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.

Galectin-9 mediates neutrophil capture and adhesion in a CD44 and ß2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of ß2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.

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.

Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity.

Endogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved.

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.

Plasma Galectin-9 Concentrations in Normal and Diseased Condition.

BACKGROUND/AIMS: Galectin-9 is a soluble immune modulator with versatile functions, including a role as an immune checkpoint molecule. Therefore, the amount of galectin-9 in the blood may reflect an individual's immunological balance. Many studies have conducted galectin-9 measurements; however, the reported galectin-9 concentration in the blood varies greatly, even within healthy controls. This study investigates the variation between the reported and actual concentrations of galectin-9. METHODS: A GalPharma ELISA and an R&D Systems ELISA kit were directly compared using the same set of plasma and a series of recombinant galectins, including degraded galectin-9. Furthermore, galectin-9 in plasma was concentrated using anti-galectin-9 antibody-conjugated beads, and subjected to western blotting to estimate the quantity and integrity of galectin-9 and assess the consistency of ELISA measurements. RESULTS: The R&D Systems' ELISA indicated a 50-fold higher median concentration of plasma galectin-9 than that indicated by the GalPharma ELISA. This variation is due to aberrantly enhanced reactivity of the R&D Systems' ELISA to degraded galectin-9 present in small quantities in the plasma. The GalPharma ELISA could detect only intact galectin-9 and its results correlated well with the plasma galectin-9 level obtained by western blotting. CONCLUSION: ELISA kits from R&D Systems reacts aberrantly higher against degraded galectin-9 than the intact galectin-9. Therefore, the existence of a small amount of degraded galectin-9 in a test sample hinders the quantification. As galectin-9 is a fragile protein, this is a serious concern when using this kit. Based on quantifications from the GalPharma ELISA, the median (25th-75th percentiles) galectin-9 concentration in healthy subjects in the current study cohort was calculated as 110 pg/mL (67 -154 pg/mL).

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.

Role of Lgals9 Deficiency in Attenuating Nephritis and Arthritis in BALB/c Mice in a Pristane-Induced Lupus Model.

OBJECTIVE: In systemic lupus erythematosus (SLE), an autoimmune disease associated with multiple organ involvement, the development of lupus nephritis determines prognosis, and arthritis impairs quality of life. Galectin 9 (Gal-9, Lgals9) is a ß-galactoside-binding lectin that has been used for clinical application in autoimmune diseases, since recombinant Gal-9, as a ligand for T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), induces apoptosis of activated CD4+TIM-3+ Th1 cells. This study was undertaken to investigate whether deficiency of Lgals9 has beneficial or deleterious effects on lupus in a murine model. METHODS: Gal-9+/+ and Gal-9-/- female BALB/c mice were injected with pristane, and the severity of arthritis, proteinuria, and levels of autoantibody production were assessed at several time points immediately following injection. At 7 months after pristane injection, renal pathologic features, the severity of joint inflammation, and formation of lipogranulomas were evaluated. Subsets of inflammatory cells in the spleen and peritoneal lavage were characterized, and expression levels of cytokines from peritoneal macrophages were analyzed. RESULTS: Lgals9 deficiency protected against the development of immune complex glomerulonephritis, arthritis, and peritoneal lipogranuloma formation in BALB/c mice in this murine model of pristane-induced lupus. The populations of T cell subsets and B cells in the spleen and peritoneum were not altered by Lgals9 deficiency in pristane-injected BALB/c mice. Furthermore, Lgals9 deficiency protected against pristane-induced lupus without altering the Toll-like receptor 7-type I interferon pathway. CONCLUSION: Gal-9 is required for the induction and development of lupus nephritis and arthritis in this murine model of SLE. The results of the current investigation provide a potential new strategy in which antagonism of Gal-9 may be beneficial for the treatment of nephritis and arthritis in patients with SLE through targeting of activated macrophages.

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.

Protective effect of Galectin-9 in murine model of lung emphysema: Involvement of neutrophil migration and MMP-9 production.

PURPOSE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow obstruction and pulmonary emphysema. Persistent inflammation and remodeling of the lungs and airways result in reduced lung function and a lower quality of life. Galectin (Gal)-9 plays a crucial role as an immune modulator in various diseases. However, its role in the pathogenesis of pulmonary emphysema is unknown. This study investigates whether Gal-9 is involved in pulmonary inflammation and changes in emphysema in a porcine pancreatic elastase (PPE)-induced emphysema model. MATERIALS AND METHODS: Gal-9 was administered to mice subcutaneously once daily from 1 day before PPE instillation to day 5. During the development of emphysema, lung tissue and bronchoalveolar lavage fluid (BALF) were collected. Histological and cytological findings, concentrations of chemokines and matrix metalloproteinases (MMPs) in the BALF, and the influence of Gal-9 treatment on neutrophils were analyzed. RESULTS: Gal-9 suppressed the pathological changes of PPE-induced emphysema. The mean linear intercept (Lm) of Gal-9-treated emphysema mice was significantly lower than that of PBS-treated emphysema mice (66.1 ± 3.3 µm vs. 118.8 ± 14.8 µm, respectively; p < 0.01). Gal-9 decreased the number of neutrophils and levels of MMP-9, MMP-2 and tissue inhibitor of metalloproteinases (TIMP)-1 in the BALF. The number of neutrophils in the BALF correlated significantly with MMPs levels. Interestingly, Gal-9 pretreatment in vitro inhibited the chemotactic activity of neutrophils and MMP-9 production from neutrophils. Furthermore, in Gal-9-deficient mice, PPE-induced emphysema progressed significantly compared with that in wild-type (WT) mice (108.7 ± 6.58 µm vs. 77.19 ± 6.97 µm, respectively; p < 0.01). CONCLUSIONS: These results suggest that Gal-9 protects PPE-induced inflammation and emphysema by inhibiting the infiltration of neutrophils and decreasing MMPs levels. Exogenous Gal-9 could be a potential therapeutic agent for COPD.

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.

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.

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.

Human Galectin-9 Is a Potent Mediator of HIV Transcription and Reactivation.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05). rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006) and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02) and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009), suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.

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.