphere-forming cells showed CSC properties. The levels of CD44, CD133
Chemoresistant and ALDH1A1 in the sphere-forming cells were increased. Moreover, Panc-1 sphere-forming cells were resistant
Apoptosis to chemotherapeutic drug gemcitabine. Combined atorvastatin with celecoxib and tipifarnib synergistically
decreased the sphere forming ability of Panc-1 cells and the drug combination also strongly inhibited cell pro­
liferation and promoted apoptosis in the sphere-forming cells. The effects of the drug combination on the Panc-1
sphere-forming cells were associated with decreases in the levels of CD44, CD133 and ALDH1A1, and sup­
pression of Akt and NF-κB activation. Results of the present study indicate that the combination of atorvastatin,
celecoxib and tipifarnib may represent an effective approach for inhibiting pancreatic CSCs.

1. Introduction
Pancreatic cancer, one of the leading causes of cancer-related death
in Western countries, has very poor prognosis (Ferlay et al., 2015; Siegel
et al., 2019). This disease is usually diagnosed at advanced stages when
little effective therapies are available (Stathis and Moore, 2010). About
60% of patients exhibit distant metastasis within 24 months after sur­
gery and the 5-year survival rate of pancreatic cancer patients is still low
(O’Reilly and Abou-Alfa, 2007; Stathis and Moore, 2010; Yan et al.,
2019). Chemotherapy with gemcitabine remains the standard treatment
for patients with advanced pancreatic cancer. Gemcitabine has a tumor
response rate of approximately 15% and treatment of patients with
gemcitabine extends survival by a mere 5 weeks on average (Wang et al.,
2011; Ina et al., 2010). The development of resistance to gemcitabine
and severe side effects associated with gemcitabine-based chemotherapy
limits its clinical success. Clearly, it holds high clinical significance to
develop novel approach for effective treatment of pancreatic cancer.
The discovery of stem cells for multiple malignant tumors has great
implications for study of tumor progression and therapy development.
The subpopulation of cancer cells commonly known as tumor-initiating
cells, or cancer stem cells (CSCs), plays a critical role in tumorigenesis
and is present in various types of cancer (Bjerkvig et al., 2005). CSCs are
characterized by extensive abilities in self-renewal and differentiation as
unidirectional cellular hierarchies and may contribute to tumor pro­
gression, recurrence and metastasis (Ajani et al., 2015; Vinogradov and
Wei, 2012; Reya et al., 2001). These characteristics suggest that CSCs
could be a critical target for cancer therapy. Development of novel
therapies that inhibit CSCs would hold great promises in improving the
clinical management of pancreatic cancer.
In an earlier study, our laboratory found that a combination of
atorvastatin (Fig. 1), celecoxib (Fig. 1) and tipifarnib (Fig. 1) strongly
inhibited the growth and induced apoptosis in human pancreatic cancer
cells (Ding et al., 2014). Atorvastatin is a statin drug that inhibits the
enzyme HMG-CoA reductase and thus decreased the cholesterol

* Corresponding author. Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersy, Piscataway, NJ, 08854,
USA
E-mail address: xizheng@pharmacy.rutgers.edu (X. Zheng).

https://doi.org/10.1016/j.ejphar.2020.173840
Received 2 June 2020; Received in revised form 21 December 2020; Accepted 22 December 2020
Available online 24 December 2020
0014-2999/© 2020 Elsevier B.V. All rights reserved.
X.-T. Xu et al.
Fig. 1. Structures of atorvastatin, celecoxib and tipifarnib.
biosynthesis (Farnier and Davignon, 1998). Celecoxib is a selective
cyclooxygenase-2 (COX-2) inhibitor and has been shown to inhibit
pancreatic cancer (Hawkey, 1999; Zuo et al., 2018). Tipifarnib is a se­
lective non-peptidomimetic inhibitor of farnesyltransferase (Karp et al.,
2009). Preclinical studies demonstrated that tipifarnib competitively
inhibits farnesylation of K-ras peptides at nanomolar concentrations.
Tipifarnib also inhibits proliferation of pancreatic cancer cells cultured
in vitro and grown as xenograft tumors in immunodeficient mice (Siegel
et al., 2013).
The present study was aimed to explore the effects and mechanisms
of the combination of atorvastatin, celecoxib and tipifarnib on pancre­
atic cancer sphere-forming cells. The Panc-1 sphere-forming cells were
isolated by the suspension sphere forming method. Our study demon­
strated for the first time that a combination of atorvastatin, celecoxib
and tipifarnib strongly inhibited the formation of Panc-1 spheres,
inhibited proliferation and promoted apoptosis in sphere-forming cells.
The effects of the triple combination on Panc-1 sphere-forming cells
were associated with suppression of Akt and NF-κB activation.
2. Materials and methods
2.1. Cells culture
Human pancreatic cancer cell line Panc-1 was purchased from the
American Type Culture Collection (ATCC, Rockville, MD, USA). Panc-1
cells sustained exponential growth by culturing in DMEM medium
supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY,
USA), penicillin (100 units/ml), streptomycin (100 mg/ml), and L-
glutamine (300 μg/ml). The cells were grown at 37 ◦ C in a humidified
atmosphere of 5% CO2. Atorvastatin and celecoxib were obtained from
Sigma-Aldrich (St. Luis, MO, USA). Tipifarnib was kindly provided by
J&J Co. Ltd. Atorvastatin, celecoxib and tipifarnib were dissolved in
DMSO, and the final concentration of DMSO in all experiments was
0.1%.
2.2. Determination of the number of viable cells
The number of viable cells after each treatment was determined by
using the trypan blue exclusion assay. After each experiment, single cell
suspension was made by trypsinized the cells. The trypan blue exclusion
assay was performed by mixing 80 μl of cell suspension and 20 μl of 0.4%
trypan blue solution for 2 min. Blue cells were marked dead and the cells
that did not absorb dye were marked alive. We used a hemacytometerto
count the number of viable cells under a Nikon light microscope
(Optiphot, Nikon, Tokyo, Japan).
2.3. Assessment of apoptotic cells
Apoptosis was determined by morphological assessment in cells
stained with propidium iodide. After each experiment, cells were tryp­
sinized and cytospin slides were prepared. The cells were fixed with
acetone/methanol (1:1) for 10 min at room temperature, followed by 10
min with propidium iodide staining (1 μg/ml in PBS), and finally
analyzed using a Nikon fluorescence microscope (Eclipse TE200, Nikon,
Tokyo, Japan). Apoptotic cells were identified by their classical
morphological features such as nuclear condensation, cell shrinkage,
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European Journal of Pharmacology 893 (2021) 173840
and formation of apoptotic bodies.
2.4. Caspase-3 activity assay
Caspase-3 activation was measured using an EnzoLyte AMC Caspase-
3 Assay Fluorimetric kit (AnaSpec, Fremont, CA) following the manu­
facturer instructions. Briefly, 1 × 105 cells were plated in triplicate in a
flat-bottomed 96-well plate. After drug treatment, caspase-3 substrate
was added to each well. Plates were incubated for 30 min at room
temperature. Fluorescence intensity was measured in a Tecan Infinite
M200 plate reader (Tecan US Inc., Durham NC).
2.5. Panc-1 sphere-forming cells
Isolation of Panc-1 sphere-forming cells was carried out using the
sphere forming assay as described elsewhere. Briefly, Panc-1 cells (1000
cells/mL) were seeded in a 24-well ultralow attachment surface culture
plate (Corning Co, NY, USA) and cultured in DMEM/F12 serum free
medium supplemented with basic fibroblast growth factor, epidermal
growth factor, insulin, selenium, transferrin, and bovine serum albumin
for 12 days. For the comparison of sphere-forming efficiency of Panc-1
cells and the sphere-forming cells, Panc-1 cells and sphere-forming cells
(collected from the 12-day sphere culture as described above) were
seeded in ultralow attachment plate and cultured in DMEM/F12 serum
free medium with atorvastatin, celecoxib and tipifarnib alone or in
combination for 12 days, and the number of spheres were counted in the
end of the experiment.
2.6. Immunofluorescence staining
The cells were fixed in 4% paraformaldehyde for 30 min and then
treated with 0.1% Triton X-100 for 10 min at room temperature. After
blocking with phosphate-buffered saline (PBS) containing 5% bovine
serum albumin for 2 h, the cells were incubated with antibodies against
CD44 (#3570, Cell Signaling Co., Beverly, MA, USA) and caspase-3
(active form, AF835, R&D System, Minneapolis, MN, USA) overnight
at 4 ◦ C. After washing with PBS, the cells were incubated with FITC-
conjugated and Rhodamine-conjugated secondary antibody for 3 h at
room temperature. The cells were counterstained with DAPI and
examined using a fluorescence cell imager (Nikon Eclipse TE200, Tokyo,
Japan).
2.7. Western blotting
After treatment, the cell lysates were prepared (Ponta et al., 2003).
Proteins were subjected to sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose mem­
branes. After blocking nonspecific binding sites with blocking buffer, the
membranes were incubated overnight at 4 ◦ C with primary antibodies
(#3570 for CD44, #5860 for CD133, #12035 for ALDH1A1, #3031 for
p-p65, #6956 for p65, #9271 for p-Akt and #4691 for Akt, Cell
Signaling Co., Beverly, MA, USA). β-actin (sc-47778, Santa Cruz
Biotechnology Inc., Dallas, TX, USA) was used as a loading control.
Following the removal of the primary antibodies, the membranes were
then washed three times with TBS (PBS containing 0.05% Tween 20)
buffer at room temperature and later incubated with
fluorochrome-conjugated secondary antibody (925–32,211, Li-Cor
Biotechnology, Lincoln, NE, USA). The membrane was then washed
with TBS three times. Final detection was done with an Odyssey infrared
imaging system (Li-Cor Biotechnology).
2.8. Statistical analyses
Statistical analyses were done by using the software InStat (Graph­
Pad Software, Inc., La Jolla, CA, USA). Comparisons of treatment
outcome were analyzed for statistical difference by ANOVA. Statistical
X.-T. Xu et al.
significance was assumed at a value of P < 0.05. Analysis of synergy was
performed using CompuSyn software. The combination index-fraction
affected (CI-Fa) curve demonstrates the relationship between the CI
value and the effective level of a certain biological indicator. If the CI is
< 1, then the compounds are considered to have synergistic effects. If the
CI is > 1 or = 1, then the compounds are thought to have an antagonistic
or additive manner.
3. Results
3.1. Sphere formation of Panc-1 cells
Cancer stem cells (CSCs) are characterized by their ability to form
three dimensional tumorspheres in vitro, and the sphere formation
method is widely used in the isolation and enrichment of CSCs in vitro. In
our studies, Panc-1 cells cultured in DMEM medium supplemented with
10% FBS showed adherent cell culture morphology (Fig. 2A). The Panc-
1 cells cultured in ultralow attachment plate with serum free medium
formed spheres (Fig. 2B).
We next investigated the expression of CSC related molecular
markers in Panc-1 sphere forming cells. The levels of CD133, CD44 and
ALDH1A1 in parental Panc-1 cells and Panc-1 sphere-forming cells were
determined by Western blotting using corresponding antibodies. Results
of the experiments showed that the abundances of CD133, CD44 and
ALDH1A1in the sphere-forming cells were higher than the parental
Panc-1 cells (Fig. 2C). Since CSCs are resistant to chemotherapeutic
drug, the Panc-1 sphere-forming cells were also tested for their sensi­
tivity to gemcitabine which is a commonly used chemotherapeutic drug
for pancreatic cancer treatment. As shown in Fig. 2D, the effect of
gemcitabine on decreasing the cell viability was significantly stronger in
the parental Panc-1 cells than the sphere-forming cells (P < 0.001)
indicating that the sphere-forming cells were partially resistant to
gemcitabine-induced growth inhibition. Taken together, the results
described above demonstrated that the sphere-forming cells derived
from Panc-1 cells through sphere-forming method exhibited both the
classical cellular and molecular properties of pancreatic CSCs.
Fig. 2. Panc-1 cells and the Panc-1 sphere-forming cells. (A) Representative
micrograph of Panc-1 cells cultured in DMEM medium with 10% FBS. (B)
Representative micrograph of Panc-1 spheres cultured in ultralow attachment
plate with serum free medium. (C) Protein levels of CSC related markers CD44,
CD133, ALDH1A1 in PCs and the SFCs. (D) Effects of gemcitabine (GEM; 5 μM)
on the viability of PCs and SFCs. Superscript a indicates significant difference
(P < 0.001) as compared to the parental Panc-1 cells.
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European Journal of Pharmacology 893 (2021) 173840
3.2. Inhibitory effect of atorvastatin, celecoxib and tipifarnib on sphere
formation
To explore whether atorvastatin, celecoxib and tipifarnib could exert
suppressive effect on formation of spheres, Panc-1 cells were cultured in
ultralow attachment plate and treated with these drugs alone or in
combination at different ratios. As shown in Fig. 3A, atorvastatin, cel­
ecoxib or tipifarnib alone dose-dependently suppressed the formation of
spheres in Panc-1 cells. Combinations of atorvastatin, celecoxib and
tipifarnib had stronger effect on suppressing the formation of spheres
than each drug used alone (Fig. 3A). Analysis of synergy based on iso­
bologram method showed that the triple combination synergistically
decreased the number of spheres in Panc-1 cells (Fig. 3B and C). The
combination index (CI) was 0.49 for the combination of atorvastatin (5
μM), celecoxib (5 μM) and tipifarnib (0.2 μM). The CI was 0.61 for the
combination of 2 μM atorvastatin, 2 μM celecoxib and 0.1 μM tipifarnib,
and the CI was 0.89 for the combination of 10 μM atorvastatin, 10 μM
celecoxib and 0.5 μM tipifarnib (Fig. 3C). Since the combination with
the ratio of 5: 5: 0.2 (μM) had the lowest CI, we used this dose ratio for
the combination in subsequent experiments. We further determined the
effect of atorvastatin, celecoxib or tipifarnib alone or in combination on
the sphere formation in Panc-1 sphere-forming cells. As shown in
Fig. 3D, the drug combination strongly inhibited the sphere formation in
the sphere-forming cells. The inhibitory effect of the combination sphere
Fig. 3. Inhibition of sphere formation by atorvastatin, celecoxib and tipifarnib.
Panc-1 cells were cultured in ultralow attachment plate with serum free me­
dium and treated with atorvastatin (LIP), celecoxib (CEL) and tipifarnib (ZAN)
alone or in combination for 12 days. The number of spheres was counted under
microscope. (A) Sphere formation in Panc-1 cells treated with LIP, CEL and ZAN
alone or in combination. (B) Effect of LIP, CEL and ZAN alone or in combina­
tionon sphere formation plotted as fraction affected (Fa). (C) Combination
index (CI) for different ratios of the three drugs in combination. (D) Effects of
LIP, CEL and ZAN alone or in combinationon sphere formation as compared
with negative control (NC) in Panc-1 cells (PCs) and the Panc-1 sphere forming
cells (SFCs). Each value represents mean ± S.D from three separate
experiments.
X.-T. Xu et al.
formation was similar in parental Panc-1 cells and Panc-1 sphere-
forming cells. Panc-1 cell population is heterogenous with a small
fraction of cancer stem cells while sphere-forming cells are enriched
with cancer stem cells. Our result of similar inhibitory effects of the
combination on sphere formation in Panc-1 and the sphere-forming cells
indicate that the combination consistently target the cancer stem cells.
3.3. Decrease in cell proliferation and increase in apoptosis in Panc-1
sphere-forming cells treated with atorvastatin, celecoxib and tipifarnib
To investigate the potential inhibitory effect of atorvastatin, cele­
coxib and tipifarnib on pancreatic CSCs, proliferation and apoptosis in
sphere-forming cells treated with these drugs alone or in combination
were determined. As shown in Fig. 4A, a strong decrease in the number
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European Journal of Pharmacology 893 (2021) 173840
of viable cells was observed in the Panc-1 sphere-forming cells after
treatment with atorvastatin, celecoxib and tipifarnib in combination
while each drug alone only had small effect on decreasing the number of
viable cells. The levels of proliferating cell nuclear antigen (PCNA) and
Cyclin D1 were also decreased in the sphere-forming cells treated with
the triple combination (Fig. 4B). Effect of the combination treatment on
apoptosis was determined by propidium iodide staining assay and
caspase-3 assay. As shown in Fig. 4C, treatment of the sphere-forming
cells with the combination resulted in a strong increase in the number
of apoptotic cells. Result of the caspase-3 assay also showed that the
combination strongly increased apoptosis in the sphere-forming cells
(Fig. 4D). Fig. 4E shows representative micrographs of immunofluo­
rescence staining in Panc-1 sphere-forming cells. Cells in the control
group showed positive staining with CSC marker CD44 and negative
Fig. 4. Effects of atorvastatin (LIP), celecoxib (CEL)
and tipifarnib (ZAN) alone or in combination (COM)
on proliferation and apoptosis in Panc-1 sphere-
forming cells. Cell viability was measured by the
trypan blue assay. PCNA and Cyclin D1 were deter­
mined by Western blotting. Apoptosis was deter­
mined by propidium iodide staining and caspase-3
assay. (A) Percent viable cells. (B) Expression levels of
PCNA and Cyclin D1. (C) Percent apoptotic cells. (D)
Caspase-3 activity. (E) Immunofluoresence staining of
CD44 and caspase-3 in the control and the
combination-treated cells. (F) Percent viable cells
Each value represents mean ± S.D from three sepa­
rate experiments. Superscript a indicates significant
differences (P < 0.001) as compared to cells treated
with LIP, CEL or ZAN alone. Superscriptb indicates
significant difference (P < 0.01) as compared to cells
treated with the triple combination (COM).
X.-T. Xu et al.
staining of caspase-3. The cells treated with the combination had posi­
tive staining for both CD44 and caspase-3 indicating apoptosis in CSCs.
The results described above indicated that the triple combination
inhibited proliferation and induced apoptosis in Panc-1 CSCs cells. Since
the sphere-forming cells were partially resistance to gemcitabine, we
further determined if the triple combination could enhance the growth
inhibitory effect of gemcitabine. As shown in Fig. 4F, atorvastatin, cel­
ecoxib or tipifarnib alone did not enhance the effect of gemcitabine on
cell viability. However, the drug combination significantly enhance the
growth inhibitory effect of gemcitabine (P < 0.01; Fig. 4F).
3.4. Down regulation of CD133, CD44 and ALDH1A1 in Panc-1 sphere-
forming cells treated with the triple combination
To evaluate the effect of atorvastatin, celecoxib and tipifarnib on the
stemness of the sphere-forming cells, we examined the levels of CSC
related markers CD133, CD44 and ALDH1A1 in these cells. In these
experiments, the sphere-forming cells were treated with the combina­
tion of atorvastatin, celecoxib and tipifarnib for 24 h. The CSC related
markers were examined by the Western blot analysis. Results of the
experiment showed small to moderate decreases in the levels of CD44,
CD133 and ALDH1A1 in the cells treated with atorvastatin, celecoxib or
tipifarnib alone (Fig. 5). As expected, the expression levels of CD44,
CD133 and ALDH1A1 were strongly reduced in the sphere-forming cells
treated with the combination of atorvastatin, celecoxib and tipifarnib
(Fig. 5).
3.5. Suppression of NF-κB and Akt in Panc-1 sphere-forming cells by the
triple combination
We further determined the effects of atorvastatin, celecoxib and
tipifarnib alone or in combination on the activity of NF-κB in the sphere-
Fig. 5. Effects of atorvastatin (LIP), celecoxib (CEL) and tipifarnib (ZAN) alone
or in combination (COM) on expression levels of CD133, CD44, ALDH1A1, Akt
and NF-κB p65. Panc-1 sphere-forming cells were treated with LIP, CEL and
ZAN alone or in combination for 24 h. The protein levels of CD133, CD44,
ALDH1, Akt, p-Akt, p65 and p-p65 were determined by the Western
blot analysis.
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European Journal of Pharmacology 893 (2021) 173840
forming cells. We found that atorvastatin, celecoxib or tipifarnib alone
had small effect on decreasing the levels of phosphorylated p65 while
the triple combination strongly blocked the phosphorylation of p65
(Fig. 5). The levels of phosphorylated Akt in the sphere-forming cells
treated with atorvastatin, celecoxib and tipifarnib alone or in combi­
nation was also determined. As shown in Fig. 5, atorvastatin alone had
little effect, and celecoxib or tipifarnib alone had moderate effect on the
level of phosphorylated Akt. However, the triple combination potently
reduced the level of phosphorylated Akt. These results demonstrated
that combined atorvastatin with celecoxib and tipifarnib strongly
inhibited the activation of NF-κB and Akt.
4. Discussion
Accumulating experimental evidence indicates that CSCs drive
tumor initiation, invasion and metastasis, and contributes to chemo­
resistance, consequently promoting unrestricted tumor progression
(Elshamy and Duhe, 2013; Koury et al., 2017; Yoshida and Saya, 2016).
The present study was carried out to test our hypothesis that combined
atorvastatin with celecoxib and tipifarnib will suppress pancreatic CSCs.
We showed in the present study that the combination of atorvastatin,
celecoxib and tipifarnib strongly inhibited Panc-1 CSCs as evidenced by
the decreases in tumorsphere formation and reduced levels of CSC
related markers in the sphere forming cells. Results of our study suggest
that the combination strategy using atorvastatin, celecoxib and tipi­
farnib may represent an effective approach for inhibiting pancreatic
CSCs.
An important step in CSC-based research is the effective isolation and
reliable identification of CSCs from a large heterogenious cancer cell
population. Pancreatic CSCs can be isolated and enriched by their
sphere-forming ability (Zeng et al., 2018). In the present study, Panc-1
cells were cultured in ultralow attachment culture plate with serum-free
medium. Panc-1 cells form spheres with this culture condition. The
sphere-forming cells were examined for expression levels of CSC related
markers. Based on existing studies, CD44, CD133 and ALDH1 are
considered pancreatic cancer CSC related markers (Raj et al., 2015; Zeng
et al., 2018; Zhou et al., 2019). Thus, these markers were chosen to
identify pancreatic cancer CSCs. We found that the levels of these
markers were much higher in the sphere-forming cells than in parental
Panc-1 cells. This result indicates that the pancreatic cancer CSCs can be
isolated and enriched by the sphere-forming method.
There is increasing interest in using combinations of different agents
for inhibiting pancreatic cancer cells. Suitable combinations may pro­
duce synergistic effect on suppressing pancreatic CSCs. In the present
study, we observed that combined atorvastatin with celecoxib and
tipifarnib synergistically suppressed Panc-1 sphere formation, while
each agent alone only had small inhibitory effect on Panc-1 sphere
formation. Moreover, the combination of atorvastatin, celecoxib and
tipifarnib effectively diminished Panc-1 CSC activity as evidenced by
decreasing proliferation and increasing apoptosis in the sphere-forming
cells. Combined atorvastatin with celecoxib and tipifarnib also strongly
downregulated CSC related markers CD44, CD133 and ALDH1.
CD44 is a transmembrane glycoprotein which possesses a particular
cell adhesion function and consequently assists the matrix adhesion and
migration of CSCs (Ponta et al., 2003). The expression of CD44 variants
was significantly correlated with poor prognosis in pancreatic cancer
(Garcea et al., 2005). In pancreatic cancer, CD44+ cells were more
tumorigenic than CD44-cells (Li et al., 2007). CD44 is one of the
important CSC markers in pancreatic cancer (Li et al., 2007). Gemcita­
bine resistant pancreatic cancer cells showed characteristics of CSC and
RNA interference of CD44 inhibited clonogenic growth of the cells
(Hong et al., 2009). CD133 (also called Prominin-1 or AC133) was the
first member verified in the prominin family of pentaspan trans­
membrane glycoproteins, and is now widely used as CSC related markers
in various types of cancer including pancreatic cancer (Gupta et al.,
2018; Hermann et al., 2007). As few as 500 patient-derived CD133+
X.-T. Xu et al.
pancreatic cancer cells were capable of forming orthotopic tumors in
immunodeficient mice. In contrast, as many as 106 CD133-pancreatic
cancer cells did not result in tumor formation (Hermann et al., 2007).
In the present study, we found that the sphere forming cells expressed
high levels of CD44 and CD133, and treatment of the cells with the triple
combination decreased the levels of CD44 and CD133. This result in­
dicates that CD44 and CD133 are useful markers for the determination
of the effect of drug combination on the stemness of pancreatic cancer
cells.
ALDH1A1 is a cytosolic enzyme belongs to the ALDH family of en­
zymes that catalyze the intracellular oxidation of different aldehydes
(Tomita et al., 2016). It plays an important role in retinoic acid signaling
and used as a marker to identify various types of CSCs (Tomita et al.,
2016; Duong et al., 2017; Jiang et al., 2009; Meng et al., 2014). A recent
study showed that accumulation of prostaglandin E2 (PGE2) resulted in
high levels of ALDH1A1 and enhanced KRAS driven pancreatic cancer
progression indicating a role of PGE2-ALDH1A1 pathway in pancreatic
cancer pathogenesis (Arima et al., 2019). PGE2 is regulated by COX-2
and the COX-2 inhibitor celecoxib may decrease the level of ALDH1A1
through the PGE2-ALDH1A1 pathway. ALDH1A1 reportedly is a target
gene of the NF-κB pathway (Moreno et al., 2004; Yan et al., 2008) and
NF-κB may act as an upstream positive regulator of ALDH1A1 (Yang
et al., 2015). In the present study, we found that combined atorvastatin
with celecoxib and tipifarnib strongly inhibited NF-κB. Our findings
raise the possibility that the strong decrease in ALDH1A1 by the com­
bination of atorvastatin, celecoxib and tipifarnib in Panc-1 sphere
forming cells was mediated by concurrent inhibition of COX-2 and
NF-κB.
Results of our study also indicate that activation of Akt was strongly
inhibited by the combination of atorvastatin, celecoxib and tipifarnib.
The PI3K/Akt signaling pathway plays an important role in cell prolif­
eration, survival and malignant transformation, and is frequently acti­
vated in human cancer (Tokunaga et al., 2008; Altomare and Testa,
2005). NF-κB and mTOR are down-stream targets of activated Akt (Deeb
et al., 2014; Huang et al., 2020), and mTOR plays critical roles in
pancreatic CSCs through specific and stemness-related functions (Mat­
subara et al., 2013). A strong inhibition of Akt activation in
sphere-forming cells by the triple combination was found in the present
study. Simultaneous suppression of Akt and NF-κB by suitable phar­
macological inhibitors may be an effective strategy for inhibition of
pancreatic CSCs.
In conclusion, results of this study demonstrate that combined
atorvastatin with celecoxib and tipifarnib synergistically inhibited the
formation of tumor sphere in Panc-1 cells. The combination also strongly
inhibited proliferation and promoted apoptosis in Panc-1 sphere-form­
ing cells. The levels of CSC related markers CD44, CD133 and ALDH1A1
were decreased in sphere-forming cells treated with this drug combi­
nation. The strong effects of the combination on sphere-forming cells
were associated with inhibition of Akt and NF-κB activation. Combined
atorvastatin with celecoxib and tipifarnib may be an effective approach
to inhibit pancreatic CSCs.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The present study was supported by grants from the Department of
Education of Guangdong Province (2016KCXTD005, 2017KZDXM084,
2017KSYS010 and 2019KZDZX2003), the Department of Science and
Technology of Jiangmen (No: 2016350100170008351,
2018110100330005446), Jiangmen Program for Innovative Research
Team (No: 2017TD02), and the Youth Team Fund of Wuyi University
(2016td01). Dr. Xi Zheng is the Unilever Chair in Nutrition and Disease
Prevention Research at Rutgers University.
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European Journal of Pharmacology 893 (2021) 173840
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2020.173840.
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