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.
Trang 1R E S E A R C H A R T I C L E 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 Yamasaki2and 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, next-generation 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
© The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the
* Correspondence: t-takami@yamaguchi-u.ac.jp
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
Trang 2Patients 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
chemother-apy, 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
Further-more, 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,
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 hepato-cellular carcinoma refractory to chemotherapy [9] Be-cause 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 mech-anism of action of iron chelators We have investigated the ability of DFX ability to suppress tumour prolifera-tion, and found that it suppresses proliferation in a concentration-dependent manner [10], it improves sensi-tivity 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)
Trang 3liver cancer [12]; we have also examined other secondary
effects of iron chelators Upon conducting a
supplemen-tary 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
path-ways, 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
Fi-nally, 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,
can-cer cells appear in peripheral blood and are a useful
pre-dictive 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
evalu-ate the effects of DFX on the invasive and migratory
abilities of pancreatic cancer cells
Methods
Cell culture
We used the pancreatic cancer cell lines BxPC-3,
Panc-1, 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
Technologies, Carlsbad, CA, USA) with 10% foetal calf serum (FBS) and 50μg/ml gentamicin Panc-1 cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) with 10% FBS and 50μg/ml gentamicin HPAF II cells were cultured in Eagle’s medium (Life Technologies) with 10% FBS and 50μg/ml gentamicin Culture was performed in a 37 °C, 5% CO2environment Reagents
The oral iron chelator DFX was obtained from Novartis (Basel, Switzerland) For in vitro studies, DFX was dis-solved 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 so-dium chloride solution (0.9% w/v; Chemix Inc., Yoko-hama, Japan)
Trypan blue exclusion assay Cell viability of pancreatic cancer cell lines under treat-ment with DFX (0, 10, 50, 100μM) was evaluated Each pancreatic cancer cell line was cultured in an environ-ment of 37 °C, 5% CO2 DFX (0, 10, 50, 100μM) was added and the cells were incubated for 48 h, after which equal amounts of 0.4% Trypan blue solution (Life
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
Trang 4Fig 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
Trang 5Technologies) 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
eval-uated using a wound-healing assay Each pancreatic
can-cer cell line was cultured at 37 °C and 5% CO2in 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
steri-lised pipette tip was then used to create a wound
(scratch) in the confluent layer Next, DFX (0, 10, 50, or
100μM), the Rac-1 inhibitor NSC 23766 (0, 50, 100,
200μM; Selleck, Houston, TX, USA), or the Cdc42
in-hibitor ML141 (0, 10, 20, 40μM; Selleck) were added to
each well, and the cells were incubated for 24 h
After-wards, the wound width was measured; the ratio of
pre-and post-culture wound widths was used as an index of
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 100μM) was added to each chamber Culture medium with 10% FBS was used in the lower well Cells were cultured at 37 °C and 5% CO2for 96 h After 96 h, the cells remaining in the upper chamber were removed, and the chamber was tilted several times in detachment solu-tion to completely detach the cells from the membrane CyQuant® was added to each well, and after 20 min of incu-bation 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
Trang 6Rho GTPase activity assay
GTP-bound Rac1 and Cdc42 were measured using
cor-responding G-LISA Activation Assay Kits (Cytoskeleton,
Denver, CO, USA) After stimulation, cells were washed
twice with cold PBS and lysed using the lysis buffer
pro-vided 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
proto-col Protein concentrations were determined, and Rho
GTPase activity was assessed according to the
manufac-turer’s instructions
Fluorescent phalloidin (F-actin) staining
DFX (0 or 50μM) was added to BxPC-3 cells, and they
were cultured at 37 °C and 5% CO2for 24 h The culture
medium was removed and cells were washed with
PBS Subsequently, 200μL of cell fixative (4%
formal-dehyde 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
(0.5% Triton X-100 in PBS) was added and incubated
at room temperature for 5 min Next, 200μL 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 non-parametric 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 pancre-atic 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
Trang 7DFX at 10μM, 50 μM, and 100 μM in all cell lines
(BxPC-3, Panc-1, and HPAFII), as compared to the
con-trol 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,
simi-lar experiments were performed with the Rac1 inhibitor
NSC 23766 and the Cdc42 inhibitor ML141 A
signifi-cant decline in migration ability was seen in the DFX
10μM, 50 μM, and 100 μM treatment groups for
BxPC-3, 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
II In Panc-1, 100μM and 200 μM NSC 23766 caused a
significant decline in migration ability (Fig.3) Similarly,
significantly declined migration ability in BxPC-3,
Panc-1, and HPAFII (Fig.3) For further evaluation of migra-tion ability in DFX treated pancreatic cancer cell lines, a Boyden chamber assay was conducted In BxPC-3, HPA-FII, and Panc-1, we confirmed a significant abrogation of
50μM, 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 expres-sion 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
group In addition, in Panc-1, a significant decline in Rac1 expression was seen in the DFX 100μM treatment group The HPAFII cells also showed a trend toward reduced ex-pression 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
Trang 8control 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 50μM and 100 μM, as compared to the control group
Panc-1 and HPAFII cells also showed a significant decline
100μM treatment groups, as compared to the control
group (Fig.6) Phalloidin staining was also performed on
BxPC3 cells treated with DFX, revealing a significant
re-duction in filopodia in the DFX treatment group,
com-pared 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
down-stream regulated gene-1, a metastasis suppression factor,
to suppress cell invasion However, the mechanisms by
understood
Rac1 and Cdc42 are Rho-family G proteins that have been linked to a variety of different cancers and are in-volved in epithelial to mesenchymal transition, cell-cycle progression, migration/invasion, tumour growth, angio-genesis, 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 tein promotes GTP hydrolysis to inactivate these
transduction molecules whose dysregulation is associ-ated with cancer occurrence and cell migration/invasion [24] Reports have shown that in cases of pancreatic can-cer, highly elevated expression of Cdc42 is significantly correlated with poor prognosis [25] It has been pre-dicted that suppressing the activity of Rac1 and Cdc42 will reduce invasive capability
This study was an in vitro analysis to evaluate the ef-fect of the iron chelator DFX, on the migration/invasion
of pancreatic cancer cell lines Our previous paper
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
Trang 9showed an elevated caspase-3 activity at 48 h after
treat-ment with 50 or 100μM DFX [10] Induction of
apop-tosis 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
ex-pression 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
sup-pression of Rac1 and Cdc42
Rac1 and Cdc42 may well be valid and effective
thera-peutic 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,
suppress-ing Rac1 or Cdc42 and thereby controllsuppress-ing invasion may
be clinically effective For example, at present, as
recom-mended 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
ad-vantage 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
hu-man pancreatic cancer should lead to a more thorough
understanding
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 re-ductions in cell migratory and invasive abilities This is the first paper to report that DFX has the ability to sup-press tumour proliferation (as we have previously re-ported), 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 2 Department 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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