Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Experimental Cell Research

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Research article

Diverse effects of G-protein-coupled free fatty acid receptors on the regulation

of cellular functions in lung cancer cells
Tsubasa Kita a, Yui Kadochi a, Kaede Takahashi a, Kaori Fukushima a, Eri Yamasaki a, Taiki Uemoto a,
Miku Hirane a, Nobuyuki Fukushima b, Kanya Honoki c, Toshifumi Tsujiuchi a,n
a
Division of Molecular Oncology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan
b
Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan
c
Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan

ar t ic l e i nf o a b s t r a c t

Article history: Free fatty acids (FFAs) are dietary nutrients which mediate a variety of biological effects through binding
Received 27 January 2016 to G-protein-coupled FFA receptors (FFARs). G-protein-coupled receptor 120 (GPR120) and GPR40 are
Received in revised form identified as FFARs for long- and medium-chain fatty acids. Here we investigated whether GPR120 and
7 March 2016
GPR40 are involved in the acquisition of malignant properties in lung cancer cells. Three lung cancer
Accepted 7 March 2016
RLCNR, LL/2 and A549 cells used in this study expressed GPR120 and GPR40 genes. The cell motile ac-
tivities of all cells were significantly suppressed by a GPR40 antagonist GW1100. In addition, GPR40
Keywords: knockdown inhibited the cell motile activity of A549 cells. In gelatin zymography, matrix metallopro-
GPR120 teinase-2 (MMP-2) activity in GPR40 knockdown was significantly lower than that in control cells. Next,
GPR40
to evaluate effects of GPR120 and GPR40 on cellular functions induced by anti-cancer drug, the long-term
Cell motility
cisplatin (CDDP) treated (A549-CDDP) cells were generated. The expression levels of GPR120 and GPR40
Invasion
Lung cancer were significantly decreased in A549-CDDP cells. While A549-CDDP cells showed the high cell motile
activity, GW1100 suppressed the cell motile activity of A549-CDDP cells. These results demonstrate that
GPR120 negatively and GPR40 positively regulate cellular functions during tumor progression in lung
cancer cells.
& 2016 Elsevier Inc. All rights reserved.

1. Introduction compared with adjacent noncancerous tissues. The activation of

G-protein-coupled receptors (GPCRs) are seven-transmembrane
receptors which mediate several cellular functions, including cell
growth, motility, differentiation and morphogenesis [1–3]. Free fatty
acid (FFA) receptors belong to a member of GPCRs and exhibit a
variety of biological responses via binding of FFAs [4–6]. GPCR 120
(GPR120) and GPR40 are identified as GPCRs for FFAs. FFAs are es-
sential dietary nutrients and classified by the length of carbon
chains. GPR120 and GPR40 are activated by long- and medium-chain
FFAs [7,8]. The distribution of GPR120 and GPR40 expressions is
closely related to tissues for metabolic controls. Moreover, GPR120
and GPR40 are involved in the regulation of hormone secretion from
pancreatic islet cells and digestive tract [9–11]. Therefore, GPR120
and GPR40 are considered as potent target molecules for the treat-
ment of metabolic disorders, such as diabetes and obesity [12–14].
Recently, it has been reported that GPR120 and GPR40 play im-
portant roles in the pathogenesis of cancer cells. The aberrant ex-
pressions of GPR120 were detected in colorectal carcinomas,
GPR120 enhanced cell motile activity and angiogenic potency of colon
cancer cells [15]. In contrast, GPR40 inhibited cell motile and invasive
activities of fibrosarcoma cells [16]. In addition, GPR120 increased and
GPR40 decreased cell motile and invasive activities of pancreatic
cancer cells [17]. Therefore, these findings suggest that opposite effects
of GPR120 and GPR40 regulate cellular functions of cancer cells.
Lung cancer is one of the most common human malignancies
[18], but the rate-limiting molecular events during the develop-
ment of lung cancer remain to be clarified. In the present study, to
assess whether intracellular signaling via GPR120 and GPR40 may
be a candidate molecule for chemotherapeutic treatment of lung
cancers, we investigated the effects of GPR120 and GPR40 on
cellular functions in three lung cancer cells.
2. Materials and methods
2.1. Cell culture and treatment

n
Corresponding author. Rat RLCNR, mouse LL/2 and human A549 cells used in this
E-mail address: ttujiuch@life.kindai.ac.jp (T. Tsujiuchi). study were cultured in Dulbecco's modified Eagle's medium

http://dx.doi.org/10.1016/j.yexcr.2016.03.008
0014-4827/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: T. Kita, et al., Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular
functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
2 T. Kita et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
(DMEM) (Wako Pure Chemical Industries Ltd., Osaka, Japan) con-
taining 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at
37 °C. To generate the long-term anti-cancer drug treated cells,
A549 cells were treated by the stepwise treatment of cisplatin
(CDDP) (Sigma, St. Louis, MO, USA) at a range of 0.01–1.0 μM for at
least 6 months [16].
2.2. Cell proliferation assay
Cells were seeded at 4000 cells/well in 96-well plates and
maintained in DMEM containing 10% FBS. The cell proliferation
rate was measured with the Cell Counting Kit-8 (CCK-8) (Dojin
Chemistry, Kumamoto, Japan). A solution from CCK-8 was added to
each plate at 0, 1 or 3 days. To examine effects of GPR120 and
GPR40 on cell proliferation, cells were cultured in DMEM con-
taining 5% charcoal stripped FBS (Sigma) and treated with
GW9508 (Sigma) and GW1100 (Sigma) at concentrations of 1–
10 μM every 24 h for 3 days [16,17].
2.3. Cell motility assay
Cells were pretreated with GW9508 (10 μM) or GW1100 (1 μM)
for 30 min and seeded into a Cell Culture Insert (BD Falcon, NJ,
USA) with 8 mm pore size at 1
105 cells in 200 μl of serum-free
DMEM (upper chamber). The filters were placed in 24-well plates
(lower chamber) containing 800 μl of DMEM supplemented with
5% charcoal stripped FBS with GW9508 (10 μM) and cultured for
20 h. The percentage of cells moved to the lower side of the filter
was counted after Giemsa staining [16,17].
2.4. Establishment of GPR40 knockdown cells from A549 cells
GPR40 knockdown (A549-40) cells were established using the
X-tremeGENE HP Transfection Reagent (Roche Diagnostics Co. Ltd.,
Mannheim, Germany). Briefly, a HuSH short hairpin RNA plasmid
(29-mer) against human GPR40 (Origene, Rockville, MD) was
transfected into A549 cells. After 3 days, the transfected cells were
treated with puromycin (Wako) for at least 2 weeks. Control
(A549-R) cells were also generated using a vector plasmid without
the target sequence [16,17].
2.5. Gelatin zymography
Cells were maintained in serum-free DMEM with or without
GW9508 (10 μM) for 48 or 72 h. The supernatants obtained from
the individual cells were loaded on a 10% SDS-PAGE containing
0.1% gelatin. After electrophoresis, the gels were washed twice
with washing buffer (50 mM Tris–HCl (pH 7.5), 100 mM NaCl and
2.5% Triton X-100) for 30 min and incubated in reaction buffer
(50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 10 mM CaCl2 and 0.02%
NaN3) at 37 °C for 16 h. The gels were stained with 0.25% Coo-
massie Brilliant Blue R250 (Wako). The densities of bands were
quantitatively determined with image analysis software (NIH Im-
age, Bethesda, MD) [16,17].
2.6. Tube formation assay
To assess effects of GPR120 and GPR40 on tube formation of
endothelial F-2 cells, cells were maintained in serum-free DMEM
for 2 days. For tube formation assay, Matrigel (BD Falcon, NJ, USA)
at 100 μl/well was plated in 96-well plates and incubated at 37 °C.
After 30 min, F-2 cells were seeded onto Matrigel-coated plates at
4
104 cells in 100 μl of supernatants from the individual cells and
cultured at 37 °C for 4 h. The length of total tube formation was
measured from three representative 100
fields/well [19].
Please cite this article as: T. Kita, et al., Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular
functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
2.7. Quantitative real-time reverse transcription-polymerase chain
reaction (RT-PCR) analysis
After RNA extraction using ISOGEN (Nippon Gene, Inc. Toyama,
Japan), cDNA was synthesized with a Transcriptor First Strand
cDNA Synthesis Kit (Roche Diagnostics). For quantitative real-time
RT-PCR analysis, the target genes were amplified with SYBR Premix
Ex Taq (Tli RNaseH Plus) (TaKaRa Bio Inc., Shiga, Japan) and the
expression values were measured using a Smart Cycler II System
(TaKaRa) [16,17].
2.8. Statistical analysis
Quantitative differences were statistically analyzed using AN-
OVA (analysis of variance) with a post-hoc test. The data were
recognized to differ significantly for values of p o0.01. The results
were given as means 7SD.
3. Results
3.1. Roles of Gpr120 and Gpr40 on cell proliferation and motility in
RLCNR and LL/2 cells
The expression patterns of Gpr120 and Gpr40 genes in RLCNR
and LL/2 cells are shown in Fig. 1A. To assess effects of Gpr120 and
Gpr40 on cell proliferation and motile activity, cells were treated
with GW9508 which is an agonist of GPR120 and GPR40 [20,21]. In
addition, GW1100 was used as a GPR40 antagonist [20–22]. In cell
proliferation assay, no effect of GW9508 and GW1100 on cell
growth rate was found in RLCNR and LL/2 cells (Fig. 1B). The cell
motile activities of RLCNR and LL/2 cells were significantly sti-
mulated by GW9508, compared with untreated cells. On the other
hand, the cell motile activities of both cells stimulated by GW9508
were markedly suppressed by GW1100 (Fig. 1C).
3.2. Roles of GPR120 and GPR40 on cell proliferation and motility in
A549 cells
The expressions of GPR120 and GPR40 genes were detected in
A549 cells (Fig. 2A). While no change of cell proliferation rate was
found in A549 cells treated with GW9508 alone, the treatment of
GW9508 and GW1100 inhibited the cell proliferation rate of A549
cells (Fig. 2B). The cell motile activity of A549 cells treated with
GW9508 was significantly lower than that of untreated cells.
Moreover, the cell motile activity of A549 cells treated with
GW9508 was markedly suppressed by GW1100 (Fig. 2C).
3.3. Effects of GPR40 knockdown on cellular functions of A549 cells
To confirm effects of GPR120 and GPR40 on cell proliferation
and motile activity of A549 cells, GPR40 knockdown (A549-40)
cells were generated from A549 cells (Fig. 3A). The cell prolifera-
tion rate of A549-40 cells was similar to that of control A549-R
cells (Fig. 3B). The cell growth activity of A549-40 cells for 3 days
was significantly inhibited by GW9508 at a concentration of
10 μM, but not A549-R cells (Fig. 3C). The cell motile activity of
A549-40 cells was markedly lower than that of A549-R cells. In
addition, GW9508 decreased the cell motile activities of both cells
(Fig. 3D). In gelatin zymography, matrix metalloproteinase-2
(MMP-2) activity of A549-40 cells was significantly lower than
that of A549-R cells. GW9508 reduced the MMP-2 activities of
both cells. No activation of MMP-9 was found in all cells (Fig. 3 E
and F).
 T. Kita et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

Fig. 1. Roles of Gpr120 and Gpr40 in cell proliferation and motile activity of RLCNR and LL/2 cells. (A) Expression patterns of Gpr120 and Gpr40 genes by semi-quantitative
RT-PCR analysis. (B) Effects of GW9508 and GW1100 on cell growth rates of RLCNR and LL/2 cells. Cells were cultured in with or without GW9508 (1 or 10 μM) and GW1100
(1 μM) and cell growth rates were measured using the CCK-8. Columns indicate the mean of three studies; bars indicate SD. (C) Cell motility assay. Cells were pretreated with
or without GW9508 (10 μM) and GW1100 (1 μM) for 60 min and seeded on the filters at 1
105 cells and incubated for 20 h. Columns indicate the mean of three studies; bars
indicate SD.*; p o 0.01 vs. untreated cells.

Fig. 2. Roles of GPR120 and GPR40 in cell proliferation and motile activity of A549 cells. (A) Expression patterns of GPR120 and GPR40 genes by semi-quantitative RT-PCR
analysis. (B) Effects of GW9508 and GW1100 on cell growth rate of A549 cells. Cells were cultured in with or without GW9508 (1 or 10 μM) and GW1100 (1 μM) and cell
growth rates were measured using the CCK-8. Columns indicate the mean of three studies; bars indicate SD.*; po 0.01 vs. untreated cells. (C) Cell motility assay. Cells were
pretreated with or without GW9508 (10 μM) or GW1100 (1 μM) for 60 min and seeded on the filters at 1
105 cells and incubated for 20 h. Columns indicate the mean of
three studies; bars indicate SD.*; p o 0.01 vs. untreated cells.

3.4. Effects of GPR120 and GPR40 on cellular functions of endothelial
F-2 cells
Using supernatants from condition medium of the individual
cells, we investigated effects of GPR120 and GPR40 on cell motility
and tube formation of F-2 cells. In both assays, F-2 cells were in-
cubated with the supernatants. The supernatant from A549 cells
treated with GW9508 showed the low cell motile activity of F-2
cells in comparison with that from untreated cells. Moreover, the
cell motile activity of F-2 cells were markedly suppressed by the
supernatants from GW9508 and GW1100 treated A549 cells
(Fig. 4A). The cell motile activity of F-2 cells incubated with su-
pernatants from A549-40 cells was significantly decreased, com-
pared with that from A549-R cells. The supernatants from
GW9508 treated cells markedly inhibited the cell motile activity of
F-2 cells (Fig. 4B). Representative results of tube formation assay
are shown in Fig. 4C. The tube formation of F-2 cells was reduced
by the supernatants from A549-40 cells, compared with that from
A549-R cells. In addition, supernatants from GW9508 treated cells
suppressed the tube formations of F-2 cells (Fig. 4D).
3.5. Roles of GPR120 and GPR40 on cellular functions of long-term
CDDP treated cells
The long-term CDDP treated (A549-CDDP) cells were generated by
culturing in condition medium containing CDDP for at least 6 months
[16,23]. In cell survival assay, A549-CDDP cells indicated the high cell
viability (LC50 ¼ 8.66 μM), compared with A549 cells (LC50 ¼ 2.96 μM)

Please cite this article as: T. Kita, et al., Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular
functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
4 T. Kita et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 3. Effects of GPR40 knockdown on cellular functions of A549 cells. (A) Expression patterns of GPR120 and GPR40 genes by semi-quantitative RT-PCR analysis. A549-R,
control cells. A549-40, GPR40 knockdown cells. (B) Cell proliferation rate of GPR40 knockdown cells. Cells were cultured in DMEM containing 10% FBS. Cell proliferation rate
was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. (C) Effects of GW9508 on cell growth of GPR40 knockdown cells. Cells were
cultured with or without GW9508 (1 or 10 μM) and cell growth rates were measured using the CCK-8. Columns indicate the mean of three studies; bars indicate SD.*;
p o0.01 vs. untreated cells. (D) Cell motility assay. Cells were pretreated with or without GW9508 (10 μM) for 60 min and seeded on the filters at 1
105 cells and incubated
for 20 h. Columns indicate the mean of three studies; bars indicate SD.*; p o 0.01 vs. untreated A549-R cells. (E) Representative results of gelatin zymography. Samples were
obtained from cells cultured in serum-free DMEM with or without GW9508 (10 μM) for 3 days. (F) Densitometric analysis of MMP-2 activity. The bands were quantitated
with NIH Image.

(data not shown). The cell proliferation rate of A549-CDDP cells was
lower than that of A549 cells (Fig. 5A). The expression levels of
GPR120 and GPR40 genes in A549-CDDP cells were significantly de-
creased, compared with A549 cells (Fig. 5B). Thus, to evaluate whe-
ther the reduced GPR120 and GPR40 expressions by CDDP treatment
affect the cell motile activity, we measured the cell motile activities of
A549 and A549-CDDP cells. The cell motile activity of A549-CDDP
cells was markedly higher than that of A549 cells. The cell motile
activities of both cells were reduced by GW9508. In addition, GW1100
suppressed the cell motile activities of both cells treated with
GW9508 (Fig. 5C). Since the cell proliferation rate of A549-CDDP cells
for 3 days was lower than that of A549 cells, the supernatants ob-
tained from the condition medium cultured for 48 h were used. In
gelatin zymography, A549-CDDP cells indicated the high activity of
MMP-2, compared with A549 cells. GW9508 and GW1100 reduced
the MMP-2 activity of A549-CDDP cells, but not A549 cells. No acti-
vation of MMP-9 was observed in all cells (Fig. 5D and E).
4. Discussion
The distribution and pattern of GPR120 and GPR40 are not uni-
form in normal tissues. While GPR120 is abundantly expressed in
lung, large intestine, adipocytes and macrophages, GPR40 expres-
sion is observed in pancreatic beta cells and endocrine cells of the
gastrointestinal tract [9,11,24]. Moreover, the expression levels of
GPR120 in colorectal carcinomas were significantly higher than
those in normal colon tissues [15]. GPR40 was highly expressed in
insulinoma cells, but not in glucagonoma cells [25]. Therefore, these
findings suggest that GPR120 and GPR40 are involved in not only
metabolic homeostasis but also the pathogenesis of tumor cells.
Three lung cancer cells used in this study expressed GPR120
and GPR40 genes. Therefore, to assess effects of GPR120 and GPR40
on cellular functions, cells were treated with GW9508 and
GW1100. GW9508 is used as an agonist of GPR120 and GPR40
[20,21]. GW1100 acts as an antagonist of GPR40 [20–22]. In

Please cite this article as: T. Kita, et al., Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular
functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
 T. Kita et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

Fig. 4. Effects of GPR120 and GPR40 on cellular functions of endothelial F-2 cells. (A) Cell motile activity of F-2 cells. F-2 cells were incubated in serum-free DMEM (upper
chamber) with supernatants (lower chamber) from A549 cells treated with or without GW9508 (10 μM) and GW1100 (1 μM) for 3 days. Columns indicate the mean of three
experiments. Bars indicate SD.*; p o 0.01 vs. untreated A549 cells. (B) Cell motile activity of F-2 cells. F-2 cells were incubated in serum-free DMEM (upper chamber) with
supernatants (lower chamber) from GPR40 knockdown cells treated with or without GW9508 (10 μM) and GW1100 (1 μM) for 3 days. Columns indicate the mean of three
experiments. Bars indicate SD.*; p o0.01 vs. untreated A549-R cells. (C) Representative results of tube formation assays. Cells were seeded onto Matrigel-coated plates at
4
104 cells in 100 μl supernatants from each GPR40 knockdown cells and cultured at 37 °C for 4 h. (D) The total tube formation length was measured from four re-
presentative 100
fields/well.

motility assay, GW9508 enhanced the cell motile activities of
RLCNR and LL/2 cells, but not A549 cells. In contrast, the cell
motile activities of all cells were suppressed by GW1100. In addi-
tion, the cell motile activity of A549 cells was inhibited by GPR40
knockdown. These results indicated that GPR120 inhibited and
GPR40 enhanced the cell motile activities of three lung cancer
cells. In recent studies, GPR120 and GPR40 exhibited the different
effects on cell motile activities of some cancer cells. Although
GPR120 positively regulated the cell motile activities of colon and
pancreatic cancer cells, the cell motile activities of pancreatic and
fibrosarcoma cells were inhibited by GPR40 [16,17]. Taken together
with these findings, the different effects of GPR120 and GPR40 on
the cell motile activities of cancer cells may be dependent of types
of cancer cells. In fact, whereas GPR120 and GPR40 were expressed
in pancreatic and lung cancer cells, no expression of GPR40 was
found in fibrosarcoma cells [16,17]. On the other hand, GW9508
showed the diverse effects on cell motile activities of human and
rodent lung cancer cells, while the cell motile activities of all cells
were suppressed by GW1100. Since GW9508 simultaneously sti-
mulates GPR120 and GPR40 [20,21], it is possible that the differ-
ence between GPR120 and GPR40 expression levels in human and
rodent lung cancer cells may result in the diverse effects of
GW9508.
It is known that MMP activation is closely related to promotion
of invasive and metastatic process of cancer cells as well as an-
giogenesis [26,27]. In pancreatic cancer cells, GPR120 enhanced
and GPR40 inhibited MMP-2 activity [17]. Moreover, GPR120 in-
duced vascular endothelial growth factor (VEGF) expression and
endothelial branching in colon cancer cells, demonstrating that
activation of GPR120 signaling promotes angiogenesis [15]. In the
present study, we showed that MMP-2 activity of A549 cells was
reduced by GPR40 knockdown. Furthermore, GPR40 knockdown
cells indicated the inhibitory effects on cell motility and tube
formation of endothelial cells. These results demonstrated that
GPR120 negatively and GPR40 positively regulate the malignant
properties of lung cancer cells. Using the soft agar assay, GPR40
knockdown enhanced the tumorigenic activity of pancreatic can-
cer, but not GPR120 knockdown [17]. However, no tumorigenic
activity of all cells used in this study was found (data not shown).
To examine whether GPR120 and GPR40 are involved in the
regulation of cellular functions induced by the long-term CDDP
treatment, A549-CDDP cells were generated from A549 cells. In
A549-CDDP cells, the cell motile and MMP-2 activities were
markedly elevated, correlating with the low expression levels of
GPR120 and GPR40 genes. Since the activated cell motility and
MMP-2 of A549-CDDP cells were suppressed by GW1100, it is
suggested that GPR120 and GPR40 contribute to the regulation of
cellular responses induced by the long-term CDDP treatment in
lung cancer cells. In fibrosarcoma cells, GPR40 inhibited MMP-2
and MMP-9 activities stimulated by the long-term CDDP treatment
[16]. The inhibition of GPR40 reduced the cell motile and MMP-2
activities of A549 cells. On the other hand, CDDP treatment en-
hanced the cell motile and MMP-2 activities, while GPR40 and
GPR120 expressions were decreased. The cell motile and MMP-2
activities of A549-CDDP cells were suppressed by GW9508, similar
as observed with A549 cells. Therefore, it is suggested that the
effects of GPR120 may be stronger than those of GPR40 in A549
cells.
In conclusion, the present results demonstrate that GPR120
negatively and GPR40 positively regulate malignant properties of

Please cite this article as: T. Kita, et al., Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular
functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
6 T. Kita et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 5. Effects of GPR120 and GPR40 on cellular functions of long-term CDDP treated cells. (A) Cell proliferation assay. Cells were cultured in DMEM containing 10% FBS. Cell
proliferation rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0.*; p o 0.01 vs. A549 cells. A549-CDDP cells, long-term CDDP-
treated A549 cells. (B) Expression levels of GPR120 and GPR40 genes by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD.
*; p o 0.01 vs. A549 cells. (C) Cell motility assay. Cells were pretreated with or without GW9508 (10 μM) and GW1100 (1 μM) for 60 min and seeded on the filters at 1
105
cells and incubated for 20 h. Columns indicate the mean of three studies; bars indicate SD.*; p o 0.01 vs. untreated A549 cells. (D) Representative results of gelatin zy-
mography. Samples were obtained from cells cultured in serum-free DMEM with or without GW9508 (10 μM) and GW1100 (1 μM) for 2 days. (E) Densitometric analysis of
MMP-2 activity. The bands were quantitated with NIH Image. Columns indicate the mean of three studies; bars indicate SD.*; p o 0.01 vs. untreated A549 cells.

lung cancer cells. Moreover, GPR120 and GPR40 play important
roles in the regulation of cellular functions in the long-term CDDP
treated cells. Previous report showed that concentrations of serum
FFAs and their metabolites were significantly higher in patients
with lung adenocarcinoma than in patients without cancer, while
no change of FFA levels was found in squamous cell carcinoma
cases [28]. Therefore, to better understand the roles of GPR120 and
GPR40 in the pathogenesis of lung cancer cells, the investigation
whether aberrant expressions of GPR120 and GPR40 occur in hu-
man lung cancer tissues should be examined.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by JSPS KAKENHI Grant number
24590493 and by Grants from the Faculty of Science and En-
gineering, Kinki University.
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functions in lung cancer cells, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.03.008i
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