Song et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00502-y(2024) 25:6

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

TonEBP/NFAT5 expression is associatedwith cisplatin resistance and migrationin macrophage-induced A549 cells
Hee Ju Song1, Young Hwan Kim1, Han Na Choi1, Taehee Kim1, Soo Jin Kim1, Min Woong Kang2 and Sang Do Lee1*
Abstract
Background Macrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly,
TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcription
factor and that of macrophages in lung cancer cells remains unclear. Therefore, this study investigated the effects ofmacrophages and TonEBP/NFAT5 expression on cisplatin resistance and migration in A549 lung adenocarcinoma cells.
Results A549 cells were cultured alone or indirectly co-cultured with THP-1-derived macrophages using a transwell
culture chamber. Cisplatin-induced cell death was markedly decreased and migration increased in co-cultured A549
cells. Macrophage-conditioned media (CM) showed a similar effect on drug resistance and migration. Cisplatininduced apoptosis, DNA fragmentation, and cleaved apoptotic proteins PARP and caspase-3 were markedly reduced
in macrophage CM-induced A549 cells. Here, ERK, p38, JNK, and NF-κB activities were increased by macrophage CM.
Furthermore, the proteins involved in cisplatin resistance and cancer cell migration were identified using specificinhibitors of each protein. ERK and NF-κB inhibition considerably reduced cisplatin resistance. The increase in
macrophage CM-induced migration was partially reduced by treatment with ERK, JNK, and NF-κB inhibitors. TonEBP/
NFAT5 expression was increased by macrophages, resulting in increased cisplatin resistance, cell migration, andinvasion. Moreover, RNAi-mediated knockdown of TonEBP/NFAT5 reduced cisplatin resistance, migration, and invasionin macrophage CM-induced A549 cells.
Conclusions These findings demonstrate that paracrine factors secreted from macrophages can change A549 cells,
resulting in the induction of drug resistance against cisplatin and migration. In addition, the TonEBP/NFAT5 ratio,
increased by macrophages, is an important regulator of the malignant transformation of cells.
Keywords A549, Cisplatin resistance, Migration, TonEBP/NFAT5*Correspondence:
Sang Do Lee
sdlee@cnu.ac.kr
1
Department of Physiology, Chungnam National University College ofMedicine, Daejeon, Republic of Korea
2
Department of thoracic surgery, Chungnam National University Schoolof Medicine, Daejeon, Republic of Korea© The Author(s) 2024. 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) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed 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 data made available
in this article, unless otherwise stated in a credit line to the data.

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Background
Lung cancer is the leading cause of cancer-related deaths
worldwide, owing to complications affecting early diagnosis, poor cell reactivity to anticancer drugs, andmetastasis [1–4]. Metastasis occurs due to cancer cell
migration and invasion. Cancer cells spread throughout
the body via the blood or lymph, and metastasis occurs as
a result of penetration and proliferation of the migrated
cancer cells into other tissues. As cancer cells migrate
through the lymph, they often metastasize to tissues
rich in blood vessels, such as the liver, lungs, and brain.
Metastatic cancer cells have a high growth rate and low
reactivity to anticancer drugs, thereby reducing patient
survival rates [5–8]. Therefore, the genes that regulate
cancer cell metastasis warrant extensive investigation.
Cancer cells are surrounded by the tumor microenvironment, which includes fibroblasts, leukocytes, theextracellular matrix, and macrophages [9]. Cancer andstromal cells in the tumor microenvironment secrete
several cytokines, chemokines, and growth factors.
Secreted soluble factors recruit macrophages from thebloodstream to the tumor periphery. Thus, the numberof macrophages surrounding a tumor can predict cancer
prognosis because they promote angiogenesis, metastasis, and drug resistance in several cancers. The infiltrationlevel of macrophages into the tumor microenvironmentis associated with poor prognosis in lung, breast, ovarian,stomach, and prostate cancers [10, 11].
Macrophages enhance the intravasation of cancer cells,
which is strongly correlated with their migration throughblood vessels [12]. Tumor-associated macrophages
release C-C motif ligand 18 (CCL18) and the granulocyte-macrophage colony-stimulating factor (GM-CSF),
which promote breast cancer invasion and metastasis[13, 14]. Furthermore, tumor-associated macrophages
promote gemcitabine resistance in pancreatic cancer bymodulating cytidine deaminase [15]. M2 type tumorassociated macrophages are associated with increased
resistance to etoposide in lung and liver cancers [16]. In
addition, tumor-associated macrophages promote cisplatin resistance in cancer stem or initiating cells by secreting milk fat globule-EGF factor 8 (MFG-E8) [17].
Cisplatin, a platinum-based drug, is one of the mostwidely used anticancer drugs [18, 19]. The efficacy of cisplatin depends on the type of cancer it is used to treat.
When cisplatin efficacy against certain types of cancer
is low, its concentration is increased to enhance its anticancer activity. However, cisplatin at higher concentrations causes adverse side effects, such as nephropathy,
nausea, vomiting, and neurotoxicity [20–22]. Therefore,
the dependency of cisplatin efficacy on cancer cell characteristics warrants further study. Further studies could
facilitate the development of drugs that can increase cell
reactivity and decrease resistance to cisplatin.

Page 2 of 11

Tonicity-responsive enhancer binding protein/nuclear
factor of activated T cells 5 (TonEBP/NFAT5) plays
a key role in the maintenance of renal function. Thistranscription factor is widely expressed in most tissues
and [23–25] is closely associated with the regulation of
inflammatory responses [26, 27]. TonEBP/NFAT5 plays
a key role in the differentiation of various cells, including adipocytes [28, 29]. It is associated with malignancy
and induces metastasis by promoting S100A4 expression in renal carcinoma and colon cancer cells [30, 31].
Furthermore, integrin-induced transcriptional activityof TonEBP/NFAT5 causes cancer cell metastasis [32].
In addition, its expression is closely associated withdecreased survival rate of patients with lung cancer [33].
Therefore, it is imperative to investigate the role of thistranscription factor in lung cancer.
In this study, we investigated the signaling pathways
that are activated by macrophages and required to promote cisplatin resistance and induce migration in lungcancer. Additionally, we investigated the relationshipbetween TonEBP/NFAT5 expression and cisplatin resistance and migration in A549 cells.

Results
Cisplatin resistance and migration in A549 cells co-cultured
with macrophages

A549 lung adenocarcinoma cells were indirectly cocultured with THP-1-derived macrophages in a Boydenchamber to investigate the effect of macrophages on cancer cells. The viability of A549 cells was 60%, 35%, and< 20% at 50 µM, 100 µM, and 200 µM cisplatin, respectively. In contrast, A549 cells co-cultured with macrophages for 24 h maintained a cell viability of 80% at all
cisplatin concentrations, indicating that macrophages
induced cisplatin resistance (Fig. 1A). The induction of
cisplatin resistance by macrophages was significant froma co-culture time of 8 h (65.7%), and the most robust
resistance was obtained at 24 h (80.2%) (Fig. 1B). Here,macrophages induced migration and cisplatin resistancein cancer cells. The wound-healing assay revealed thatthe migration of A549 cells co-cultured with macrophages was increased compared to that of cells that were
not co-cultured (Fig. 1C).
Induction of cisplatin resistance and migration bymacrophage CM in A549 cells

In this study, indirect co-culture with macrophages indicated that soluble factors secreted from these effector
cells might act on cancer cells to induce cisplatin resistance and migration. To confirm this, conditioned media(CM) was prepared from the culture supernatants ofmacrophages and used to treat A549 cells. Similar to the
co-culture, this macrophage CM considerably increased
cisplatin resistance (8 h:66.4%, 24 h:86.4%). In contrast,

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Page 3 of 11

Fig. 1 Effect of macrophage co-culture on cisplatin resistance and migration. (A) A549 cells were cultured alone or co-culutred with THP-1 derived macrophages for 24 h and then treated with various concentrations (0 ~ 400 µM) of cisplatin for 24 h. (B) A549 cells were cultured alone or co-culutred with
THP-1 derived macrophages for 8 or 24 h and then treated with 200 µM cisplatin for 24 h. The cell viability was determined using MTT assay. (C) Migration
ability was determined using a wound healing assay in A549 cells co-cultured with macrophages for 24 h. Data are average values ± SD of at least six
independent experiments. ** p < 0.01, *** p < 0.001 (compared with alone and 0 h)

monocyte CM (47.8%) did not show any effect (Fig. 2A
and B).
To confirm whether macrophage CM-induced cisplatin
resistance was caused by apoptosis, early apoptotic cells
were observed using Annexin-V staining. Early apoptotic
cells increased when treated with cisplatin (41.5%). However, these apoptotic cells were substantially reduced inmacrophage CM-induced cells (11.7%) (Fig. 2C). Thus,
DNA fragmentation that occurs during apoptosis was
subsequently investigated. DNA fragmentation was notobserved in macrophage CM-induced cells, even after
cisplatin treatment (Fig. 2D). In addition, the cleavageof PARP and caspase-3, which are apoptosis markers,
was investigated. Cleaved PARP and caspase-3 levels,
which were increased by cisplatin in non-induced cells,
decreased in macrophage CM-induced cells (Fig. 2E).
In a previous experiment, migration increased in A549
cells co-cultured with macrophages. This increase inmigration was also induced in macrophage CM. In thisstudy, increased cell migration was observed with macrophage CM in the transwell migration assay (423.2%).
However, monocyte CM did not induce migration(94.0%) (Fig. 3A). The increase in migration by macrophage CM was also confirmed through the wound healing assay (Fig. 3B).
Induction of cisplatin resistance and migration bymacrophage CM in the NSCLC cell line

We investigated whether cisplatin resistance inducedby macrophage CM was specifically induced in A549
cells or typically induced in non-small cell lung cancer(NSCLC) cell lines. Similar to A549 cells (68.6%), cisplatin resistance was significantly induced in Calu-3 (58.2%)
and H460 (46.9%) cells following macrophage CM treatment. However, cisplatin resistance was not induced in
H1299 cells (8.2%), even when cultured in macrophage

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Page 4 of 11

Fig. 2 Induction of cisplatin resistance by macrophage CM. (A) A549 cells were incubated with macrophage conditioned media (CM) for 8 or 24 h andthen treated with 200 µM cisplatin for 24 h. (B) A549 cells were incubated with monocyte or macrophage conditioned media (CM) for 24 h and thentreated with 200 µM cisplatin for 24 h. Cell viability was evaluated using a MTT assay. (C-E) A549 cells were induced with macrophage CM and treatedwith 200 µM cisplatin for 8 h. Cisplatin induced early apoptotic cells were identified using Annexin V and PI staining. Cisplatin induced DNA fragmentation was identified using DAPI staining. Cleavage of apoptotic proteins, PARP and caspase-3 were determined by immunoblotting. Full-length blots arepresented in Supplementary Fig. [1]. Data are average values ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01 (compared with 0 h
and no cisplatin-treated group), ## p < 0.01 (compared with CM-induced cisplatin-treated group)

CM (Fig. 4A). Migration induced by macrophage CM
was also investigated in NSCLC cell lines. Migration wasincreased by macrophage CM only in Calu-3 (172.2%)
and H1299 cells (118.5%), whereas it was not increased in
H460 cells (180.3%) (Fig. 4B).
Induction of cisplatin resistance and migration bymacrophage CM via the ERK, JNK, and NF-κB pathways

Several cytokines have been reported to increase thesurvival and migration of cancer cells by activating themitogen-activated protein kinase (MAPK) and NF-κB
signaling pathways. Therefore, we examined the effect ofmacrophage CM on the activation of these pathways in

A549 cells. As shown in Fig. 5A, phosphorylation of ERK,
p38, and JNK and degradation of IκBα were markedly
increased by macrophage CM but not by monocyte CM.
The maximal activation of these pathways was observed
after 15–30 min. Macrophage-induced phosphorylationof ERK was maintained for 2 h, and activation of p38,
JNK, and NF-κB disappeared after 1 h. Furthermore,
NF-κB promoter activity was considerably increased byincubating cells with macrophage CM for 8 h (234%).
However, monocyte CM had no effect (74.7%) (Fig. 5B).
A549 cells were treated with specific inhibitors of eachsignaling pathway during induction with macrophage CM
to assess the participation of ERK, p38, JNK, and NF-κB

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Page 5 of 11

Fig. 3 Induction of migration by macrophage CM. (A)A549 cells were incubated with monocyte or macrophage conditioned media (CM) for 24 h. Migration ability was determined using a wound healing assay. (B) Migrated cells were quantitated using a trans migration assay. Data are average values ± SD
of at least five independent experiments. *** p < 0.001 (compared with Non-induced)

Fig. 4 Macrophage CM induces cisplatin resistance and migration in NSCLC. (A) A549, Calu-3, H460, and H1299 cells were incubated with macrophage
conditioned media (CM) for 24 h and then treated with 200 µM cisplatin for another 24 h. Cell viability was evaluated using a MTT assay. (B) Migrated cells
were quantitated using a trans migration assay. Data are average values ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01 (comparedwith Non-induced)

signaling in macrophage-induced cisplatin resistance.
The specific inhibitor, PD98059, blocks ERK phosphorylation. SB203580 and SP600125 inhibit p38 and JNK
activity, respectively. IKK2 inhibitor IV inhibits IKK2,
an upstream factor of IκBα signaling. Inhibition of ERK(33%) or NF-κB (47.2%) activity substantially suppressed
the induction of cisplatin resistance by macrophage CM.
However, p38 (95.8%) and JNK (101.4%) inhibition did
not affect cisplatin-induced cell death (Fig. 5C). To confirm the involvement of the ERK and NF-κB pathways
in macrophage-induced cisplatin resistance, the effectof each inhibitor on cisplatin-induced apoptosis was
examined. Cells were induced with macrophage CM containing ERK or NF-κB inhibitors and then exposed tocisplatin for 8 h. However, caspase-3 and PARP cleavage
was completely inhibited in A549 cells incubated withmacrophage CM. Inhibition of ERK activation during the
incubation of A549 cells with macrophage CM induced

levels of apoptosis similar to those observed in the control cells, whereas NF-κB inhibition did not (Fig. 5D).
To investigate the involvement of the MAPK and
NF-κB signaling pathways in the enhanced migration,
treatment with each inhibitor was performed during theinduction of A549 cells with macrophage CM. The number of transmigrated cells was considerably reduced byinhibiting ERK and JNK activation, but not by the p38
inhibitor (Fig. 5E). Inhibition of macrophage-induced
NF-κB signaling also suppressed the macrophageinduced transmigration ability of A549 cells (Fig. 5E). The
participation of ERK, JNK, and NF-κB in macrophageenhanced migration was confirmed using wound healing
assays (Fig. 5F).

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Page 6 of 11

Fig. 5 Signaling pathways are involved in macrophage-induced cisplatin resistance and migration. (A) A549 cells were incubated in monocyte or macrophage CM for the indicated times. Phosphorylation or the expression level of each protein was detected by western blotting using specific antibodies.
The experiment was repeated with similar results. (B) A549 cells were transfected with an NF-κB-dependent luciferase expression plasmid and thenincubated in CM or treated with 15 ng/ml TNF-α for 8 h. (C) A549 cells were induced with macrophage CM in the presence or absence of each inhibitorfor 24 h, respectively. Viable cells in each condition were measured and compared with the corresponding control. (D) A549 cells were incubated withmacrophage CM containing PD98059 or IKK2 inhibitor IV (IKK2 i). Cisplatin was added into each cell for 8 h. Whole cell lysates were subjected to western
blotting. (E, F) A549 cells were treated with macrophage CM containing each inhibitor for 24 h. Migration ability was determined using a trans migration
assay and a wound healing assay. The wound healing assay was repeated six times and representative images are shown. Full-length blots are presented
in Supplementary Fig. (2). Data are average values ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (compared with
Non-induced and vehicle)

Effect of TonEBP/NFAT5 expression of cisplatin resistance,
migration, and invasion in macrophage-induced A549 cells

TonEBP/NFAT5 expression is closely associated with thesurvival rate of patients with lung cancer. Therefore, we
confirmed that TonEBP/NFAT5 expression was affectedby macrophages. TonEBP/NFAT5 expression increased
after 4 h of incubation with macrophage CM and was thehighest after 24 h of incubation (Fig. 6A). The increased
TonEBP/NFAT5 expression in macrophage-conditioned
medium was inhibited using siRNA (Fig. 6B).
To investigate the effects of TonEBP/NFAT5 expression on cisplatin resistance in macrophage CM-induced
A549 cells, the cells were treated with cisplatin for 12 hafter suppressing TonEBP/NFAT5 expression. In noninduced cells, apoptotic cells accounted for 24.0% of thetotal cell count after 200 µM cisplatin treatment for 12 h.
In contrast, the percentage of apoptotic cells significantly
increased to 53.0% in the cells with suppressed TonEBP/
NFAT5 expression. When A549 cells induced by macrophage CM were treated with cisplatin, the percentage
of apoptotic cells was 15.2%, which was lower than thatof non-induced cells. When TonEBP/NFAT5 expression

was inhibited in macrophage CM-induced A549 cells, thepercentage of apoptotic cells significantly increased to54.7% (Fig. 6C).
The migration ability of cells with suppressed TonEBP/
NFAT5 expression was determined using wound healing assays to investigate the effect of TonEBP/NFAT5 on
cell migration. Cells migrated in the control group when
cultured 24 h after wounding, but not when TonEBP/
NFAT5 expression was suppressed. However, TonEBP/
NFAT5-suppressed cells did not show increased cell
migration ability in macrophage CM (Fig. 6D). The effectof TonEBP/NFAT5 expression on cell invasion was investigated using Matrigel. After wounding, Matrigel wasadded to the upper layer, and invasion was measured byobserving the cells infiltrated with Matrigel after 48 h.
Invasion was observed in the control group but not after
the suppression of TonEBP/NFAT5 expression. Furthermore, increased invasion by macrophages decreased
when TonEBP/NFAT5 expression was suppressed(Fig. 6E).

Song et al. BMC Molecular and Cell Biology(2024) 25:6

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Fig. 6 Effect of TonEBP expression of cisplatin resistance, migration and invasion in macrophage-induced A549 cells. (A) A549 cells were incubated inmacrophage CM for the indicated times. The expression of TonEBP was analyzed by immunoblotting. (B) A549 cells were transfected with negative control or TonEBP siRNA. TonEBP knocked down cells were incubated in macrophage CM for 24 h. (C) Apoptosis was measured by Annexin-V assay. TonEBP
knocked down cells were treated with 200 µM cisplatin for 8 h. Annexin-V and PI-stained cells were analyzed using flow cytometry. (D-E) TonEBP knocked
down cells were incubated in macrophage CM for 24 h. Migration ability was determined using wound healing assay. Invasion ability was measured byadding 200 µL Matrigel after wounding. Full-length blots are presented in Supplementary Fig. (3). Data are average values ± SD of at least three independent experiments. * p < 0.05, (compared with siNC)

Discussion
Macrophages around cancer tissues are differentiated byvarious soluble factors secreted by cancer cells, which
promote cancer growth and progression. They specifically affect the migration and invasion of cancer cells andthe formation of new blood vessels during cancer progression [34]. In this study, cisplatin resistance occurred
when induced by macrophages for at least 8 h in A549
cells. In addition, macrophage-induced A549 cells
showed increased migration compared to non-induced
A549 cells.
Induction of cisplatin resistance and cell migration bymacrophages was not observed in A549 cells alone. Cisplatin resistance and cell migration were induced by macrophages in the Calu-3 and H460 NSCLC cell lines and in
Calu-3 and H1299 cells, respectively. Cisplatin resistanceor cell motility depends on the type of cell line because
the receptors expressed in each cell are different [35, 36].
In addition, the signal transduction of cells that increase
cisplatin resistance or cell motility may be different even
if the cell receptors are the same [37, 38].
In human cancer tissues, necrosis occurs due to a lowoxygen environment and nutrient deficiency because newblood vessels do not reach the inside of the early cancer
tissue [39]. Necrotic cells secrete various inflammatory
factors that induce other macrophages. As a result, many
macrophages surround cancer tissue, thereby accelerating cancer progression [40].
The involvement of the ERK, p38, JNK, and NF-κB
pathways in macrophage-induced cisplatin resistanceand migration of A549 cells was investigated. Inhibitionof macrophage-stimulated ERK signaling considerably

Song et al. BMC Molecular and Cell Biology(2024) 25:6

suppressed the induction of cisplatin resistance and protected cells from cisplatin-induced apoptosis. Similarly,
suppression of the ERK, JNK, and NF-κB signaling pathways effectively inhibited macrophage-induced migration. These results suggest that macrophage-induced
cisplatin resistance and migration are regulated by different mechanisms. Macrophages secrete not only interleukin family members, but also TNF, CXCL, and growth
factors [41, 42]. In IL-6 knockout mice, tumor growthand formation were downregulated, and proliferation
markers and MMP-9 were reduce [43]. In addition, IL-6
prevents etoposide- and melphalan-induced apoptosis
in neuroblastoma [44]. Transforming growth factor-β(TGF-β) can increase the resistance of lung adenocarcinoma cells to gefitinib [45]. Moreover, EGF increases
breast cancer cell migration in synergy with IL-1β [46].
Similarly, TNF-α enhances migration and metastasis
through several signaling pathways [47]. Chemokines,
including CXCL10, CXCL12, CX3CL1, CCL2, CCL5,
CCL18, and CCL20, induce chemotaxis to promote cell
migration and invasion in ovarian, lung, breast, andcolorectal cancers [14, 48–52]. The involvement of these
secretory factors in macrophage-induced cisplatin resistance or migration can be determined through genetargeting in macrophages or inhibition of the action ofreceptors in cancer cells. Cancer cells can become resistant to multiple drugs through several mechanisms.
ATP-binding cassette (ABC) transporters such as ABCA,
ABCB, ABCC, and ABCG mediate resistance to multiple
drugs [53]. ABCB 1 and ABCC 3 are also regulated by
ERK, which affects the response of mesothelioma cells
to doxorubicin [54]. DNA repair pathways also confer
resistance to chemotherapy in cancer cells [55]. Platinum chemotherapeutic agents, such as carboplatin, cisplatin, and oxaliplatin, induce DNA damage in cells.
However, drug resistance by DNA repair proteins canoccur. Excision repair cross-complementation group 1(ERCC1) plays an important role in nucleotide excision
repair [56]. Increased ERCC1 expression by ERK causes
cisplatin resistance in melanoma [57]. In addition, breast
cancer genes 1 and 2 (BRCA 1 and BRCA 2), which are
DNA repair genes, are also regulated by ERK to modulate
the response to doxorubicin [54]. As ERK regulates ABC
transporters and DNA repair genes, the contribution of
ERK downstream factors to macrophage-induced cisplatin resistance warrants further investigation.
Metastasis is a multifaceted process involving the
migration and invasion of cancer cells through thedegraded extracellular matrix (ECM). MMP-2 and
MMP-9 are ECM-degrading enzymes, and u-PA activates
this enzymatic cascade that can eventually initiate metastasis. Activation of these proteins is frequently detectedin cancers [58–60]. MMP-9 and u-PA can be controlled
by EGF through JNK/AP-1 signaling, consequently

Page 8 of 11

promoting cell migration [59]. Downregulation of ERK
and NF-κB signaling pathways by gypenosides, used
as folk medicine in China, inhibits activation of MMP2, MMP-9, and u-PA. This results in the suppression of
migration and invasion of human oral cancer cells [61].
Furthermore, the downregulation of MMP-2 and MMP-9
by inhibiting ERK and NF-κB activity reduces colon cancer cell migration and invasion [62]. Thus, the identification of the downstream target could help elucidate therelationship between ERK, JNK, and NF-κB signals andthe macrophage-mediated migration effects on MMPs
and u-PA.
A recent study showed that the survival rate of patientswith lung cancer is linked to TonEBP/NFAT5 expression.
The survival rate of patients with lung cancer was considerably lowered with increased TonEBP/NFAT5 expression [33]. Moreover, disease-free survival is closely linked
to tumor cell proliferation, cell reactivity to anticancer
drugs, and metastasis [63, 64]. Therefore, we investigated
the relationship between macrophages and TonEBP/
NFAT5 expression. TonEBP/NFAT5 expression increased
with incubation time in the macrophage culture medium.
In addition, the suppression of TonEBP/NFAT5 expression by macrophages decreased cell migration and invasion. These results showed that macrophages increased
TonEBP/NFAT5 expression in cancer cells, thereby
increasing cell mobility and invasiveness. A previous
study focused on macrophage polarization by TonEBP/
NFAT5 [65]. The results of this study showed that macrophages contribute to cell malignancy by regulating
TonEBP/NFAT5 expression.

Conclusions
In conclusion, paracrine factors secreted from macrophages can alter lung cancer A549 cells, resulting in the
induction of drug resistance against cisplatin via ERK
signaling. Contrastingly, ERK, JNK, and NF-κB signalingenhance migration. In addition, macrophages increase
TonEBP/NFAT5 expression, which is an important regulator of the malignant transformation of lung cancer cells.
Our findings could help develop therapies that inhibit themetastasis of cancer cells by regulating TonEBP/NFAT5
expression.
Methods
Cell culture and preparation of macrophage conditioned
media

Human lung cancer A549 cells and monocytic leukemia
THP-1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Both cell
types were cultured in RPMI 1640 medium (Welgene,
Daegu, South Korea) supplemented with antibiotics(Life Technologies, Carlsbad, CA, USA) and heat-inactivated 10% fetal bovine serum (FBS; GE Healthcare Life

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Sciences, Victoria, Australia) in a 5% CO2 atmosphere at
37 °C. The cells were routinely subcultured at approximately 80% confluence. Furthermore, THP-1 cells weretreated with 30 nM phorbol myristate acetate (PMA;
Sigma-Aldrich, Saint Louis, MO, USA) and cultured for
24 h to obtain macrophages. THP-1 cells that differentiated into macrophages were cultured in fresh culture
medium for 24 h. Then, only the supernatant (macrophage-conditioned media) was collected and added to
A549 cells.
Measurement of cell viability

Cell viability was measured using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The cells were seeded in 24-well plates at a densityof 40,000 cells/well. After induction with conditioned
medium (CM) for indicated time, various concentrations of cisplatin were treated. Then, 20 µL MTT solution(5 mg/mL in phosphate-buffered saline; Sigma, St. Louis,
MO, USA) was added to each well and the samples incubated at 37 °C for 3 h to obtain formazan crystals. The
formazan crystals were dissolved in 500 µL dimethyl
sulfoxide. Finally, cell viability was measured at 570 nm
using a microplate reader (TECAN, Hombrechtikon,
Switzerland).
Measurement of apoptosis

Apoptosis was measured by staining cells with Annexin
V-FITC (BD Biosciences, San Jose, CA, USA) and propidium iodide. Cells were plated at a density of 1 × 106 cells
in a 100-mm dish. And incubated for 24 h in the presenceof conditioned media the cells were treated with 200µM
cisplatin for 8 h and then trypsinized for FACs analysis.
The cells were then resuspended in 500 µL DPBS containing Annexin V-FITC and propidium iodide. The fluorescence intensity of each cell plate was measured using
a BD FACSCalibur instrument (BD Biosciences), and thedata were analyzed using FlowJo software (Tree Star Inc.,
Ashland, OR, USA).
Assessment of apoptosis by DAPI staining

Cells were plated in 6-well plate. After 24 h, cells weretreated with various concentrations of cisplatin for 8 and12 h. Cells were fixed in methanol/DMSO (4:1) solution
at 4 °C. Cells were washed with PBS and then stainedwith DAPI. Stained cells were observed under the fluorescence microscope (Zeiss, Oberkochen, Germany).
Measurement of cell migration and invasion

Cell migration was measured using wound healing andtransmigration assays. In the wound healing assay, cells
were scraped using a micropipette tip to ensure disruption. Subsequently, images were captured at 100× magnification using the Nikon Eclipse Ti inverted microscope

Page 9 of 11(Nikon, Tokyo, Japan). Cell invasion was then measured
by adding 200 µL Matrigel (BD Biosciences), and theculture medium was replaced with a fresh culture solution. The transmigration assay was performed in a Boyden chamber with an 8-µm pore size and 24-well insert(Corning Inc., Corning, NY, USA). The treated cells were
washed with DPBS and seeded into the upper chamber
in serum-free RPMI medium. A medium containing 10%
FBS was placed in the bottom well. The cells were stainedwith 30 µg/mL Hoechst 33,342. Then, images of stained
cells on the membrane were captured using a fluorescence microscope (Zeiss, Oberkochen, Germany), andthe cell count was determined using ImageJ software.
Similar inserts coated with 100 µL Matrigel were used tomeasure cell invasion.
Small interfering RNA (siRNA) transfection

Dicer-substrate siRNA targeting TonEBP/NFAT5 RNA
was used to knockdown TonEBP/NFAT5 expression. The
siRNA sequence used to target TonEBP/NFAT5 RNA
was
5’-​A​U​G​U​U​U​C​U​G​A​U​G​A​A​A​A​C​U​C​U​U​G​G​A​A-3’,
3-‘​U​U​U​A​C​A​A​A​G​A​C​U​A​C​U​U​U​U​G​A​G​A​A​C​C​U​U-5’. All
siRNA was purchased from Integrated DNA Technologies (DS ScrambledNeg; Coralville, IA, USA). A549 cells
were transfected with TonEBP/NFAT5 RNA or negative
control siRNA for 48 h using the Lipofectamine™ 2000
transfection reagent (Invitrogen, Waltham, MA, USA)according to the manufacturer’s instructions.
Western blot analysis

Cells were lysed using 200 µL radio-immunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1%
NP-40, 0.5% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate, pH 8.0). Equal amounts of protein wereseparated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro transferred onto nitrocellulose membranes. Membranes were
blocked using Tris-buffered saline (20 mM Tris-HCl and
140 mM NaCl, pH 7.6) containing 0.1% Tween 20 and
5% non-fat dry milk at room temperature for 2 h. Subsequently, the membranes were incubated with primary
antibodies at 4 °C overnight. They were then washed andincubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Signals were detected using achemiluminescence assay kit (Thermo Fisher Scientific,
Waltham, MA, USA).
Statistical analysis

Statistical significance was determined using Tukey’s
multiple comparison test (SigmaStat; Jandel, San Rafael,
CA, USA). Data are expressed as mean ± standard error.
Finally, statistical significance was set at P < 0.05.

Song et al. BMC Molecular and Cell Biology(2024) 25:6

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-024-00502-y.
Additional file 1 of “TonEBP/NFAT5 expression is associated with cisplatin
resistance and migration in macrophage-induced A549 cells”
Acknowledgements
We would like to thank Editage (www.editage.co.kr) for editing and reviewing
this manuscript for English language.
Author contributions
HJS performed the data analysis, interpreted the results and wrote themanuscript. YHK and HNC performed cell viability, migration and invasion. TK
conducted the flow cytometry analysis, DAPI staining, western blot analysis
and analyzed the data. SJK contributed to study design and interpretation ofdata. MWK contributed to the critical revision of article. SDL contributed tofunding acquisition, study design, study coordination, interpretation of thedata and helped to draft the manuscript. All authors read and approved thefinal manuscript.
Funding
This work was supported by research fund of Chungnam National University.
Data availability
Not applicable.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 7 June 2023 / Accepted: 26 February 2024

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