Galectin-9 suppresses the proliferation of gastric cancer cells in vitro

  • Authors:
    • Jitsuko Takano
    • Asahiro Morishita
    • Shintaro Fujihara
    • Hisakazu Iwama
    • Fuyuko Kokado
    • Keiko Fujikawa
    • Koji Fujita
    • Taiga Chiyo
    • Tomoko Tadokoro
    • Teppei Sakamoto
    • Takako Nomura
    • Joji Tani
    • Hisaaki Miyoshi
    • Hirohito Yoneyama
    • Hideki Kobara
    • Hirohito Mori
    • Toshihiro Niki
    • Mitsuomi Hirashima
    • Tsutomu Masaki
  • View Affiliations

  • Published online on: November 26, 2015     https://doi.org/10.3892/or.2015.4452
  • Pages: 851-860
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Abstract

Gastric cancer is the second-leading cause of cancer-related mortality worldwide, and the prognosis of advanced gastric cancer remains poor. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been demonstrated to exert anti-proliferative effects on various types of cancer cells. The aim of our present study was to evaluate the effects of Gal-9 on human gastric cancer cells and the expression levels of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. In our initial experiments, Gal-9 suppressed the proliferation of gastric cancer cell lines in vitro. Our data further revealed that Gal-9 increased caspase-cleaved keratin 18 (CCK18) levels in gastric cancer cells. Additionally, Gal-9 reduced the phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR-3) and insulin-like growth factor-1 receptor (IGF-1R). Furthermore, miRNA expression levels were markedly altered with Gal-9 treatment in vitro. In conclusion, Gal-9 suppressed the proliferation of human gastric cancer cells by inducing apoptosis. These findings suggest that Gal-9 could be a potential therapeutic target in the treatment of gastric cancer.

Introduction

Gastric cancer is the second-leading cause of cancer-related mortality worldwide, and the prognosis of advanced gastric cancer remains poor (1). Despite advances in the treatment of gastric cancer, no standard palliative chemotherapy has been accepted for patients with metastatic gastric cancer (2,3). Thus, there is a strong demand for new treatment options to address advanced stages of gastric cancer.

Galectins are classified according to their carbohydrate-recognition domain (CRD) and are subdivided into three groups: prototype galectins (galectins-1, -2, -7, -10, -13, and -14) and the chimera-type galectin (galectin-3) have a single CRD, while tandem-repeat-type galectins (galectins-4, -8, -9, and -12) have two CRDs joined by a flexible peptide linker (46). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that is known for its key roles in eosinophil chemoattraction and activation (79). Similar to other galectins, Gal-9 regulates various cellular functions, such as aggregation, adhesion and apoptosis (10,11). In recent studies, Gal-9 has been shown to induce apoptosis and thereby suppress cell proliferation and tumor growth in various hematologic malignancies, such as human melanoma (12,13) and chronic myelogenous leukemia (14). Additionally, our recent studies revealed the antitumor effects of recombinant Gal-9 in various solid malignancies, such as hepatocellular carcinoma (15) and cholangiocarcinoma (16).

However, it is unclear whether Gal-9 suppresses gastric cancer cell proliferation. The aim of this study was to determine the effectiveness of Gal-9 against gastric cancer cell proliferation. Possible mechanisms associated with the antitumor effect of Gal-9 were also explored, including the activation of receptor tyrosine kinases, angiogenesis and microRNAs (miRNAs).

Materials and methods

Reagents and chemicals

Recombinant stable and mutant forms of human Gal-9 lacking the entire linker region were expressed and purified as described in our previous studies (17). Lactose, sucrose, and fetal bovine serum (FBS) were purchased from Wako Chemicals (Osaka, Japan). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan), and all other chemicals were obtained from Sigma Chemical (Tokyo, Japan).

Cell lines and culture

The human gastric cancer cell lines MKN1, MKN7, MKN45, and MKN74 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were cultured in RPMI-1640 (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin (100 µg/ml; Invitrogen) in a humidified atmosphere with 5% CO2 at 37°C.

Cell proliferation assay

We performed cell proliferation assays using a CCK-8 according to the manufacturer's instructions. Samples of each cell line (1×104 cells/well) were seeded into a 96-well plate and cultured in 100 µl of RPMI-1640 supplemented with 10% FBS. Twenty-four hours after seeding, the cells were treated with 0.01, 0.03, 0.1 or 0.3 µmol/l Gal-9 in the culture medium and cultured for an additional 48 h. To inhibit the galactoside binding of Gal-9, 30 mM lactose was added. Sucrose was added as a control. CCK-8 reagent (10 µl) was then added to each well, and the 96-well plate was incubated at 37°C for 3 h. The absorbance of each well was measured at 450 nm using an auto-microplate reader.

Enzyme-linked immunosorbent assay (ELISA)

Cell apoptosis assays were conducted by measuring the amounts of caspase-cleaved keratin-18 (CCK-18) using an M30 Apoptosense ELISA kit (Previva, Bromma, Sweden) (18). Samples of each cell type (5×103 cells/well) were seeded into a 96-well plate and cultured in 100 µl of culture medium for 24 h. The seeded cells were then treated with 0.3 µmol/l Gal-9. The remaining steps of the assay were carried out according to the manufacturer's instructions. The amounts of antigen in the controls and samples were calculated by interpolation from a standard curve.

Cell and tissue lysates

The preparation of lysates was conducted according to the methods previously described by us (19). All the steps were carried out at 4°C. Protein concentrations were measured using a dye-binding protein assay based on the Bradford method (20).

Antibody arrays of phosphorylated receptor tyrosine kinases (phospho-RTKs)

Human phospho-RTKs were assayed using human phospho-RTK array kits (R&D Systems) according to the manufacturer's instructions. Briefly, phospho-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h and incubated with 2 ml of cell line lysates after normalization so that the amounts of proteins were equal. After 3 washes for 10 min each with TBS plus 0.1% v/v Tween-20 and 2 washes for 10 min with TBS alone to remove unbound materials, the membranes were incubated with anti-phosphotyrosine-HRP antibody for 2 h at room temperature. Unbound HRP-conjugated antibody was washed out with 0.1% Tween-20 in TBS. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.).

Angiogenic profile analysis using an antibody array

A RayBio Human Angiogenesis Antibody Array (RayBiotech, Inc.) was used according to the manufacturer's protocol. This method is a dot-based assay enabling the detection and comparison of 20 angiogenesis-specific cytokines. Each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.).

Analysis of a miRNA microarray

Samples of each cancer cell line were processed for total RNA extraction with an miRNeasy Mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. Using an Agilent 2100 Bioanalyzer (Agilent Technologies), the RNA samples typically had A260/280 ratios between 1.9 and 2.1.

After measuring RNA levels with an RNA 6000 Nano kit (Agilent Technologies), the samples were labeled using a miRCURY Hy3/Hy5 Power labeling kit and were hybridized to a human miRNA Oligo chip 10, version 19.0 (Toray Industries, Tokyo, Japan). Scanning was conducted with a 3D-Gene Scanner 3000 (Toray Industries). 3D-Gene Extraction version 1.2 software (Toray Industries) was used to read the raw intensities of the images. To determine changes in miRNA expression between the Gal-9-treated and control samples, the raw data were analyzed via GeneSpring GX version 10.0 (Agilent Technologies). The samples were first normalized relative to 28S RNA and baseline-corrected to the median of all the samples.

Replicate data were consolidated into two groups, i.e., those from the galectin-9-treated cells and those from the control cells, and were organized using the hierarchical clustering and ANOVA functions in GeneSpring software. Hierarchical clustering was performed using the clustering function (condition tree) and the Euclidean correlation as a distance metric. A two-way ANOVA was conducted, and asymptotic p-values (<0.05) were determined with no error corrections to identify the miRNAs that varied most prominently across the different groups. Only changes >50% for at least one of the time-points for each sample were considered significant. All the analyzed data were globally normalized. The statistical significance of differentially expressed miRNAs was analyzed using Student's t-test.

Statistical analysis

All analyses were conducted using JMP8.0 (SAS Institute, Cary, NC, USA). Paired analyses between the groups were conducted using Student's t-test. A p<0.05 was considered to indicate a significant difference between groups.

Results

Gal-9 suppresses the proliferation of human gastric cancer cells

To evaluate the effect of Gal-9 on the in vitro growth of human gastric cancer cells, we examined the effect of Gal-9 on the proliferation of 4 gastric cancer cell lines, MKN1, MKN7, MKN45 and MKN74. Cells were grown in 10% FBS and treated with 0.01, 0.03, 0.1 or 0.3 µmol/l Gal-9 or without Gal-9 as a control. The cell proliferation assay was conducted 48 h after the addition of the agents. As depicted in Fig. 1, treatment with Gal-9 led to a strong, dose-dependent inhibition of cell proliferation in the MKN74 and MKN45 cells, which are Gal-9-sensitive gastric cancer cell lines (Fig. 1). However, no anti-proliferative effects of Gal-9 were detected in the MKN7 and MKN1 cells (Fig. 1).

The anti-proliferative effect of Gal-9 is inhibited by lactose in Gal-9-sensitive gastric cancer cells

The growth of the MKN74 and MKN45 cells was inhibited by Gal-9 with 30 mM sucrose but not with 30 mM lactose (Fig. 2). These data suggest that the binding of β-galactoside is essential for Gal-9 to inhibit the proliferation of the MKN74 and MKN45 cells, which are Gal-9-sensitive gastric cancer cells.

Gal-9 induces apoptosis in Gal-9-sensitive gastric cancer cells (MKN74 cells) but not Gal-9-resistant cells (MKN7 cells)

To determine the mechanism(s) of the Gal-9 anti-proliferative effects, the level of caspase-cleaved cytokeratin 18 (CCK18), which is specifically produced during apoptosis, was measured using ELISAs in gastric cancer cells treated with 0.3 µM Gal-9. Remarkably, Gal-9 increased the levels of CCK18 in Gal-9-sensitive cells (MKN74 cells) but not in Gal-9-resistant cells (MKN7 cells) (Fig. 3). This result suggests that apoptosis is involved in Gal-9-induced cytostasis in the Gal-9-sensitive cells.

Differences in phospho-RTKs in Gal-9-sensitive versus Gal-9-resistant gastric cancer cells treated with and without Gal-9

We next used a phospho-RTK array system to identify the key receptor tyrosine kinases (RTKs) associated with the anti-proliferative effects of Gal-9. Using this antibody array (Fig. 4A), we simultaneously screened the expression levels of 42 different RTKs activated in MKN74 and MKN7 cells with or without 0.3 µM Gal-9. Gal-9 reduced the expression levels of vascular endothelial growth factor receptor-3 (VEGFR-3) and phosphorylated insulin-like-growth factor-1 receptor (IGF-1R) in the MKN74 cells (Fig. 4B). In contrast, no activated RTKs were upregulated in the Gal-9-resistant MKN1 cells.

Densitometric analyses revealed that the ratios of the p-VEGFR-3 and p-IGF-1R spots of the Gal-9-treated cells to those of the untreated cells were 28.2 and 55.1%, respectively (Fig. 4C).

Effects of Gal-9 on angiogenesis in Gal-9-sensitive versus Gal-9-resistant gastric cancer cells

To examine the relationship between angiogenesis and Gal-9, an angiogenesis antibody array analysis was conducted regarding the anti-tumor effects of Gal-9 (Fig. 5A). Using the antibody array, we simultaneously screened the expression levels of 20 different angiogenesis-related proteins in MKN7 and MKN74 cells with or without Gal-9. The expression levels of interleukin-8 (IL-8), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, growth-related oncogene (GRO) and regulated-on-activation normal-T-cell-expressed and secreted (RANTES) were induced by Gal-9 treatment in the MKN74 cells as detected by the protein array (Fig. 5B). In the Gal-9-resistant MKN1 cells, no altered expression of angiogenesis molecules was detected with Gal-9 treatment.

Densitometric analyses indicated that the ratios of the IL-8, TIMP-1, TIMP-2, GRO, and RANTES spots of the Gal-9-treated cells to those of the untreated cells were 356.1, 355.3, 389.36, 231.1 and 201.0%, respectively (Fig. 5C).

miRNA profiles of the cell lines treated in vitro with or without Gal-9

Using a custom microarray platform, we analyzed the in vitro expression levels of 2,555 human miRNA probes in gastric cancer cell lines that were treated with or without Gal-9. As presented in Table I, when the expression levels of miRNAs were measured in the MKN74 cells treated in vitro with or without 0.3 µmol/l Gal-9, 153 miRNAs were significantly upregulated 24 h after the Gal-9 treatment, whereas 18 miRNAs were downregulated.

Table I

Changes in expression compared with untreated cells and chromosomal locations of miRNAs in MKN74 cells treated with galectin-9.

Table I

Changes in expression compared with untreated cells and chromosomal locations of miRNAs in MKN74 cells treated with galectin-9.

miRNAFold (treated/control) means ± SDP-valueChromosomal localization
Upregulated
hsa-miR-204-3p12.41±3.0110.000239q21.12
hsa-miR-468811.27±2.9830.0003211
hsa-miR-4731-5p10.05±4.2360.0009117
hsa-miR-44598.36±2.170.000165
hsa-miR-47307.78±2.4320.0040617
hsa-miR-42947.54±3.6540.0016410
hsa-miR-31967.39±2.8290.0019120
hsa-miR-36487.32±1.060.0004921
hsa-miR-4687-3p6.74±1.9380.0001111
hsa-miR-31976.46±1.1530.0040821
hsa-miR-45306.35±0.7380.0005819
hsa-miR-6386.33±1.6760.002319p13.2
hsa-miR-19086.31±1.5950.0011611
hsa-miR-44426.28±1.7810.002813
hsa-miR-44886.19±1.8390.0005711
hsa-miR-61256.16±0.6990.0004812
hsa-miR-4787-5p5.98±1.5670.000313
hsa-miR-60885.79±1.1240.0009119
hsa-miR-44665.78±1.5620.006336
hsa-miR-36215.6±1.2710.00089
hsa-miR-1237-5p5.6±1.9020.0014211
hsa-miR-3940-5p5.54±1.0680.0010619
hsa-miR-1227-5p5.52±1.7850.0102319
hsa-miR-47925.49±1.0650.00123
hsa-miR-45085.41±0.5950.0001915
hsa-miR-36655.4±1.6820.0006613
hsa-miR-28615.38±1.9460.000579
hsa-miR-61265.36±1.5170.0073516
hsa-miR-57875.29±0.7980.000493
hsa-miR-36565.15±1.0830.0005311
hsa-miR-60755.14±1.7570.000365
hsa-miR-12465.08±1.8960.034582q31.1
hsa-miR-1229-5p4.92±1.8540.000225
hsa-miR-4667-5p4.84±0.8910.002949
hsa-miR-4756-5p4.68±4.770.0076220
hsa-miR-61324.63±0.5460.007127
hsa-miR-1268a4.63±0.4020.0007115q11.2
hsa-miR-60854.28±1.4560.0027315
hsa-miR-44844.17±1.3540.0133110
hsa-miR-652-5p4.15±0.760.001Xq23
hsa-miR-4800-5p4.1±0.030.025764
hsa-miR-61314.08±0.4460.020935
hsa-miR-6724-5p4.07±1.8750.004121
hsa-miR-4745-5p4.04±0.8830.0170519
hsa-miR-642a-3p4±0.9010.011419q13.32
hsa-miR-19733.97±0.4220.003644
hsa-miR-663a3.88±0.3360.0018520p11.1
hsa-miR-4749-5p3.87±0.7220.0005319
hsa-miR-47413.8±0.1680.0036118
hsa-miR-14693.8±0.6520.0003415q26.2
hsa-miR-1228-5p3.8±0.5640.0004712
hsa-miR-44633.78±0.1310.000746
hsa-miR-45053.73±0.4180.0008114
hsa-miR-1915-3p3.72±1.5710.0021910p12.31
hsa-miR-4763-3p3.7±0.4110.0018622
hsa-miR-491-5p3.69±0.8660.000149p21.3
hsa-miR-46513.66±0.8840.001367
hsa-miR-44353.62±1.3080.03972
hsa-miR-12753.62±1.0310.002376
hsa-miR-31803.53±1.9890.0099816
hsa-miR-31-5p3.44±1.150.000199p21.3
hsa-miR-1909-3p3.34±0.8250.0049819p13.3
hsa-miR-3620-5p3.3±0.4720.001821
hsa-miR-149-3p3.21±0.3070.003552q37.3
hsa-miR-4725-3p3.18±0.7110.0003617
hsa-miR-132-3p3.17±2.0480.0024217p13.3
hsa-miR-15873.13±0.5570.0024Xp11.4
hsa-miR-43273.12±0.8430.000821
hsa-miR-4726-5p3.12±0.4540.0008417
hsa-miR-1247-3p3.11±1.0690.0229714q32.31
hsa-miR-486-3p3.1±0.2670.014758p11.21
hsa-miR-1268b3.1±0.5610.0004917
hsa-miR-44973.08±0.3950.0009212
hsa-miR-642b-3p3.06±0.5650.0066719
hsa-miR-937-5p3.06±0.3370.0058q24.3
hsa-miR-4728-5p3.03±1.1610.0487417
hsa-miR-365a-5p3.03±0.2160.0029616p13.12
hsa-miR-3180-3p3.01±0.6060.0003216
hsa-miR-675-5p2.97±1.0270.0274411p15.5
hsa-miR-46562.89±0.2750.003587
hsa-miR-42702.83±0.6150.016173
hsa-miR-5001-5p2.83±0.4520.017922
hsa-miR-42572.81±0.2630.000511
hsa-miR-44502.79±1.0730.015474
hsa-miR-45072.79±0.3560.0106814
hsa-miR-39372.77±1.0630.00578X
hsa-miR-513a-5p2.74±0.4420.023Xq27.3
hsa-miR-12022.7±0.0820.000846
hsa-miR-7602.69±0.6340.02431p22.1
hsa-miR-4638-3p2.67±0.3940.019865
hsa-miR-5585-3p2.67±1.3410.006791p35.2
hsa-miR-671-5p2.66±0.8920.006237q36.1
hsa-miR-4655-5p2.65±0.380.001987
hsa-miR-212-3p2.64±0.4480.0200617p13.3
hsa-miR-42992.56±1.7270.0156811
hsa-miR-47062.54±0.7810.0011514
hsa-miR-132-5p2.52±0.4690.0182617p13.3
hsa-miR-4722-5p2.49±0.1160.0195516
hsa-miR-3135b2.48±0.0960.000616
hsa-miR-4732-5p2.48±0.2160.0073717
hsa-miR-4758-5p2.46±0.4870.000220
hsa-miR-23922.4±0.7490.0109214
hsa-miR-44492.37±0.5820.045874
hsa-miR-29a-3p2.36±0.6850.004387q32.3
hsa-miR-6722-3p2.34±0.2820.001759
hsa-miR-37142.31±0.1860.033063
hsa-miR-744-5p2.3±1.1040.0267617p12
hsa-miR-5006-5p2.23±0.6940.0122413
hsa-miR-31412.22±0.6770.002995
hsa-miR-1185-1-3p2.21±0.6980.0312814
hsa-miR-4436b-5p2.19±0.7120.012952
hsa-miR-44172.14±0.5360.006431
hsa-miR-5196-5p2.13±0.420.0044819
hsa-miR-296-5p2.12±0.3830.0072520q13.32
hsa-miR-6722-5p2.11±0.2550.018749
hsa-miR-6142.09±0.5970.0017912p13.1
hsa-miR-3678-3p2.08±0.8050.0444217
hsa-miR-1914-3p2.03±0.4740.0258720q13.33
hsa-miR-4640-5p2.03±0.3710.037836
hsa-miR-44982.03±0.5220.0446212
hsa-miR-194-5p1.97±0.5880.008491q41
hsa-miR-29b-1-5p1.97±0.340.004447q32.3
hsa-miR-4695-5p1.95±0.3520.000971
hsa-miR-4707-5p1.93±0.20.0428614
hsa-miR-4690-5p1.92±0.0980.0181411
hsa-miR-92b-5p1.91±0.4990.005371q22
hsa-miR-44651.9±0.1480.007536
hsa-miR-3679-3p1.88±0.1850.001012
hsa-miR-7111.87±0.5360.01243
hsa-miR-31851.84±0.1610.0103617
hsa-miR-192-5p1.83±0.4640.0209611q13.1
hsa-miR-3701.82±0.5470.0213114q32.2
hsa-miR-6581.81±0.5770.0359822q13.1
hsa-miR-222-3p1.76±0.6140.01237Xp11.3
hsa-miR-31951.71±0.1310.0237820
hsa-miR-5571.67±0.4150.016781q24.2
hsa-miR-221-3p1.59±0.4060.03976Xp11.3
hsa-miR-46341.54±0.3670.048685
hsa-miR-1225-5p1.52±0.1890.0367616p13.3
hsa-miR-36091.52±0.0020.00847
hsa-miR-30c-1-3p1.48±0.0650.001711p34.2
hsa-miR-6515-3p1.45±0.1060.016119
hsa-miR-21-3p1.44±0.3430.0275917q23.1
hsa-miR-3144-5p1.44±0.1240.008246
hsa-miR-22-3p1.41±0.0280.0130117p13.3
hsa-miR-6221.38±0.0720.0157613q31.3
hsa-miR-4650-3p1.38±0.0960.002667q11.21
hsa-miR-877-5p1.38±0.1560.022976p21.33
hsa-miR-660-5p1.3±0.2470.03583Xp11.23
hsa-miR-39071.25±0.1280.015677
hsa-miR-31611.24±0.0090.0021211
hsa-miR-4714-3p1.24±0.040.0431515
hsa-miR-532-5p1.22±0.1350.01241Xp11.23
Downregulated
hsa-miR-30d-3p0.36±0.1370.015868q24.22
hsa-miR-47850.44±0.1440.045522
hsa-miR-45210.46±0.1850.0238417
hsa-miR-4722-3p0.5±0.1710.0303416
hsa-miR-46830.56±0.0890.0165110
hsa-miR-200b-5p0.58±0.1370.016791p36.33
hsa-miR-345-5p0.6±0.190.0438914q32.2
hsa-miR-590-5p0.6±0.0770.003267q11.23
hsa-miR-103a-2-5p0.61±0.1160.035520p13
hsa-miR-7610.65±0.0170.04271
hsa-miR-5692c0.65±0.1320.015025
hsa-miR-299-3p0.68±0.020.0376314q32.31
hsa-miR-126-5p0.69±0.0790.007589q34.3
hsa-miR-43010.7±0.1520.0320111
hsa-miR-45220.72±0.1610.0350617
hsa-miR-6270.74±0.0340.0068615q15.1
hsa-miR-13230.75±0.0660.0428319q13.42
hsa-miR-2681-5p0.76±0.0550.0338713

[i] miRNAs, microRNAs; SD, standard deviation.

An unsupervised hierarchical clustering analysis and a Pearson's correlation analysis indicated that MKN74 cells treated in vitro with Gal-9 clustered together and separately from the untreated MKN74 cells (Fig. 6).

Discussion

Gastric cancer is a very common disease worldwide and is the second most frequent cause of cancer-related deaths (21,22). Although the overall incidence and mortality have dramatically declined over the past few decades, gastric cancer remains a major health problem (23). More than half of gastric cancer patients have lymph node metastasis when initially diagnosed or after surgical resection, which results in a poor prognosis (2426). Patients with advanced stages of gastric cancer are treated with chemotherapy and radiation, either singly or in combination. However, none of these therapies is fully curative. Therefore, new therapeutic strategies are urgently required for the treatment of advanced gastric cancer. In recent studies, higher levels of Gal-9 expression were observed in patients without lymph-vascular invasion, lymph node metastases or distant metastases, and Gal-9 expression is closely associated with better survival rates in gastric cancer (27). Additionally, the downregulation of Gal-9 mRNA levels was observed in gastric cancer tissues; therefore, the loss of Gal-9 expression may be involved in the progression of gastric cancers (28). In the present study, we found that Gal-9 inhibited the proliferation of gastric cancer cell lines. In addition, we identified miRNAs associated with the antitumor effect of Gal-9 in gastric cancer.

Recombinant Gal-9 inhibits proliferation in various cancers such as melanoma (13) and chronic myelogenous leukemia (14) by inducing apoptosis. Our present results revealed that Gal-9 suppresses the proliferation of human gastric cancer cell lines in vitro. Gal-9 strongly inhibited cell proliferation in a dose-dependent manner in MKN45 and MKN74 cells but not in MKN1 and MKN7 cells. Notably, the histological type of the MKN74 and MKN7 cell lines is intestinal, while the MKN45 cells are a diffuse type, and the MKN1 cells are an adenosquamous carcinoma (29). Furthermore, the MKN7 and MKN1 cells were established from metastatic foci in the lymph nodes (29,30), whereas the MKN74 and MKN45 cells were derived from liver metastases (29,30). Accordingly, these data suggest that the liver metastatic type of gastric cancer cells (MKN74 and MKN45 cells) are more sensitive to Gal-9 treatment compared with the lymph node metastatic type of cells (MKN7 and MKN1 cells), regardless of their respective histological differentiation. These results suggest that Gal-9 may be a potential targeting moiety useful in distant metastasis in the advanced stages of gastric cancer.

The cleavage of cytokeratin by caspase (CCK18) occurs as an early event during apoptosis following the activation of apoptosis executioners, particularly effector caspases. However, cytokeratin remains intact during other types of cell death, such as autophagy or necrosis (31). In the present study, Gal-9 increased the levels of CCK18 in the MKN74 cells, which are sensitive to Gal-9, but not the MKN1 cells, which are resistant to Gal-9. These data indicate that Gal-9 suppresses the proliferation of Gal-9-sensitive gastric cancer cells by inducing apoptosis.

Since the discovery of these proteins, RTKs have been investigated as key regulators of the proliferation, differentiation, and metastasis of gastric cancer cells (32). Our present study used phospho-RTK arrays to demonstrate that Gal-9 treatment reduced the expression levels of IGF-1R and VEGFR-3 in Gal-9-sensitive cells (MKN74) when compared with those in Gal-9-resistant cells (MKN7). IGF-1R is involved in cell proliferation and the prevention of apoptosis (32,33). Galamb et al found via immunohistochemistry that IGF-1R was overexpressed in gastric cancer tissues when compared with normal mucosa (34). Additionally, the mRNA expression levels tended to be higher in cancer tissues than in normal mucosa (35). The overexpression of IGF-1R is associated with a poor response to chemotherapy and poor outcomes in stage I–IV gastric cancers (36). Gal-9 may reduce the expression levels of phospho-IGF-1R in gastric cancer and thereby improve chemosensitivity and the disease prognosis.

Galectins are important regulators of tumor progression that influence tumor cell transformation, immune escape and angiogenesis (46). Using angiogenesis-related protein arrays, we observed that IL-8, GRO, TIMP-1, TIMP-2 and RANTES were enhanced in Gal-9-sensitive gastric cancer cells. The enhanced expression of IL-8, in particular, may be associated with a poor prognosis, as determined by stage and histology, and indicate a more aggressive gastric cancer (3739). These data suggest that Gal-9-sensitive gastric cancer cells immune to the antitumor effect of Gal-9 may produce angiogenesis-related proteins and thereby regulate angiogenesis in the in vivo microenvironment of gastric cancer tissues.

MicroRNAs, small non-coding RNA sequences, have been shown to regulate the development and progression of various cancers (40). To identify the miRNAs associated with the antitumor effects of Gal-9, we used miRNA expression arrays to determine variations in the miRNA profiles of the MKN74 cell line in cultures treated with or without Gal-9. The cluster-based analysis clearly demonstrated that Gal-9 treatment affected the expression of numerous miRNAs. In the analysis, we selected sets of miRNAs with significantly altered expression levels with or without Gal-9 treatment. These altered miRNAs may provide clues to the molecular basis for the anticancer effects of Gal-9 in gastric cancer. Our data revealed that miR-204-3p and miR-1246 were upregulated in MKN74 cells treated with Gal-9. miR-204 targets Bcl-2, brain-derived neurotrophic factor (BDNF), SIRT1 and IGFBP5 expression and leads to apoptosis and cell cycle arrest in gastric cancer, neuroblastoma, ovarian cancer and papillary thyroid carcinoma. Furthermore, miR-204-3p has also been reported to act on fibronectin, inhibit the proliferation of hepatocellular carcinoma (HCC) endothelial cells and promote apoptosis (41). In contrast, miR-1246 has been reported to be a target of the p53 family, to inhibit Down syndrome-associated DYRK1A, and consequently activate NFTA1c and induce apoptosis (39). Our previous studies have shown that Gal-9 treatment upregulated miR-1246 in hepatocellular carcinoma (15) and cholangiocarcinoma (16), suggesting that miR-1246 may be associated with the anti-proliferative effects of Gal-9 in various cancer cells.

In conclusion, our results revealed that Gal-9 suppresses human gastric cancer cell proliferation, possibly by inducing apoptosis, regulating RTK pathways and angiogenesis-related molecules, and altering miRNA expression profiles. These findings suggest that Gal-9 may be a novel therapeutic target in the treatment of gastric cancer.

Acknowledgments

We thank Ms. Ryoko Unose, Ms. Kana Ogawa, Ms. Kayo Hirose, Ms. Miwako Watanabe, Ms. Kayo Endo, and Ms. Noriko Murao for providing technical assistance.

Abbreviations:

Gal-9

galectin-9

CRDs

carbohydrate-recognition domains

miRNAs

microRNAs

CCK-8

Cell Counting Kit-8

phospho-RTKs

phosphorylated receptor tyrosine kinases

VEGR-3

vascular endothelial growth factor receptor-3

IGF-1R

insulin-like growth factor-1 receptor

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February 2016
Volume 35 Issue 2

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APA
Takano, J., Morishita, A., Fujihara, S., Iwama, H., Kokado, F., Fujikawa, K. ... Masaki, T. (2016). Galectin-9 suppresses the proliferation of gastric cancer cells in vitro. Oncology Reports, 35, 851-860. https://doi.org/10.3892/or.2015.4452
MLA
Takano, J., Morishita, A., Fujihara, S., Iwama, H., Kokado, F., Fujikawa, K., Fujita, K., Chiyo, T., Tadokoro, T., Sakamoto, T., Nomura, T., Tani, J., Miyoshi, H., Yoneyama, H., Kobara, H., Mori, H., Niki, T., Hirashima, M., Masaki, T."Galectin-9 suppresses the proliferation of gastric cancer cells in vitro". Oncology Reports 35.2 (2016): 851-860.
Chicago
Takano, J., Morishita, A., Fujihara, S., Iwama, H., Kokado, F., Fujikawa, K., Fujita, K., Chiyo, T., Tadokoro, T., Sakamoto, T., Nomura, T., Tani, J., Miyoshi, H., Yoneyama, H., Kobara, H., Mori, H., Niki, T., Hirashima, M., Masaki, T."Galectin-9 suppresses the proliferation of gastric cancer cells in vitro". Oncology Reports 35, no. 2 (2016): 851-860. https://doi.org/10.3892/or.2015.4452