Effects of galectin-9 on apoptosis, cell cycle and autophagy in human esophageal adenocarcinoma cells
- Authors:
- Published online on: June 1, 2017 https://doi.org/10.3892/or.2017.5689
- Pages: 506-514
Abstract
Introduction
Esophageal carcinoma is the sixth most common type of cancer worldwide and has remained an aggressive cancer because it is commonly diagnosed at later stages (1). Esophageal carcinoma is divided into two major types [squamous cell carcinoma (SCC) and adenocarcinoma], and the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in Western countries (2). The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC (3). A poor prognosis has been associated with diagnosis at an advanced stage and metastasis (4,5).
Galectin-9 (Gal-9) is a tandem-repeat type galectin with two carbohydrate recognition domains (CRDs); it was first identified as an eosinophil chemoattractant and activation factor (6–8). Similar to other galectins, Gal-9 regulates various cellular functions in eosinophils, including cell aggregation, adhesion and apoptosis (9,10). Gal-9 also enhances antitumor immunity by initiating CRD-independent dendritic cell maturation and Th1-mediated antitumor immunity (11). Treatment with recombinant Gal-9 prolonged survival in a murine melanoma model by increasing the number of CD8+ cytotoxic T cells (CTLs), natural killer (NK) cells and macrophages (12). Furthermore, the Gal-9 receptor T cell immunoglobulin mucin-3 (Tim-3) negatively regulated T cell responses by promoting CD8+ T cell exhaustion and inducing the expansion of myeloid-derived suppressor cells (13,14).
Recombinant Gal-9 induces dose-dependent apoptosis in various leukemic T cell lines in the presence of a functional CRD (15,16). Additionally, Gal-9 inhibits the growth of myeloma (17), chronic myeloid leukemia (18) and human melanoma both in vitro and in vivo (19,20). Moreover, we have recently reported that recombinant Gal-9 exerts antitumor effects on various solid malignancies by affecting the phosphorylation of various proteins, angiogenesis and the expression of microRNAs (miRNAs) (21–23).
However, little is known about the antitumor effects of Gal-9 on EAC cells or the miRNAs associated with these effects. Therefore, the present study evaluated the effects of Gal-9 on the growth of EAC cell lines, the mechanism of action and the miRNAs associated with its antitumor effects.
Materials and methods
Reagents and antibodies
A mutant form of Gal-9 lacking the entire linker region was recombinantly produced and purified as described in our previous report (24). Fetal bovine serum (FBS) was purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan), Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan), and all other chemicals were obtained from Sigma Chemical Corp. (Tokyo, Japan). Z-VAD-FMK and Z-DEVD-FMK were purchased from AdooQ Bioscience (Irvine, CA, USA).
The primary antibodies used in the present study included monoclonal anti-β-actin (A5441, 1:3,000; Sigma-Aldrich, St. Louis, MO, USA), anti-cyclin D1 (RB-9041, 1:1,000; Thermo Fisher Scientific, Waltham, MA, USA), anti-cyclin E (1:1,000; Thermo Fisher Scientific), anti-Cdk6 (sc-177, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Cdk4 (sc-749, 1:1,000; Santa Cruz Biotechnology), anti-Cdk2 (sc-163, 1:2,000; Santa Cruz Biotechnology) and anti-phosphorylated retinoblastoma protein (558385, 1:1,000 Rb; BD Biosciences, San Jose, CA, USA). Antibodies to caspase-3 (#9665), cleaved caspase-3 (#9664), caspase-7 (#12827), caspase-9 (#9508), cleaved caspase-9 (#7237), PARP (#9542), cleaved PARP (#5625), LC3 (#12741) and SQSTM1/p62 (#8025) were purchased from Cell Signaling Technology (Boston, MA, USA).
The secondary antibodies used in the present study included horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies purchased from Cell Signaling Technology (1:2,000 each).
Cell culture and cell proliferation assay
Four human EAC cell lines (OE19, OE33, SK-GT4 and OACM5.1c) were obtained from the European Collection of Authenticated Cell Cultures (ECACC). All cell lines were grown in RPMI-1640 medium (Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 9100 mg/l of penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2.
Cell proliferation was assayed using a Cell Counting kit-8 (CCK-8) according to the manufacturers instructions. Briefly, 5×103 cells were seeded into each well of a 96-well plate and cultured in 100 µl of RPMI-1640 medium supplemented with 10% FBS. After 24 h, Gal-9 (0, 0.1, 0.3 or 1.0 µM) was added to each well, and the cells were cultured for an additional 48 h. Then, the CCK-8 reagent (10 µl) was added to each well, and the plates were incubated at 37°C for 3 h. The absorbance of each well was measured at 450 nm using a microplate reader.
ELISA to assess apoptosis
Caspase-cleaved cytokeratin 18 (CCK18) expression was evaluated using an M30 Apoptosense ELISA kit obtained from Peviva AB (Bromma, Sweden) according to the manufacturers instructions (25). Cells (5×103/well) were seeded into 96-well plates, cultured in 100 µl of culture medium for 24 h and then treated with 0.3 µM Gal-9. The antigen concentrations in the control and treated samples were calculated via interpolation from a standard curve.
Cell cycle and apoptosis analyses
The cell cycle and apoptosis analyses were performed separately using a Cell Cycle Phase Determination kit (Cayman Chemical, Ann Arbor, MI, USA) and an Annexin V-FITC Early apoptosis detection kit (Cell Signaling Technology), respectively.
SK-GT4 cells (1.0×106 cells in a 100-mm dish) were treated with 0.3 µM Gal-9 for 48 h, and untreated cells were used as the controls. The cell cycle profiles were analyzed by measuring the propidium iodide (PI)-labeled DNA content in ethanol-fixed cells. Fixed cells were washed with phosphate-buffered saline (PBS) and then stored at −20°C prior to flow cytometric analysis. On the day of the analysis, the cells were washed with cold PBS, suspended in 100 µl of PBS with 10 µl of RNase A (250 µg/ml) and incubated for 30 min. Then, 110 µl of PI stain (100 µg/ml) was added to each cell suspension, and the cells were incubated at 4°C for at least 30 min prior to the analysis. Apoptotic and necrotic cell death were analyzed by performing double staining with FITC-conjugated Annexin V and PI; this staining method is based on the binding of Annexin V to apoptotic cells with exposed phosphatidylserine residues and the binding of PI to late apoptotic/necrotic cells with membrane damage. Tumor cells were treated with 0.3 µM Gal-9 for either 12 or 24 h and untreated cells were used as the controls. Staining was performed according to the manufacturers instructions. Flow cytometry was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA), and the proportions of stained cells were analyzed using the Kaluza software (Beckman Coulter). All experiments were performed in triplicate.
Gel electrophoresis and western blotting
SK-GT4 cells were seeded at a density of 1.0×106 cells/100-mm dish and cultured for 24 h. Then, 0.3 µM Gal-9 was added, and the cells were cultured for an additional 24–48 h. Next, the cells were lysed in a protease inhibitor cocktail (Complete protease inhibitor mixture; iNtRON Biotechnology, Sungnam, Korea) on ice for 20 min. Suspensions of lysed cells were centrifuged at 13,000 × g at 4°C for 5 min, and supernatants containing soluble cellular proteins were harvested and stored at −80°C until use. The protein concentrations were measured using a NanoDrop 2000 fluorospectrometer (Thermo Fisher Scientific). Protein aliquots (1–10 µg) were resuspended in sample buffer, separated on 10% Tris-glycine gradient gels by SDS-PAGE (26), and then transferred to nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies (27). Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Perkin-Elmer Co., Waltham, MA, USA) on X-ray film.
Angiogenic profile analysis
SK-GT4 cells were seeded (6.0×106 cells/100 mm-diameter dish) and treated with 0.3 µM Gal-9, while control cells remained untreated; all cells were cultured with RPMI-1640 medium supplemented with 10% FBS for 24 h and then lysed in PRO-PREP. A RayBio Human Angiogenesis Antibody array (RayBiotech, Inc., Norcross, GA, USA) was employed according to the manufacturers protocol. This protocol includes a spot-based assay that facilitates the detection and comparison of 20 angiogenic cytokines. Each array membrane was exposed to X-ray film, and the signals were detected using a chemiluminescence detection system (Perkin-Elmer). The immunoreactive band densities obtained with this array were analyzed by densitometric scanning (TIc scanner; Shimizu, Co., Ltd., Kyoto, Japan).
Phosphorylated receptor tyrosine kinase (p-RTK) antibody arrays
SK-GT4 cells were treated with 0.3 µM Gal-9 for 24 h and then lysed in PRO-PREP. Human phospho-RTKs were assayed using a Human Phospho-RTK Array kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturers instructions. Each array membrane was exposed to X-ray film, and the signals were detected using a chemiluminescence detection system (Perkin-Elmer).
Analysis of miRNA arrays
SK-GT4 cells were treated with 0.3 µM Gal-9 for 24 h and then stored in the RNAprotect reagent (Qiagen, Venlo, The Netherlands). Total RNA was extracted from each cell line using a miRNeasy Mini kit (Qiagen) according to the manufacturer's instructions, and the RNA quantity and quality were measured using an RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). The samples were labeled using a miRCURY Hy3 Power Labeling kit (Exiqon A/S, Vedbaek, Denmark) and hybridized to a human miRNA oligo chip (v.21; Toray Industries, Tokyo, Japan). Scanning was performed using a 3D-Gene Scanner 3000 (Toray Industries). The 3D-Gene extraction version 1.2 software (Toray Industries) was used to read the raw intensities from the images. To detect differences in miRNA expression between the Gal-9-treated and control samples, the raw data were analyzed using the GeneSpring GX 10.0 software (Agilent Technologies). Quantile normalization was performed for raw data above background levels, and differentially expressed miRNAs were identified using the Mann-Whitney U test. Hierarchical clustering was performed using the farthest neighbor method with the absolute uncentered Pearson's correlation coefficient as a metric. A heat map was produced with the base-2 logarithms of the intensities median-centered for each row to depict the relative expression intensity of each miRNA.
Statistical analysis
All statistical analyses were performed using the GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between the treated and control groups were performed using two-tailed paired or unpaired Students t-tests. A P<0.05 was considered significant.
Results
Gal-9 suppresses human esophageal adenocarcinoma cell proliferation
The effects of Gal-9 on the proliferation of four EAC cell lines (OE19, OE33, SK-GT4 and OACM5.1c) were evaluated. The cells were grown in 10% FBS and treated with 0, 0.1, 0.3 or 1.0 µmol/l Gal-9 for 48 h. Gal-9 inhibited the proliferation of all four EAC cell lines in a dose-dependent manner (Fig. 1).
Gal-9 exhibits antitumor effects in EAC cells by inducing apoptosis
To determine whether Gal-9 induces apoptosis, SK-GT4, OE19 and OE33 cells were treated with 0.3 µM Gal-9, and the CCK18 levels were measured following treatment using an M30 ELISA kit. The results of Annexin V-FITC/PI staining and the flow cytometric analysis demonstrated that Gal-9 significantly increased the CCK-18 levels in the three EAC cell lines (Fig. 2A) and also induced apoptosis of the SK-GT4 cells in a dose- and time-dependent manner. The different quadrants presented in Fig. 2B represent living cells (lower left quadrant), early apoptotic cells (lower right quadrant), and late apoptotic cells (upper right quadrant). The increased numbers of early- and late-phase apoptotic cells indicated that the antitumor effects of Gal-9 involved induction of apoptosis (Fig. 2C). This apoptotic induction was accompanied by increases in the cleaved caspase-3, cleaved caspase-9 and cleaved PARP levels (Fig. 2D). The blockade of caspase activation by treatment with either the pan-caspase inhibitor Z-VAD-FMK or caspase-3 inhibitor Z-DEVD-FMK did not protect the cells from Gal-9-induced cell death (Fig. 2E). Thus, Gal-9 may suppress the proliferation of EAC cells by inducing apoptosis via a caspase-independent pathway. Because apoptosis often occurs simultaneously with autophagy, this process is also involved in the antitumor effect of Gal-9. Therefore, we evaluated the levels of SQSTM1/p62 and LC3-II, which are key proteins involved in autophagy regulation, after treatment with Gal-9 for 24 h. As shown in Fig. 2F, the accumulation of LC3-II and upregulation of SQSTM1/p62 were observed in the treated SK-GT4 cells, indicating that Gal-9 may inhibit the autophagic flux.
No specific effects of Gal-9 are observed on cell cycle regulatory proteins in SK-GT4 cells
The effects of Gal-9 on the expression of various cell cycle-related molecules in SK-GT4 cells were evaluated by western blotting. SK-GT4 cells were treated with 0.3 µM Gal-9 for 48 h. Gal-9 treatment resulted in progressive decreases in the cyclin D1, cyclin E and Cdk4 levels but had no effects on the levels of other cell cycle regulatory proteins (Fig. 3A).
To elucidate the mechanism of action of Gal-9 in the control of SK-GT4 cell proliferation, cell cycle progression was examined by flow cytometry. No changes were observed in the cell cycle profiles of SK-GT4 cells treated with 0.3 µM Gal-9 (Fig. 3B), suggesting that Gal-9 suppressed EAC cell growth through tumor cell apoptosis but not through cell cycle arrest.
Gal-9 treatment affects the expression of angiogenesis-related molecules
We used an angiogenesis array system (Fig. 4A) to identify the key angiogenesis-related molecules associated with the antitumor effects of Gal-9 in SK-GT4 cells. Of the 20 angiogenesis molecules screened, only the interleukin (IL)-8 levels increased in vitro following Gal-9 treatment (Fig. 4B). The densitometric ratio of IL-8 spots for Gal-9-treated vs. untreated cells was 58.2-fold (Fig. 4C).
Effects of Gal-9 on p-RTKs in SK-GT4 cells
A p-RTK array system was used to identify the key RTKs associated with the antitumor effects of Gal-9. The use of an antibody array (Fig. 5A) enabled the evaluation of the expression of 49 activated RTKs in SK-GT4 cells and tumors in the presence and absence of Gal-9. The activated RTK levels did not change following Gal-9 treatment (Fig. 5B).
Effects of Gal-9 on miRNA expression
Using a custom microarray platform, we analyzed the expression of 2,555 miRNA probes in cell lines cultured in the presence and absence of Gal-9. Treatment of SK-GT4 cells with 0.3 µmol/l Gal-9 for 48 h resulted in the significant upregulation of 31 miRNAs and in the significant downregulation of 10 miRNAs (Table I).
 | Table I.Relative expression levels and chromosomal locations of miRNAs in SK-GT4 cells cultured with or without Gal-9. |
An unsupervised hierarchical clustering analysis performed by calculating Pearsons correlation coefficient revealed clustering of the cell lines treated with Gal-9 in vitro; the miRNA expression patterns of the treated cells were distinct from the patterns of the untreated cell lines (Fig. 6).
Discussion
Based on the results of the present study, Gal-9 suppresses the cell proliferation and tumor growth of human EAC cell lines in vitro. The antitumor effects of Gal-9 on T cell homeostasis, cell aggregation and metastasis are well known (13,14). Additionally, Gal-9 inhibits the proliferation of hematologic malignancies, such as multiple myeloma (17) and chronic myeloid leukemia (18) and significantly retards the growth of myeloma xenografts in mice (17). Furthermore, cell surface-associated Gal-9 triggers the aggregation of melanoma cells, which is indicative of the Gal-9-mediated activation of cellular adhesion and inhibition of cell detachment (19,20). Although Gal-9 may suppress the proliferation and tumor growth of hematologic malignancies in vitro and in vivo, Gal-9 exerts different effects on solid malignancies. For example, breast cancer cell lines with high endogenous Gal-9 levels exhibit a strong tendency to aggregate, whereas cells with low Gal-9 levels do not (28). Importantly, ectopic expression of endogenous Gal-9 and treatment with recombinant Gal-9 trigger the formation of tight cellular clusters (19,28). Therefore, Gal-9 directly suppresses cell proliferation and tumor growth and has therapeutic potential for several solid tumors.
Recombinant Gal-9 induces apoptosis and cell death through apoptotic signaling pathways (17,18). In multiple myeloma cells, apoptotic signaling is induced via the activation of the MAP kinases JNK and p38 (17). Additionally, Gal-9 induces the pro-apoptotic Bcl-2 family member Noxa via activation of transcription factor 3, leading to the death of chronic myeloma cells (18). Moreover, various hematologic malignancies are sensitive to apoptotic elimination by recombinant Gal-9. Cleavage of cytokeratin 18 occurs as an early event during apoptosis following the activation of apoptosis executioners, particularly effector caspases, but remains intact during other types of cell death, such as autophagy and necrosis (29).
Gal-9 induces apoptosis through both caspase-dependent and caspase-independent mechanisms (17,20). In previous studies, Gal-9 increased the levels of cleaved cytokeratin-18 in various cancer cell lines in a dose- and time-dependent manner (21–23,31). Based on our data, Gal-9 also increased CCK18 levels in the three EAC cell lines. Additionally, Gal-9 increased the activated caspase-3, caspase-9 and PARP levels. The death receptor and mitochondrial pathways are the two major pathways that initiate apoptotic responses, and caspase-3 is the key executioner caspase in both pathways (32). The present study revealed that the apoptosis of EAC cells was initiated through caspase-independent pathways. Recently, Wiersma et al (30) showed an association of Gal-9 with impaired lysosomal function and fatal frustrated autophagy. Gal-9 converts LC3-I to LC3-II, whereas SQSTM1/p62 is increased after Gal-9 treatment for 24–48 h. Moreover, the inhibition of autophagosome-lysosome fusion and LC3-II-SQSTM1/p62 accumulation by the lysosomal inhibitor chloroquine has previously been reported. Combination therapies using autophagy inhibitors and standard chemotherapies have been proposed for many cancer types, and these findings indicate that Gal-9 may have a synergic effect in such a combination therapy for acquired therapeutic resistance.
The expression levels of cell cycle-related proteins were unchanged or only slightly altered 48 h after the addition of Gal-9. Additionally, the results of the flow cytometric analysis revealed no effects of Gal-9 on the G0 to G1 transition in EAC cells in vitro. Thus, the antitumor effects of Gal-9 may not be related to a reduction in the levels of various cell cycle-related proteins.
IL-8 in tumors and the tumor microenvironment contributes to tumor progression by regulating angiogenesis and cancer cell growth and survival (33). In patients with esophageal cancer, the elevated expression of IL-8 and its receptor CXCR-2 has been associated with a poor prognosis (34). Based on our data, Gal-9 increased IL-8 expression in Gal-9-treated SK-GT4 cells. Acquired Gal-9 resistance in EAC cells may be attributable to Gal-9-induced IL-8 expression; thus, the applicability of Gal-9 for EAC treatment may be limited to the tumor microenvironment and angiogenesis. Conversely, the levels of 49 pRTKs did not change following treatment of the human EAC cell lines with Gal-9.
The miRNAs associated with the antitumor effects of Gal-9 were assessed using miRNA expression arrays. The cluster analysis clearly showed the effects of Gal-9 treatment on the miRNA expression levels in cancer cells. We identified 41 miRNAs that were differentially expressed in the Gal-9-treated EAC cells. These miRNAs are important candidates to gauge the effectiveness of Gal-9 treatment and provide insights into the molecular basis of Gal-9-mediated antitumor effects, particularly those mediated by miRNAs.
miR-200c expression was upregulated in hepatocellular carcinoma (35) and ovarian cancer (36) tissues compared with their respective normal tissues. Additionally, the overexpression of miR-200c in esophageal cancer was associated with unfavorable responses to chemotherapy and poor prognoses (37) because this miRNA supported tumor growth by directly suppressing PPP2R1 and promoting Akt activation (38). The present study was not able to determine whether miR-200c acted as an oncogenic or tumor-suppressive molecule. However, Gal-9 treatment downregulated miR-200c expression in EAC cells, which may be associated with the antitumor effects of Gal-9.
In conclusion, Gal-9 suppresses human EAC cell proliferation, possibly by inducing apoptosis in a miRNA-dependent manner.
Acknowledgements
We thank Ms. Noriko Murao and Ms. Kana Ogawa for providing technical assistance.
Glossary
Abbreviations
Abbreviations:
|
Gal-9 |
galectin-9 |
|
EAC |
esophageal adenocarcinoma |
|
CRDs |
carbohydrate recognition domains |
|
miRNAs |
microRNAs |
|
CCK-8 |
Cell Counting kit-8 |
|
IL-8 |
interleukin-8 |
|
phospho-RTKs |
phosphorylated receptor tyrosine kinases |
|
CCK18 |
caspase-cleaved cytokeratin 18 |
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