Invasion inhibition in pancreatic cancer using the oral iron chelating agent deferasirox

Amano et al. BMC Cancer
(2020) 20:681
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RESEARCH ARTICLE

Open Access

Invasion inhibition in pancreatic cancer
using the oral iron chelating agent
deferasirox
Shogo Amano1, Seiji Kaino1, Shuhei Shinoda1, Hirofumi Harima1, Toshihiko Matsumoto2, Koichi Fujisawa1,
Taro Takami1* , Naoki Yamamoto1, Takahiro Yamasaki2 and Isao Sakaida1

Abstract
Background: Iron is required for cellular metabolism, and rapidly proliferating cancer cells require more of this
essential nutrient. Therefore, iron regulation may well represent a new avenue for cancer therapy. We have
reported, through in vitro and in vivo research involving pancreatic cancer cell lines, that the internal-use, nextgeneration iron chelator deferasirox (DFX) exhibits concentration-dependent tumour-suppressive effects, among
other effects. After performing a microarray analysis on the tumour grafts used in that research, we found that DFX
may be able to suppress the cellular movement pathways of pancreatic cancer cells. In this study, we conducted
in vitro analyses to evaluate the effects of DFX on the invasive and migratory abilities of pancreatic cancer cells.
Methods: We used pancreatic cancer cell lines (BxPC-3, Panc-1, and HPAF II) to examine the efficacy of DFX in
preventing invasion in vitro, evaluated using scratch assays and Boyden chamber assays. In an effort to understand
the mechanism of action whereby DFX suppresses tumour invasion and migration, we performed G-LISA to
examine the activation of Cdc42 and Rac1 which are known for their involvement in cellular movement pathways.
Results: In our scratch assays, we observed that DFX-treated cells had significantly reduced invasive ability
compared with that of control cells. Similarly, in our Boyden chamber assays, we observed that DFX-treated cells
had significantly reduced migratory ability. After analysis of the Rho family of proteins, we observed a significant
reduction in the activation of Cdc42 and Rac1 in DFX-treated cells.
Conclusions: DFX can suppress the motility of cancer cells by reducing Cdc42 and Rac1 activation. Pancreatic
cancers often have metastatic lesions, which means that use of DFX will suppress not only tumour proliferation but
also tumour invasion, and we expect that this will lead to improved prognoses.

Keywords: Pancreatic cancer, Iron chelation, Cancer therapy, Rho family protein, Deferasirox, Invasion

* Correspondence:
1
Department of Gastroenterology and Hepatology, Yamaguchi University
Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi
755-8505, Japan
Full list of author information is available at the end of the article
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Amano et al. BMC Cancer

(2020) 20:681

Background
Patients with pancreatic cancer have exceedingly poor
prognoses, and in the United States, the 5-year survival
rate of the disease is 6%—a staggeringly low number [1].
In Japan, the condition in nearly half of all pancreatic
cancer patients is detected at the metastatic state [2],
and the fact that pancreatic cancer often exhibits strong
invasive and metastatic tendencies is thought to be one

reason for poor patient prognoses [1]. The first-choice
therapy for unresectable pancreatic cancer is chemotherapy, and over the last 20 years, gemcitabine (GEM) has
come to be used as the primary standard therapy [3]. In
recent years, FOLFIRINOX therapy [4], a combination
of fluorouracil, irinotecan, oxaliplatin, and leucovorin
[4], and GEM plus nab-paclitaxel [5] has been reported
to be useful. However, while these kinds of combination
chemotherapies have comparatively higher therapeutic
effects than GEM monotherapy, they also have higher
incidence rates of side effects like cytopenia. Furthermore, more than half of all pancreatic cancer patients
are diagnosed at age 65 or older [6]. Consequently, it is
vital, especially for elderly pancreatic cancer patients,

Page 2 of 10

that new chemotherapies with low side-effect incidence
rates be studied.
Iron is required for cellular replication, metabolism,
and proliferation [7]. Cancer cells proliferate rapidly,
causing them to need more iron than normal cells; thus,
iron regulation therapy may represent a new avenue for
cancer therapy [8]. Iron chelators are existing drugs that
are prescribed for iron overload. Because they are not
anticancer drugs, they have very few side effects. We
were the first to report the clinical effectiveness of the
iron chelator deferoxamine (DFO) on advanced hepatocellular carcinoma refractory to chemotherapy [9]. Because DFO is an intravenously administered drug, the
orally administrable, outpatient-suited, next-generation
iron chelator deferasirox (DFX) has begun to be used in
recent years. Much is still unknown regarding the mechanism of action of iron chelators. We have investigated
the ability of DFX ability to suppress tumour proliferation, and found that it suppresses proliferation in a

concentration-dependent manner [10], it improves sensitivity to GEM [11], and that the combination of DFX
and sorafenib is better than DFX alone at suppressing

Fig. 1 Cell viability of pancreatic cancer cell lines treated with DFX. BxPC3, Panc1, and HPAFIIwere treated with DFX (0, 10, 50, or 100 μM) for 48 h
and stained with trypan blue to evaluate cell viability (n = 3)


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Page 3 of 10

liver cancer [12]; we have also examined other secondary
effects of iron chelators. Upon conducting a supplementary microarray analysis of the in vivo samples used [10],
we observed that the expression of Rho-family genes like
Rac1 and Cdc42, involved in cellular movement pathways, was altered in DFX-treated cells, and have begun
to consider the possibility that DFX could reduce the
metastatic and invasive capabilities of cancer cells. Finally, in recent years, it has been reported that, even in
stage II and III pancreatic cancer according to the
American Joint Committee on Cancer, 8th Edition, cancer cells appear in peripheral blood and are a useful predictive indicator of patient prognosis [13]. Thus, the
importance of elucidating the mechanisms of invasion
and their prevention and treatment led to the idea of
this study. Here, we conducted in vitro analyses to evaluate the effects of DFX on the invasive and migratory
abilities of pancreatic cancer cells.

Technologies, Carlsbad, CA, USA) with 10% foetal calf
serum (FBS) and 50 μg/ml gentamicin. Panc-1 cells were
cultured in Dulbecco’s modified Eagle’s medium (Life
Technologies) with 10% FBS and 50 μg/ml gentamicin.

HPAF II cells were cultured in Eagle’s medium (Life
Technologies) with 10% FBS and 50 μg/ml gentamicin.
Culture was performed in a 37 °C, 5% CO2 environment.

Methods

Trypan blue exclusion assay

Cell culture

Cell viability of pancreatic cancer cell lines under treatment with DFX (0, 10, 50, 100 μM) was evaluated. Each
pancreatic cancer cell line was cultured in an environment of 37 °C, 5% CO2. DFX (0, 10, 50, 100 μM) was
added and the cells were incubated for 48 h, after which
equal amounts of 0.4% Trypan blue solution (Life

We used the pancreatic cancer cell lines BxPC-3, Panc1, and HPAF II, purchased from the American Type
Culture Collection (Manassas, VA, USA). All of these
cell lines are epithelial cells derived from cancer cells.
BxPC-3 cells were cultured in RPMI-1640 (Life

Reagents

The oral iron chelator DFX was obtained from Novartis
(Basel, Switzerland). For in vitro studies, DFX was dissolved in dimethyl sulphoxide at a stock concentration
of 100 mM and was used at the concentrations indicated
in the results and figures by dilution in culture medium
containing 10% FBS (172,012; Sigma-Aldrich, St. Louis,
MO, USA). For in vivo studies. DFX was dissolved in sodium chloride solution (0.9% w/v; Chemix Inc., Yokohama, Japan).

Fig. 2 Effect of DFX on migratory ability of pancreatic cancer cells. Pancreatic cancer cell lines (BxPC-3, Panc-1, HPAFII) were treated with DFX (0,

10, 50, 100 μM) and incubated for 24 h. a-c Migrated cells were visualized via phase-contrast microscopy. d-f % wound closure was measured and
the ratio to wound width at the start of the incubation was used as an index of cell migration, compared to control group (n = 3 each). Data are
presented as mean ± SD. *P < 0.01 vs control


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Fig. 3 Effect of NSC 23766 and ML141 on the invasion ability of pancreatic cancer cells. Pancreatic cancer cell lines (BxPC-3, Panc-1, HPAFII) were
treated with either NSC 23766 (0, 50, 100, 200 μM) or ML141 (0, 10, 20, 40 μM), and incubated for 24 h. a-c, g-i Migrated cells were visualised via
phase-contrast microscopy. d-f, j-l % wound closure was measured, and the ratio to wound width at the start of the culture was used as an
index of cell migration to conduct a comparative evaluation against the control group (n = 3). Data are presented as mean ± SD. *P < 0.05, **P < 0.01
vs control


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Technologies) was added to the cell suspensions, and
the viability of each pancreatic cancer cell line was
assessed using the Countess Automated Cell Counter
(Invitrogen, CA, USA).
Wound-healing scratch assay

The migratory ability of pancreatic cancer cells was evaluated using a wound-healing assay. Each pancreatic cancer cell line was cultured at 37 °C and 5% CO2 in 6-well
culture plates (BD Biosciences, San Jose, CA, USA) to

80% confluence. The suspended cells were removed with
three washes of phosphate-buffered saline (PBS). A sterilised pipette tip was then used to create a wound
(scratch) in the confluent layer. Next, DFX (0, 10, 50, or
100 μM), the Rac-1 inhibitor NSC 23766 (0, 50, 100,
200 μM; Selleck, Houston, TX, USA), or the Cdc42 inhibitor ML141 (0, 10, 20, 40 μM; Selleck) were added to
each well, and the cells were incubated for 24 h. Afterwards, the wound width was measured; the ratio of preand post-culture wound widths was used as an index of

Page 5 of 10

cell migration, and comparisons were made with the
control group.
Boyden chamber assay

To assess invasion ability, we used 24-well Boyden chamber
assays (CytoSelect 24-Well Cell Invasion Assay Kits, CELL
BIOLABS, San Diego, CA, USA). Each pancreatic cancer
cell line was cultured in the upper chamber (at 1.0 × 106
cells/insert), and serum-free medium containing DFX (0,
10, 50, or 100 μM) was added to each chamber. Culture
medium with 10% FBS was used in the lower well. Cells
were cultured at 37 °C and 5% CO2 for 96 h. After 96 h, the
cells remaining in the upper chamber were removed, and
the chamber was tilted several times in detachment solution to completely detach the cells from the membrane.
CyQuant® was added to each well, and after 20 min of incubation at room temperature, a multimode reader (Infinite
200 PRO, Tecan Trading, AG, Switzerland) was used to
measure fluorescence at 480 nm/520 nm, which was then
compared to that of the control group.

Fig. 4 Effect of DFX on invasive ability of pancreatic cancer cells. a–c Pancreatic cancer cells were cultured in the upper chamber, and serum-free
medium containing DFX (0, 10, 50, or 100 μM) was added to each chamber. After 96 h of incubation, a multimode reader was used to measure

fluorescence at 480 nm/520 nm, which was then compared to that of the control group (n = 3). Data are presented as mean ± SD. *P < 0.05


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Rho GTPase activity assay

GTP-bound Rac1 and Cdc42 were measured using corresponding G-LISA Activation Assay Kits (Cytoskeleton,
Denver, CO, USA). After stimulation, cells were washed
twice with cold PBS and lysed using the lysis buffer provided with the kits for 15 min on ice. The lysates were
centrifuged at 10,000×g for 1 min at 4 °C. Supernatants
were aliquoted, snap-frozen in liquid nitrogen, and
stored at − 80 °C according to the manufacturer’s protocol. Protein concentrations were determined, and Rho
GTPase activity was assessed according to the manufacturer’s instructions.
Fluorescent phalloidin (F-actin) staining

DFX (0 or 50 μM) was added to BxPC-3 cells, and they
were cultured at 37 °C and 5% CO2 for 24 h. The culture
medium was removed and cells were washed with
PBS. Subsequently, 200 μL of cell fixative (4% formaldehyde in PBS) was added, and cells were left for 10
min at room temperature for fixing. Afterwards, cells
were washed with PBS and permeabilisation buffer

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(0.5% Triton X-100 in PBS) was added and incubated
at room temperature for 5 min. Next, 200 μL of 100
nM Acti-stain™ 488 phalloidin (Cytoskeleton, Denver,

CO, USA) was added, and after 30 min of dark-room
incubation, cells were viewed using a multi-confocal
laser microscope (Zeiss, LSM710 system, Oberkochen,
Germany).
Statistical analyses

Analyses performed were the Student’s t test or nonparametric ANOVA test, using the statistical analysis
software JMP13 (SAS Institute Inc., Cary, NC, USA).
Results are expressed as mean ± standard deviation
(SD). P values < 0.05 were deemed significant.

Results
DFX does not affect the cell viability of pancreatic cancer
cells in vitro

We assessed the effect of DFX on the viability of pancreatic cancer cells treated with DFX for 48 h. No obvious
decline was seen in the survival rates for treatments with

Fig. 5 Effect of DFX on Rac1 activation in pancreatic cancer cells. Pancreatic cancer cells were treated with DFX (0, 10, 50, or 100 μM); after 48 h
of incubation, Rac1 activation was measured using G-LISA in (a) BxPC-3 cells(n = 3), b Panc-1 cells (n = 3), and (c) HPAF II cells (n = 3). Data are
presented as mean ± SD. *P < 0.05, **P < 0.01


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DFX at 10 μM, 50 μM, and 100 μM in all cell lines
(BxPC-3, Panc-1, and HPAFII), as compared to the control group (Fig. 1).
DFX attenuates the migratory and invasive abilities of

pancreatic cancer cells in vitro

To evaluate the effect of DFX on pancreatic cancer cell
line migration ability, a scratch assay was conducted. In
addition, because the results of a micro-array analysis
showed a decline in expression of Rac1 and Cdc42, similar experiments were performed with the Rac1 inhibitor
NSC 23766 and the Cdc42 inhibitor ML141. A significant decline in migration ability was seen in the DFX
10 μM, 50 μM, and 100 μM treatment groups for BxPC3, Panc-1, and HPAFII, as compared to the control
group (Fig. 2). In addition, compared to the control cells,
a significant decline in migration ability was achieved
with as low as 50 μM NSC 23766 in BxPC-3 and HPAF
II. In Panc-1, 100 μM and 200 μM NSC 23766 caused a
significant decline in migration ability (Fig. 3). Similarly,
treatment with up to 40 μM ML141 resulted in a

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significantly declined migration ability in BxPC-3, Panc1, and HPAFII (Fig. 3). For further evaluation of migration ability in DFX treated pancreatic cancer cell lines, a
Boyden chamber assay was conducted. In BxPC-3, HPAFII, and Panc-1, we confirmed a significant abrogation of
migration following treatment with DFX at 10 μM,
50 μM, and 100 μM, as compared to the control cells
(Fig. 4).
DFX reduces activation of rho family proteins Rac1 and
Cdc42 in vitro

To assess the contributions of Rho/Rac1/Cdc42 signaling
in DFX-suppressed cell migration, we analysed the expression of Rho family proteins in DFX-treated pancreatic
cancer cell lines using G-LISA. We observed a significant
decline in Rac1 expression in BxPC-3 cells treated with
50 μM and 100 μM DFX, as compared to the control

group. In addition, in Panc-1, a significant decline in Rac1
expression was seen in the DFX 100 μM treatment group.
The HPAFII cells also showed a trend toward reduced expression of Rac1 in the DFX groups as compared to the

Fig. 6 Effect of DFX on Cdc42 activation in pancreatic cancer cells. Pancreatic cancer cells were treated with DFX (0, 10, 50, or 100 μM); after 48 h
of incubation, Cdc42 activation was measured using G-LISA in (a) BxPC-3 cells (n = 3), b Panc-1 cells (n = 3), and c HPAF II cells (n = 3). Data are
presented as mean ± SD. *P < 0.05, **P < 0.01


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Fig. 7 BxPC-3 cells stained with phalloidin-rhodamine. DFX (0, 50 μM) was added to BxPC-3 cells and incubated for 24 h. Phalloidin staining was
performed and cells observed with a multi-confocal microscope to measure the number of filopodia (n = 13). Scale bars indicate 50 μm. Data are
presented as mean ± SD. *P < 0.01

control group, though this difference was not significant
(Fig. 5). Assessment of Cdc42 levels showed a significantly
declined expression in the BxPC-3 cells treated with DFX
at 50 μM and 100 μM, as compared to the control group.
Panc-1 and HPAFII cells also showed a significant decline
in Cdc42 expression in the DFX 10 μM, 50 μM and
100 μM treatment groups, as compared to the control
group (Fig. 6). Phalloidin staining was also performed on
BxPC3 cells treated with DFX, revealing a significant reduction in filopodia in the DFX treatment group, compared with the control cells (Fig. 7).

Discussion

The effectiveness of iron chelators on cancer was first
reported in leukaemia in 1986 [14, 15], and since then,
the effectiveness of DFX on a variety of carcinomas has
been reported [9, 10, 16–19]. Furthermore, in recent
years, reports [20, 21] have indicated that administration
of iron chelators in prostate and colon cancer inhibits
TGF-β and promotes the expression of N-myc downstream regulated gene-1, a metastasis suppression factor,
to suppress cell invasion. However, the mechanisms by

which these effects are achieved is still poorly
understood.
Rac1 and Cdc42 are Rho-family G proteins that have
been linked to a variety of different cancers and are involved in epithelial to mesenchymal transition, cell-cycle
progression, migration/invasion, tumour growth, angiogenesis, and oncogenic transformation. Rac1 and Cdc42
are generally overexpressed or overactivated in cancer
cells [22]. In Rho-family G proteins, guanine nucleotide
exchange factors work to allow GDP-GTP exchange and
activate the protein. In contrast, GTPase-activating protein promotes GTP hydrolysis to inactivate these proteins [23]. Rac1 and Cdc42 are important signal
transduction molecules whose dysregulation is associated with cancer occurrence and cell migration/invasion
[24]. Reports have shown that in cases of pancreatic cancer, highly elevated expression of Cdc42 is significantly
correlated with poor prognosis [25]. It has been predicted that suppressing the activity of Rac1 and Cdc42
will reduce invasive capability.
This study was an in vitro analysis to evaluate the effect of the iron chelator DFX, on the migration/invasion
of pancreatic cancer cell lines. Our previous paper


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(2020) 20:681

showed an elevated caspase-3 activity at 48 h after treatment with 50 or 100 μM DFX [10]. Induction of apoptosis by DFX treatment may thus have contributed to
suppress migration/invasion, though we confirmed the
higher cellular viability of pancreatic cancer cells at 48 h
after DFX treatment (Fig. 1). Our results also suggested
that DFX may not only demonstrate the tumor growth
inhibitory effect that we reported previously, but may
also reduce the migration/invasion of pancreatic cancer
cells by abrogating the expression of invasion-related
Rho-family G proteins, Rac1 and Cdc42. Previous studies
have reported that iron chelator treatment suppresses
ROCK expression with consequent reduction of actin
polymerization [20], and suppression of N-cadherin expression that resulted in blocking the invasiveness of
esophageal cancer cells [26]. However, this is the first
study to report the changes caused by iron chelators in
cell shape and in reducing migration ability through suppression of Rac1 and Cdc42.
Rac1 and Cdc42 may well be valid and effective therapeutic targets in the treatment of cancer. The literature
shows that administration of Rac1 and Cdc42 inhibitors
suppresses migration and invasion in breast cancer
models [27]. Additionally, reports indicate that inhibition
of Rac1 increases the susceptibility of pancreatic and
breast cancers to radiation therapy [28, 29]. In any case,
many studies are being performed on these compounds
and their links with cancer.
The strong invasive tendency of pancreatic cancer is a
factor that leads to its poor prognosis [1]; thus, suppressing Rac1 or Cdc42 and thereby controlling invasion may
be clinically effective. For example, at present, as recommended by the National Comprehensive Cancer Network
guidelines, preoperative adjuvant therapies are actively
performed in resectable, border region (BR) pancreatic
cancers [30]. However, almost no evidence recommends a
specific preoperative adjuvant therapy regimen for BR

cases. While FOLFIRINOX or GEM + albumin-bound
paclitaxel therapies are the most approved [31, 32], the
addition of an iron chelator like DFX would contribute
the tumour-proliferation-suppressive effects of this class
of drug as well as its suppressive effects on the emergence
of metastatic lesions during chemotherapy treatment,
owing to the iron chelator’s ability to reduce Rac1 and
Cdc42 activation. Furthermore, because iron chelators are
known medications that are commonly used to treat iron
overload, the fact that they are not anticancer drugs
means that their addition to anticancer drug regimens
should cause little to no adverse effects; this is another advantage of this class of therapies. However, because this
study was an in vitro analysis, future evaluations of protein
expression changes and invasive ability in vivo or in human pancreatic cancer should lead to a more thorough
understanding.

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Conclusions
In this study, after administration of DFX to pancreatic
cancer cell lines, we confirmed significant reductions in
the activation of Rac1 and Cdc42. In scratch assays and
Boyden chamber assays, we also observed significant reductions in cell migratory and invasive abilities. This is
the first paper to report that DFX has the ability to suppress tumour proliferation (as we have previously reported), as well as to reduce the abilities of pancreatic
cancer cells to change shape and migrate by reducing
the activation of Rac1 and Cdc42, Rho-family G proteins
involved in cancer invasion.
Abbreviations
GEM: Gemcitabine; DFO: Deferoxamine; DFX: Deferasirox; FBS: Foetal calf
serum; PBS: Phosphate-buffered saline; SD: Standard deviation; BR: Border

region
Acknowledgements
Not applicable.
Authors’ contributions
SA performed all of the experiments. SS performed G-LISA, scratch assay, and
Boyden chamber assay. TM, KF, and NY performed histology and fluorescent
phalloidin (F-actin) staining. SA, SK, HH, and TT designed the study, analysed
the data, and wrote the paper. SS, TY, and IS provided financial support and
final approval of the manuscript. All authors approved and commented on
the manuscript.
Funding
This work was supported by Grants-in-Aid for Scientific Research from the
Japan Society for the Promotion of Science(19 K17434), (16H05287), the
Japan Science and Technology Agency, and the Ministry of Health, Labor,
and Welfare.
Availability of data and materials
The microarray data have been deposited in the NCBI’s Gene Expression
Omnibus (GEO) under GEO series accession no. GSE81363 [10]. The other
datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Ethics approval and consent to participate
We used the pancreatic cancer cell lines (BxPC-3, Panc-1, and HPAF II) which
were intended for research use only from the American Type Culture Collection
(Manassas, VA, USA).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1

Department of Gastroenterology and Hepatology, Yamaguchi University
Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi
755-8505, Japan. 2Department of Oncology and Laboratory Medicine,
Yamaguchi University, Graduate School of Medicine, Ube, Yamaguchi, Japan.
Received: 2 September 2019 Accepted: 12 July 2020

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