Antitumor Effect of Galectin-9 on Cell Proliferation and Tumor Growth of Human Duodenal Adenocarcinoma Cells.

BACKGROUND/AIM: Galectin-9 (Gal-9) induces tumor cell apoptosis in lymphoma and other malignant cell types. Duodenal adenocarcinoma is a rare malignancy, and there are insufficient data to determine a standard therapeutic approach. Here, we investigated the antitumor effect of Gal-9 in HuTu-80 duodenal adenocarcinoma cells. MATERIALS AND METHODS: Cell proliferation was examined in HuTu-80 cells using a Cell Counting Kit-8 assay. Cell cycle analysis, apoptosis array, and microRNA expression analysis were performed to identify the effect of Gal-9 on HuTu-80 cells. The antitumor effect of Gal-9 was also examined using xenograft mouse models. RESULTS: Gal-9 suppressed the proliferation of HuTu-80 via blockade of the G0 to G1 cell cycle transition. This blockade was accompanied by a strong decrease in cyclin D1 and phosphorylated Rb, suggesting a G1 arrest. Additionally, Gal-9 induced apoptosis, and the expression of cleaved caspase-3 was increased in Gal-9-treated HuTu-80 cells according to the apoptosis array. MiRNA microarrays revealed that Gal-9 altered the expression of miRNAs in HuTu-80 cells. CONCLUSION: These data demonstrate the therapeutic potential of Gal-9 and provide molecular mechanistic insights into its antitumor effect in HuTu-80 cells.

Galectin­9 suppresses the tumor growth of colon cancer in vitro and in vivo.

Colon cancer is the second leading cause of cancer­related mortality worldwide, and the prognosis of advanced colon cancer has remained poor in recent years. Galectin­9 (Gal­9) is a tandem­repeat type galectin that has recently been shown to exert antiproliferative effects on various types of cancer cells. The present study aimed to assess the effects of Gal­9 on human colon and colorectal cancer cells in vitro and in vivo, as well as to evaluate the microRNAs (miRNAs/miRs) associated with the antitumor effects of Gal­9. We examined the ability of Gal­9 to inhibit cell proliferation via apoptosis, and the effects of Gal­9 on cell cycle­related molecules in various human colon and colorectal cancer cell lines. In addition, Gal­9­mediated changes in activated tyrosine kinase receptors and angiogenic molecules were assessed using protein array chips in colon and colorectal cancer cells. Moreover, miRNA array analysis was performed to examine Gal­9­induced miRNA expression profiles. We also elucidated if Gal­9 inhibited tumor growth in a murine in vivo model. We found that Gal­9 suppressed the cell proliferation of colon cancer cell lines in vitro and in vivo. Our data further revealed that Gal­9 increased caspase­cleaved keratin 18 levels in Gal­9­treated colon cancer cells. In addition, Gal­9 enhanced the phosphorylation of ALK, DDR1, and EphA10 proteins. Furthermore, the miRNA expression levels, such as miR­1246, miR­15b­5p, and miR­1237, were markedly altered by Gal­9 treatment in vitro and in vivo. In conclusion, Gal­9 suppresses the cell proliferation of human colon cancer by inducing apoptosis, and these findings suggest that Gal­9 can be a potential therapeutic target in the treatment of colon cancer.

Galectin-9 Induces Mitochondria-Mediated Apoptosis of Esophageal Cancer In Vitro and In Vivo in a Xenograft Mouse Model.

Galectin-9 (Gal-9) enhances tumor immunity mediated by T cells, macrophages, and dendritic cells. Its expression level in various cancers correlates with prognosis. Furthermore, Gal-9 directly induces apoptosis in various cancers; however, its mechanism of action and bioactivity has not been clarified. We evaluated Gal-9 antitumor effect against esophageal squamous cell carcinoma (ESCC) to analyze the dynamics of apoptosis-related molecules, elucidate its mechanism of action, and identify relevant changes in miRNA expressions. KYSE-150 and KYSE-180 cells were treated with Gal-9 and their proliferation was evaluated. Gal-9 inhibited cell proliferation in a concentration-dependent manner. The xenograft mouse model established with KYSE-150 cells was administered with Gal-9 and significant suppression in the tumor growth observed. Gal-9 treatment of KYSE-150 cells increased the number of Annexin V-positive cells, activation of caspase-3, and collapse of mitochondrial potential, indicating apoptosis induction. c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) phosphorylation were activated and could be involved in apoptosis. Therefore, Gal-9 induces mitochondria-mediated apoptosis of ESCC and inhibits cell proliferation in vitro and in vivo with JNK and p38 activation.

Characterization of neutralizing antibodies reacting with the 213-224 amino-acid segment of human galectin-9.

Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.

MicroRNA profiles during galectin-9-induced apoptosis of pancreatic cancer cells.

Pancreatic cancer is the eighth-leading cause of cancer-associated mortality in males and the ninth-leading cause in females worldwide. Even when diagnosed early enough to be potentially resectable, the prognosis of invasive pancreatic cancer is poor. Galectin-9 (Gal-9) is a tandem-repeat type galectin that has recently been demonstrated to possess an anti-proliferative effect on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human pancreatic cancer cells and examined the microRNAs that are associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of multiple pancreatic cancer cell lines. In addition, Gal-9 treatment increased the levels of caspase-cleaved keratin 18 and the expression of cytochrome c in pancreatic cancer cell lines. This data suggests that Gal-9 induces intrinsic apoptosis in pancreatic cancer cell lines through the caspase-dependent and caspase-independent pathways. In addition, Gal-9 reduced the expression levels of phosphorylated epidermal growth factor receptor and numerous receptor tyrosine kinases (RTKs). In conclusion, Gal-9 may suppress the growth of human pancreatic cancer cells in vitro. These findings suggest that Gal-9 may be a new therapeutic agent for the treatment of pancreatic cancer.

Correlation between serum galectin-9 levels and liver fibrosis.

BACKGROUND AND AIM: Chronic liver diseases progress from chronic inflammation to fibrosis to tumorigenesis. Galectin-9, a ß-galactoside-specific animal lectin, is indicated to contribute to all three steps of progression. The aim of this study was to determine which of the three steps was most dominant in elevating the serum galectin-9 concentration and to test the possibility of galectin-9 as a serum biomarker. METHODS: Japanese patients with chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), non-alcoholic fatty liver disease, or alcoholic liver disease who provided informed consent were enrolled in this study. Serum galectin-9 levels were measured using a sandwich ELISA. Multiple regression analyses were performed using ezr to identify factors that determined serum galectin-9 concentration. RESULTS: One hundred one patients with 50 of chronic hepatitis and 51 of liver cirrhosis were enrolled; the cohort included 45 cases of hepatitis C virus infection, 13 cases of hepatitis B virus infection, and 46 cases with HCC-related complications. The median serum galectin-9 concentration was 77.54 pg/mL (interquartile range: 18.89-241.9 pg/mL). Multiple linear regression analyses proved Fibrosis-4 index and aspartate aminotransferase to platelet ratio index, indexes of liver fibrosis, were able to predict the serum galectin-9 levels with statistical significance. A multiple logistic regression analysis determined 10 pg/mL increase in the serum galectin-9 concentration presented an odds ratio of 3.90 for liver fibrosis progression. CONCLUSIONS: The serum galectin-9 concentration represents a potential biomarker of liver fibrosis in patients with chronic liver diseases, regardless of chronic inflammation or the presence of HCC complications. Furthermore, higher serum galectin-9 levels are a predictor for liver fibrosis progression.

Regulatory T Cell-Mediated Suppression of Inflammation Induced by DR3 Signaling Is Dependent on Galectin-9.

Stimulation of several TNF receptor family proteins has been shown to dampen inflammatory disease in murine models through augmenting the number and/or activity of regulatory T cells (Tregs). We recently found that one molecule, 4-1BB, used binding to Galectin-9 to exert its immunosuppressive effects and drive expansion of CD8+Foxp3- Tregs. We now show that ligation of another TNFR family molecule, DR3, which has previously been found to strongly expand CD4+Foxp3+ Tregs and suppress inflammation, also requires Galectin-9. We found that the extracellular region of DR3 directly binds to Galectin-9, and that Galectin-9 associates with DR3 in Tregs. From studies in vitro with Galectin-9-/- CD4+ T cells and Tregs, we found that stimulatory activity induced by ligating DR3 was in part dependent on Galectin-9. In vivo, in a model of experimental autoimmune encephalomyelitis, we show that an agonist of DR3 suppressed disease, correlating with expansion of CD4+Foxp3+ Tregs, and this protective effect was lost in Galectin-9-/- mice. Similar results were seen in an allergic lung inflammation model. Thus, we demonstrate a novel function of Galectin-9 in facilitating activity of DR3 related to Treg-mediated suppression.

X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

Galectin-9 (G9) is a tandem-repeat type ß-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.

Induction of apoptosis by Galectin-9 in liver metastatic cancer cells: In vitro study.

Liver metastasis from gastrointestinal cancer defines a patient's prognosis. Despite medical developments, pancreatic cancer with liver metastasis confers a very poor prognosis. Galectin-9 (Gal­9) is a tandem-repeat-type galectin that has recently been demonstrated to exert antitumor effects on various types of cancer cells by inducing apoptosis. However, the apoptotic pathway of Gal­9 in solid tumors is unclear. The aim of the present study was to evaluate the effects of Gal­9 on human liver metastasis from pancreatic cancer. Gal­9 suppressed cell proliferation in metastatic liver cancer cell lines derived from pancreatic cancer (KMP2, KMP7, and KMP8) and increased the levels of caspase-cleaved keratin 18 and fluorescein isothiocyanate (FITC)-conjugated Annexin V. Furthermore, expression of apoptosis-related molecules such as caspase-7, cleaved caspase-3, cleaved PARP, cytochrome c, Smac/Diablo and HtrA2/Omi was enhanced. However, Gal­9 did not affect expression of various cell cycle-related proteins. The microRNA (miRNA) expression profile was markedly altered by Gal­9, and various miRNAs might contribute to tumor growth suppression. Our data reveal that Gal­9 suppresses the growth of liver metastasis, possibly by inducing apoptosis through a mechanism involving mitochondria and changes in miRNA expression. Thus, Gal­9 might serve as a therapeutic agent for the treatment of liver metastasis from pancreatic cancer.

Effects of galectin-9 on apoptosis, cell cycle and autophagy in human esophageal adenocarcinoma cells.

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. 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. Galectin-9 (Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8 (IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.

Galectin­9 ameliorates fulminant liver injury.

Fulminant hepatitis is a severe liver disease resulting in hepatocyte necrosis. Galectin­9 (Gal­9) is a tandem­repeat­type galectin that has been evaluated as a potential therapeutic agent for various diseases that regulate the host immune system. Concanavalin A (ConA) injection into mice results in serious, immune­mediated liver injury similar to human viral, autoimmune and fulminant hepatitis. The present study investigated the effects of Gal­9 treatment on fulminant hepatitis in vivo and the effect on the expression of microRNAs (miRNAs), in order to identify specific miRNAs associated with the immune effects of Gal­9. A ConA­induced mouse hepatitis model was used to investigate the effects of Gal­9 treatment on overall survival rates, liver enzymes, histopathology and miRNA expression levels. Histological analyses, TUNEL assay, immunohistochemistry and miRNA expression characterization, were used to investigate the degree of necrosis, fibrosis, apoptosis and infiltration of neutrophils and macrophages. Overall survival rates following ConA administration were significantly higher in Gal­9­treated mice compared with control mice treated with ConA + PBS. Histological examination revealed that Gal­9 attenuated hepatocellular damage, reduced local neutrophil infiltration and prevented the local accumulation of macrophages and liver cell apoptosis in ConA­treated mice. In addition, various miRNAs induced by Gal­9 may contribute to its anti­apoptotic, anti­inflammatory and pro­proliferative effects on hepatocytes. The results of the present study demonstrate that Gal­9 may be a candidate therapeutic target for the treatment of fulminant hepatitis.

Cancer Therapy Due to Apoptosis: Galectin-9.

Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described.

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

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.

Galectin-9: An anticancer molecule for gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. There are various histological types of GBC, and the vast majority of GBC cases are adenocarcinomas. Squamous and adenosquamous carcinomas are rare GBC subtypes that are traditionally considered to be more aggressive and to be associated with a poorer prognosis than adenocarcinoma. Galectin-9 (Gal-9), a tandem-repeat-type galectin, has been reported to induce apoptosis-mediated elimination of various cancers, including hepatocellular carcinoma, cholangiocarcinoma, and hematologic malignancies. Therefore, we investigated the antitumor effects of Gal-9 on GBC in vitro and in vivo. In our in vitro experiments, Gal-9 suppressed cell proliferation in various GBC cell lines but not in the OCUG-1 cell line, which represents a poorly differentiated type of adenosquamous carcinoma. Gal-9 induced the apoptosis of Gal-9-sensitive GBC cells by increasing the levels of caspase-cleaved keratin 18 and phosphorylated p53. However, Gal-9 did not affect the expression of various cell cycle-related proteins. In addition, Gal-9 suppressed tumor growth by implanted human GBC cells in a xenograft model. Furthermore, Gal-9 induced the phosphorylation of the Ephrin type-B receptor, and the microRNA (miRNA) expression profile was markedly altered by Gal-9. Based on these results, various miRNAs might contribute to the suppression of tumor growth. Our data reveal that Gal-9 suppresses the growth of GBC, possibly by inducing apoptosis and altering miRNA expression. Thus, Gal-9 might serve as a therapeutic agent for the treatment of GBC.

Galectin-9 suppresses cholangiocarcinoma cell proliferation by inducing apoptosis but not cell cycle arrest.

Cholangiocarcinoma is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma (HCC). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been shown to exert antiproliferative effects on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human cholangiocarcinoma cells in vitro as well as the microRNAs (miRNAs) associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of cholangiocarcinoma cell lines in vitro and the growth of human cholangiocarcinoma cell xenografts in nude mice. Our data further revealed that Gal-9 increased caspase­cleaved keratin 18 (CCK18) levels, and the expression of cytochrome c increased in Gal-9-treated cholangiocarcinoma cell lines. These data suggested that Gal-9 induced cholangiocarcinoma cell apoptosis via the intrinsic apoptosis pathway mediated by caspase-dependent or -independent pathways. In addition, Gal-9 reduced the phosphorylation of the epidermal growth factor receptor (EGFR), insulin-like growth factor and insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor and fibroblast growth factor receptor 3 (FGFR3). These findings suggest that Gal-9 can be a candidate of therapeutic target in the treatment of cholangiocarcinoma.

The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux.

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRAS(mut)) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRAS(mut) CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAF(mut)). In KRAS(mut) CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRAS(mut) cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRAS(mut) CRC cells that may be exploitable for therapeutic use.

MicroRNA profiles in cisplatin-induced apoptosis of hepatocellular carcinoma cells.

Cisplatin [cis-diamminedichloroplatinum (II)], is a platinum coordination compound that is commonly used to treat hepatocellular carcinoma (HCC). It is also one of the most compelling anticancer drugs. Recent studies suggest that cisplatin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of cisplatin in several types of cancers, including HCC, has not been elucidated. The goal of the present study was to evaluate the effects of cisplatin on the proliferation of HCC cells in vitro and to determine which microRNAs (miRNAs) are associated with the anticancer effects of cisplatin in vitro. We used various human HCC-derived cell lines to study the effects of cisplatin on human HCC cells. Cisplatin led to a strong dose- and time- dependent inhibition of cell proliferation in HLE, HLF, HuH7, Li-7, Hep3B and HepG2 cells in vitro. Cisplatin also blocked the progression of the cell cycle in the G0/G1 phase, which inhibited cyclin D1 and induced apoptosis. In addition, miRNA expression was markedly altered by treatment with cisplatin in vitro. Therefore, various miRNAs induced by cisplatin may also contribute to the suppression of cellular proliferation and apoptosis. Our results demonstrate that cisplatin inhibits the growth of HCC, possibly through the induction of G1 cell cycle arrest and apoptosis through the alteration of microRNA expression.

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION.

Galectin-9 (gal-9) is a multifunctional ß-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca(2+)) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca(2+) mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca(2+) mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4(+) T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca(2+) mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4(+) T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.

Galectin-9 suppresses the growth of hepatocellular carcinoma via apoptosis in vitro and in vivo.

Galectin-9, a soluble ß-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.