Galectin-1 Knockdown Research Articles

Diosgenin upregulates axonal guidance partner molecules, Galectin-1 and Secernin-1

Galectin-1, a β-galactosides-binding protein, is widely expressed in various tissues and exhibits diverse biological activities. We previously obtained following findings; 1) Diosgenin, a steroid sapogenin, promoted axonal regeneration in the brain and recovered memory deficits in a model of Alzheimer’s disease (AD), 5XFAD mouse; 2) Neuron-specific overexpression of Galectin-1 protein in the hippocampus recovered memory impairment and promoted axonal regeneration in the brain in 5XFAD mice; 3) Secernin-1, a counterpart and axonal guidance molecule for Galectin-1-expressing axons, was secreted from the prefrontal cortical neurons to promote axonal guidance from the hippocampus to the prefrontal cortex. However, it has never been elucidated that diosgenin signaling increase Galectin-1 and Secernin-1 or not. Here, we found that diosgenin treatment upregulated the protein level of Galectin-1 in the hippocampus both in primary cultured neurons and in 5XFAD mouse brains. In addition, diosgenin-induced upregulation of Galectin-1 was diminished by treatment of a neutralizing antibody of 1,25D3-membrane-associated rapid response steroid-binding receptor (1,25D3-MARRS), a direct binding receptor for diosgenin. Importantly, knockdown of Galectin-1 in hippocampal neurons inhibited axonal growth activity of diosgenin. Furthermore, the expression level of Secernin-1 was also increased in prefrontal cortical neurons by administration of diosgenin to 5XFAD mice. These findings suggest that diosgenin is a suitable compound to facilitate Galectin-1-Secernin-1-mediated axonal growth in AD brains.

Upregulation of galectin-3 in influenza A virus infection promotes viral RNA synthesis through its association with viral PA protein

BackgroundInfluenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the β-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice.MethodsWe used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells.ResultsGalectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells.ConclusionWe demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza.

Galectin-1 knockdown inhibits proliferation, migration, invasion and promotes apoptosis of lung adenocarcinoma cells in vitro

To investigate the effect of galectin-1 on biological behaviors of lung adenocarcinoma cells and the underlying mechanism. The expression levels of galectin-1 mRNA were detected in 8 pairs of lung adenocarcinoma tissues and adjacent normal tissues and in lung adenocarcinoma cell lines A549 and H1299 and normal bronchial epithelial cell line BEAS-2b using qRT-PCR. The effect of galectin-1 knockdown by RNA interference on the proliferation, invasion and migration abilities and apoptosis of lung adenocarcinoma cells were examined using CCK8 assay, Transwell assay, scratch assay and flow cytometry. Western blotting was performed to detect the expressions of apoptosis-related proteins BAX, BCL-2, and caspase-3 and the proteins involved in the AKT and ERK pathways. The mRNA expression of galectin-1 was significantly increased in lung cancer tissues and lung adenocarcinoma cell lines (P < 0.05). In lung adenocarcinoma cells, galectin-1 knockdown significantly inhibited cell proliferation, migration and invasion and obviously increased cell apoptosis rate (P < 0.05), causing also significant reduction of the phosphorylation level of ERK signaling pathway (P < 0.05). Galectin-1 knockdown inhibits proliferation, migration, and invasion and promotes apoptosis of lung adenocarcinoma cells, and this effect is mediated probably by inhibition of the phosphorylation levels of the ERK pathway.

Inhibition of Src improves cardiac fibrosis in AngII-induced hypertrophy by regulating the expression of galectin-3.

The expression of Src is upregulated in the vasculature associated with cardiac hypertrophy events. Here, we aimed to explore the underlying mechanism of Src in angiotensin II (AngII)-mediated cardiac fibrosis and hypertrophy. The heart conditional Src knockout mouse model was established and administrated with AngII. The effects of Src on the AngII-mediated cardiac hypertrophy were assessed by Hematoxylin and Eosin (HE), Masson's trichrome, immunohistochemical staining, Annexin V-FITC/PI apoptosis detection assay and Western blot analysis. The expression levels of galectin-3, Src and the hypertrophy marker brain natriuretic peptide (BNP), as well as the phosphorylation of Src were all elevated in heart tissues of mice with AngII-induced cardiac hypertrophy and fibrosis. Heart conditional Src knockout attenuated AngII-activated cardiac fibrosis and hypertrophy in mice. Consistently, AngII could promote the expression of Src in a dose-dependent manner and the knockout of Src impaired Ang II-mediated apoptosis and fibrosis in the cardiomyocytes. In addition, Src inhibition suppressed the expression of galectin-3 in vivo and in vitro. Specifically, AngII could upregulate the expression of galectin-3, and knockdown of galectin-3 (Gal-3) remarkably inhibited AngII-enhanced apoptosis and fibrosis in the cardiomyocytes. Furthermore, overexpression of galectin-3 reinforced Ang II-induced cell apoptosis and fibrosis that was attenuated by knockout of Src. Our findings indicate that Src and Gal-3 play an important role in AngII-mediated cardiac structural remodeling. Src and galectin-3 might serve as potential targets for the treatment of AngII-induced cardiac fibrosis and hypertrophy.

Galectin-3 Mediates Thrombin-Induced Vascular Smooth Muscle Cell Migration.

Vascular smooth muscle cell (VSMC) migration is an important step in the progression and development of vulnerable plaques. Thrombin is involved in both physiological and pathological processes of atherosclerosis. Therefore, the elucidation of the mechanisms underlying thrombin-induced VSMC migration is essential for devising effective treatments aimed at the prevention of plaque instability. In this study, we found that thrombin activated MAPK signaling pathways and increased the expression of galectin-3, which was also a well-known factor in atherosclerosis. Knockdown of galectin-3 by specific small interfering RNA (siRNA) blocked thrombin-induced activation of ERK1/2 and p38 MAPK, but not JNK MAPK. Src/FAK phosphorylation was also shown to be activated by thrombin. FAK autophosphorylation at Y397 was most significantly inhibited by galectin-3 siRNA. Galectin-3 siRNA or specific inhibitor (P38 MAPK inhibitor and ERK1/2 inhibitor) effectively prevented thrombin-induced VSMC migration via reducing paxillin expression. These findings demonstrate, for the first time, that thrombin stimulation of VSMC migration and paxillin expression are regulated by galectin-3, and ERK1/2, p38 MAPK, and Src/FAK signaling pathways are involved in this process. These results are beneficial to clarify the role of galectin-3 in thrombin-induced advanced lesions in atherosclerosis and shed new insights into the regulatory mechanism of VSMC migration in combating plaque rupture.

Galectin-1 Contributes to Vascular Remodeling and Blood Flow Recovery After Cerebral Ischemia in Mice.

Galectin-1 is found in the vasculature and has been confirmed to promote angiogenesis in several cancer models. Furthermore, galectin-1 has been demonstrated to improve the recovery of cerebral ischemia. However, whether vascular remodeling contributes to this improvement is still unknown. In the present study, photochemical cerebral ischemia was induced in both galectin-1-treated (2μg/day, i.c.v, 3days) and galectin-1 knockout mice. Laser speckle imaging and immunofluorescent staining demonstrated that circulation and vascular remodeling in the ischemic cortex were improved by galectin-1 treatment but disrupted in galectin-1 knockout mice. Western blot analysis showed that the expression of matrix metallopeptidase-9 and vascular endothelial growth factor (VEGF) was regulated by galectin-1 in vivo. To determine how galectin-1 influences endothelial cells, the expression of galectin-1 in bEnd.3 cells was increased by transfection with an expression plasmid and knocked down by siRNA. As demonstrated by quantitative RT-PCR and western blot analysis, the expression of metallopeptidase-9, VEGF, and VEGF receptors was upregulated by galectin-1 overexpression but downregulated after galectin-1 knockdown. Flow cytometry, Transwell assay, and capillary-like tube formation assay were performed on cells after gene manipulation as well as cells treated by exogenous galectin-1 after anoxia. It demonstrated that galectin-1 potentiated the cell proliferation, migration capacity, and tube formation ability. Taken together, these data suggest that by targeting vascular remodeling, galectin-1 contributes to the restoration of blood flow, which promotes the recovery of mice after cerebral ischemic insults.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche

BackgroundAs a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated.MethodsTo generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit−CD29+CD49f+/lowCD45−Ter-119− cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs.ResultsFollowing liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies.ConclusionsOur findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Abstract 1241: Galectin-3 and the modulation of autophagy in the pancreatic cancer microenvironment

Abstract Galectin-3, a member of the β-galactoside binding proteins family was found up- regulated in pancreatic cancer and may be affected by microenvironment factors, such as hypoxia and/or nutritional deprivation. In this study, we aimed to analyze the effects of hypoxia and nutritional deprivation on the Galectin-3 protein levels and the possible influence of this galectin on the autophagy of pancreatic cancer cells. Nutritional deprivation was performed through partial or total reduction of fetal bovine serum (FBS) or through complete removal of serum, glucose and amino acids. Hypoxia was established with 1% oxygen tension. Proteins Galectin-3, Beclin- 1 and LC3 were accessed through Western blot and immunofluorescence. Induction of LC3 stable production was performed through transfection of pEGFP-LC3 and evaluated by confocal microcopy. Degradative vacuole quantification was done by transmission electron microscopy and knockdown of Galectin-3 was performed using siRNA transfection. According to the analyzes, the most effective conditions for the induction of autophagy were partial FBS deprivation for PANC-1 (p = 0.0035) and complete FBS/amino acid/glucose deprivation for Mia PaCa 2 cells (p = 0.0362). PANC-1 cells presented higher levels of Galectin-3 when they were submitted to 24 hours of FBS deprivation and simultaneously FBS deprivation and hypoxia. Hypoxia alone caused increases in LC3 levels for Mia PaCa-2 and for PANC-1 cells. Knockdown of Galectin-3 caused decreases of LC3 levels in almost all experimental conditions for both pancreatic cell lines with exception of concomitant nutritional deprivation and hypoxia for PANC-1. These results show that the Galectin-3 and LC-3 protein are modulated by hypoxia and nutritional deprivation. Cells incubated under hypoxia presented higher levels of LC3, what arguments in favor of the pro-autophagic aspects of hypoxia. Moreover, inhibition of Galectin-3 causes reduction of LC3, which implies Galectin-3 as an important regulator of the autophagic process in pancreatic cancer cells. Key-words: Pancreatic Ductal Adenocarcinoma, LC3, Hypoxia, Nutritional deprivation. Citation Format: Antonio Felix, Silvio Consonni, Maira Pitta, Hernandes Carvalho, Michelly Pereira, Moacyr Rêgo. Galectin-3 and the modulation of autophagy in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1241.

Galectin-3 Released by Pancreatic Ductal Adenocarcinoma Suppresses γδ T Cell Proliferation but Not Their Cytotoxicity.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment with a dense desmoplastic stroma. The expression of β-galactoside-binding protein galectin-3 is regarded as an intrinsic tumor escape mechanism for inhibition of tumor-infiltrating T cell function. In this study, we demonstrated that galectin-3 is expressed by PDAC and by γδ or αβ T cells but is only released in small amounts by either cell population. Interestingly, large amounts of galectin-3 were released during the co-culture of allogeneic in vitro expanded or allogeneic or autologous resting T cells with PDAC cells. By focusing on the co-culture of tumor cells and γδ T cells, we observed that knockdown of galectin-3 in tumor cells identified these cells as the source of secreted galectin-3. Galectin-3 released by tumor cells or addition of physiological concentrations of recombinant galectin-3 did neither further inhibit the impaired γδ T cell cytotoxicity against PDAC cells nor did it induce cell death of in vitro expanded γδ T cells. Initial proliferation of resting peripheral blood and tumor-infiltrating Vδ2-expressing γδ T cells was impaired by galectin-3 in a cell-cell-contact dependent manner. The interaction of galectin-3 with α3β1 integrin expressed by Vδ2 γδ T cells was involved in the inhibition of γδ T cell proliferation. The addition of bispecific antibodies targeting γδ T cells to PDAC cells enhanced their cytotoxic activity independent of the galectin-3 release. These results are of high relevance in the context of an in vivo application of bispecific antibodies which can enhance cytotoxic activity of γδ T cells against tumor cells but probably not their proliferation when galectin-3 is present. In contrast, adoptive transfer of in vitro expanded γδ T cells together with bispecific antibodies will enhance γδ T cell cytotoxicity and overcomes the immunosuppressive function of galectin-3.

Galectin-1 Overexpression Activates the FAK/PI3K/AKT/mTOR Pathway and Is Correlated with Upper Urinary Urothelial Carcinoma Progression and Survival.

Galectin-1 (GAL1) is a β-galactoside-binding protein involved in multiple aspects of tumorigenesis. However, the biological role of GAL1 in upper tract urothelial carcinoma (UTUC) has not been entirely understood. Herein, we investigated the oncological effects of GAL1 expression in tumor specimens and identified related gene alterations through molecular analysis of GAL1. Clinical parameter data and tumor specimens were collected from 86 patients with pT3N0M0 UTUC who had undergone radical nephroureterectomy. We analyzed the difference in survival by using Kaplan–Meier analyses and Cox proportional regression models and in GAL1 expression by using immunohistochemical (IHC) methods. Public genomic data from the Cancer Genome Atlas (TCGA) and GSE32894 data sets were analyzed for comparison. Using four urothelial carcinoma (UC) cell lines (BFTC-909, T24, RT4, and J82) as in vitro models, we evaluated the functions of GAL1 in UC cell growth, invasiveness, and migration and its role in downstream signaling pathways. The study population was classified into two groups, GAL1-high (n = 35) and GAL1-low (GAL1 n = 51), according to IHC interpretation. Univariate analysis revealed that high GAL1 expression was significantly associated with poor recurrence-free survival (RFS; p = 0.028) and low cancer-specific survival (CSS; p = 0.025). Multivariate analysis revealed that GAL1-high was an independent predictive factor for RFS (hazard ratio (HR) 2.43; 95% confidence interval (CI) 1.17–5.05, p = 0.018) and CSS (HR 4.04; 95% CI 1.25–13.03, p = 0.019). In vitro studies revealed that GAL1 knockdown significantly reduced migration and invasiveness in UTUC (BFTC-909) and bladder cancer cells (T24). GAL1 knockdown significantly reduced protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9, which increased tissue inhibitor of metalloproteinase-1 (TIMP-1) and promoted epithelial–mesenchymal transition (EMT). Through gene expression microarray analysis of GAL1 vector and GAL1-KD cells, we identified multiple significant signaling pathways including p53, Forkhead box O (FOXO), and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). We validated microarray results through immunoblotting, thus proving that downregulation of GAL1 reduced focal adhesion kinase (FAK), p-PI3K, p-AKT, and p-mTOR expression. We concluded that GAL1 expression was highly related to oncological survival in patients with locally advanced UTUC. GAL1 promoted UC invasion and metastasis by activating the FAK/PI3K/AKT/mTOR pathway.

Treg Cells Attenuate Neuroinflammation and Protect Neurons in a Mouse Model of Parkinson's Disease.

Regulatory T cells (Tregs), which secrete transforming growth factor (TGF)-β and interleukin (IL)-10, have essential role in anti-inflammatory and neurotrophic functions. Herein, we explore the neuroprotection of Tregs in Parkinson's disease (PD) by adoptive transfer of Tregs. Tregs, isolated by magnetic sorting, were activated in vitro and then were adoptively transferred to 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mice. Neuroinflammation, dopaminergic neuronal loss and behavioral changes of PD mice were evaluated. Live cell imaging system detected a dynamic contact of Tregs with MN9D cells that were stained with CD45 and galectin-1, respectively. Tregs prevented MPTP-induced dopaminergic neuronal loss, behavioral changes, and attenuated the inflammatory reaction in the brain. When blockade the LFA-1 activity in Tregs or the ICAM-1 activity in endothelial cells, the percentage of Tregs in substantia nigra (SN) decreased. CD45 and galectin-1 were expressed by Tregs and MN9D cells, respectively. CD45-labeled Tregs dynamically contacted with galectin-1-labeled MN9D cells. Inhibiting CD45 in Tregs impaired the ability of Tregs to protect dopaminergic neurons against MPP+ toxicity. Similarly, galectin-1 knockdown in MN9D cells reduced the ability of Tregs neuroprotection. Adoptive transfer of Tregs protects dopaminergic neurons in PD mice by a cell-to-cell contact mechanism underlying CD45-galectin-1 interaction. Graphical Abstract.

Induction of Trop-2 expression through the binding of galectin-3 to MUC1

Both mucin 1 (MUC1) and trophoblast cell surface antigen 2 (Trop-2) are overexpressed in various epithelial tumor cells, and their high expression is correlated with a poor prognosis. Both proteins were expressed in a human breast cancer cell line, MCF-7 cells, but neither was in a human colon cancer cell line, HCT116 cells. When MUC1 cDNA was introduced into HCT116 cells (HCT116/MUC1), expression of Trop-2 was induced. Reciprocally, treatment of MCF-7 cells with MUC1 siRNA reduced the level of Trop-2. Mithramycin A, an inhibitor of specificity protein 1 (Sp1) transcription factor, effectively inhibited the expression of Trop-2. Consistently, treatment with Sp1 siRNA reduced the expression of Trop-2. To reveal the relationship between MUC1 and Sp1, coimmunoprecipitation assays were performed. Sp1 was coimmunoprecipitated with MUC1 and the level of coimmunoprecipitated Sp1 increased in relation to the level of induced Trop-2. It is known that galectin-3 is an endogenous ligand of MUC1. Binding of galectin-3 to MUC1 elevated the recruitment of Sp1 to MUC1, and knockdown of galectin-3 reduced the level of Trop-2. These results suggest that the binding of galectin-3 to MUC1 enhances the recruitment of Sp1, leading to promotion of the transcription of Trop-2.

IL (Interleukin)-10-STAT3-Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction.

Both osteopontin (OPN) and galectin-3 have been implicated in phagocytic clearance of dead cells and reparative fibrosis during wound healing. CD206+ macrophages are involved in tissue repair through phagocytosis and fibrosis after myocardial infarction (MI). However, the relationship among OPN, galectin-3, and macrophage polarization in the context of MI remains unclear. The time course of Spp1 (encoding OPN) expression in the heart after MI showed a strong activation of Spp1 on day 3 after MI. To identify where in the body and in which cells the transcriptional activity of Spp1 increased after MI, we analyzed EGFP (enhanced green fluorescent protein)- Spp1 knockin reporter mice on day 3 after MI. The transcriptional activity of Spp1 increased only in CD206+ macrophages in the infarct myocardium, and most of CD206+ macrophages have strong transcriptional activation of Spp1 after MI. The temporal expression pattern of Lgal3 (encoding galectin-3) in cardiac macrophages after MI was similar to that of Spp1, and OPN is almost exclusively produced by galectin-3hiCD206+ macrophages. Although both interleukin (IL)-4 and IL-10 were reported to promote CD206+ macrophage-mediated cardiac repair after MI, IL-10- but not IL-4-stimulated CD11b+Ly6G- cells could differentiate into OPN-producing galectin-3hiCD206+ macrophages and showed enhanced phagocytic ability. Inhibition of STAT3 tyrosine phosphorylation suppressed IL-10-induced expression of intracellular galectin-3 and transcriptional activation of Spp1. Knockdown of galectin-3 suppressed their ability to differentiate into OPN-producing cells, but not STAT3 activation. The tyrosine phosphorylation of STAT3 and the appearance rate of galectin-3hiCD206+ cells on cardiac CD11b+Ly6G- cells in Spp1 knockout mice were the same as those in wild-type mice. Spp1 knockout mice showed vulnerability to developing post-MI left ventricular chamber dilatation and the terminal deoxynucleo-tidyltransferase 2'-Deoxyuridine-5'-triphosphate nick-end labeling (TUNEL)-positive cells in the infarcted myocardium after MI remained higher in number in Spp1 knockout mice than in wild-type mice. OPN is almost exclusively produced by galectin-3hiCD206+ macrophages, which specifically appear in the infarct myocardium after MI. The IL-10-STAT3-galectin-3 axis is essential for OPN-producing reparative macrophage polarization after myocardial infarction, and these macrophages contribute to tissue repair by promoting fibrosis and clearance of apoptotic cells. These results suggest that galectin-3 may contribute to reparative fibrosis in the infarct myocardium by controlling OPN levels.

Knockdown of galectin-1 facilitated cisplatin sensitivity by inhibiting autophagy in neuroblastoma cells

Neuroblastoma (NB) is a type of solid extracranial tumor that usually occurs in babies and children. Chemotherapy is a common method for NB treatment, however, the drug resistance exerts during the chemotherapy of NB. Galectin-1 is a member of galectin family and plays a potent role in the development of chemotherapy and radiotherapy resistance. However, the effect of galectin-1 on cisplatin resistance in NB remains unknown. The present study aimed to investigate the role of galectin-1 in cisplatin resisitance and the potential mechanism. Human neuroblastoma SH-SY5Y and SK-N-SH cells were treated with cisplatin and/or galectin-1/siRNA targeting galectin-1 (si-Gal-1). The cell viability was measured by MTT assay. The IC50 values for cisplatin of neuroblastoma cells were calculated. The expression levels of autophagy markers including microtubule-associated protein light chain 3 (LC3B), Beclin-1, and p62 were detected by western blot. We found that cisplatin inhibited cell viability of SH-SY5Y and SK-N-SH in a dose-dependent manner. Cisplatin induced the ratio of LC3B-II/LC3B-I and Beclin-1 expression, and inhibited the p62 expression. Knockdown of galectin-1 decreased the IC50 for cisplatin of SH-SY5Y and SK-N-SH cells and inhibited cisplatin-induced autophagy. Moreover, inhibition of autophagy suppressed galectin-1-induced increase in IC50 for cisplatin. In conclusion, galectin-1 knockdown enhanced cisplatin sensitivity of neuroblastoma cells by inhibiting autophagy. The findings might provid a novel therapeutic target to overcome cisplatin resistance in chemotherapy of NB.

Ablation of Galectin-1 Reduces Adipogenesis and Obesity

Galectin-1 is a member of the animal lectin family that contains a carbohydrate-recognition binding domain (CRD) that binds a B-galactoside. Many studies are reported about the role of galectin-1 in cancer and immune disorders. But, the role of galectin-1 in metabolic dysfunction is not fully understood. We were interested in the role of galectin-1 on metabolic diseases and investigated the mechanism how galectin-1 regulates adipocyte differentiation and high fat diet (HFD) induced obesity. The level of galectin-1 increased during adipocyte differentiation and was predominantly expressed in mouse fat tissues. Galectin-1 knockdown significantly reduced adipocyte differentiation in 3T3-L1 cells and also decreased the expression of peroxisome proliferator-activated receptor (PPAR)-γ, ccaat enhancer binding protein (C/EBP)-α. fatty acid binding protein (FABP) 4 and fatty acid synthase (FASN). When lactose was treated to inhibit function of extracellular galectin-1, there was no effect on adipocyte differentiation. After 10-week high-fat diet (60% fat), lgals1-/- mice had lower body weight and WAT mass than wild type (lgals1+/+) mice. The expression levels of lipogenic genes were significantly down-regulated in liver and gonadal WAT of lgals1-/- mice. In addition, lgals1-/- mice had elevated expression of genes involved in thermogenesis in inguinal WAT and BAT. These data suggest that galectin-1 is an important regulator of adipogenesis. We also suppose that galectin-1 might be potential therapeutic target in obesity and fat liver diseases and further study is needed for clinical application. Disclosure J. Baek: None.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

Epithelial cells require a precise intracellular transport and sorting machinery to establish and maintain their polarized architecture. This machinery includes β-galactoside-binding galectins for targeting of glycoprotein to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analysed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, knockout of galectin-3 results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells.

Proteomic Identification of the Galectin-1-Involved Molecular Pathways in Urinary Bladder Urothelial Carcinoma

Among various heterogeneous types of bladder tumors, urothelial carcinoma is the most prevalent lesion. Some of the urinary bladder urothelial carcinomas (UBUCs) develop local recurrence and may cause distal invasion. Galectin-1 de-regulation significantly affects cell transformation, cell proliferation, angiogenesis, and cell invasiveness. In continuation of our previous investigation on the role of galectin-1 in UBUC tumorigenesis, in this study, proteomics strategies were implemented in order to find more galectin-1-associated signaling pathways. The results of this study showed that galectin-1 knockdown could induce 15 down-regulated proteins and two up-regulated proteins in T24 cells. These de-regulated proteins might participate in lipid/amino acid/energy metabolism, cytoskeleton, cell proliferation, cell-cell interaction, cell apoptosis, metastasis, and protein degradation. The aforementioned dys-regulated proteins were confirmed by western immunoblotting. Proteomics results were further translated to prognostic markers by analyses of biopsy samples. Results of cohort studies demonstrated that over-expressions of glutamine synthetase, alcohol dehydrogenase (NADP+), fatty acid binding protein 4, and toll interacting protein in clinical specimens were all significantly associated with galectin-1 up-regulation. Univariate analyses showed that de-regulations of glutamine synthetase and fatty acid binding protein 4 in clinical samples were respectively linked to disease-specific survival and metastasis-free survival.

1136 Galectin-3 regulates UVB-induced inflammation in skin

Galectin-3, a β-galactoside binding lectin, can be detected in diverse types of cells, including epidermal keratinocytes. Regulatory roles of galectin-3 in the pathogenesis of various malignant and inflammatory skin disorders have been reported, but the known roles of galectin-3 in UVB-induced inflammation in skin are still limited and the underlying molecular mechanisms remain elusive. In this study, we aimed to evaluate the regulatory roles of galectin-3 in UVB-induced inflammatory responses in skin by using primary normal human epidermal keratinocytes (NHEKs) and galectin-3 knockout mice as the models. Our results demonstrated knockdown of galectin-3 in NHEKs attenuated the UVB-induced production of active IL-. UVB-induced ASC cross-linking and active caspase-1 formation were reduced but LDH release was not affected after galectin-3 knockdown. In addition, after galectin-3 knockdown, UVB-induced IL-8, TNF-α and COX-2 expression in NHEKs were also down-regulated. Furthermore, UVB-induced production of reactive oxygen species in NHEKs was also reduced after galectin-3 knockdown. In the animal study, UVB-induced skin inflammation and damage were less severe in galectin-3 knockout mice than in wild type mice grossly and histologically. When measuring the physiological parameters, UVB-induced transepidermal water loss and skin erythema index were also reduced in galectin-3 knockout mice compared with wild type mice. In conclusion, current findings indicate that galectin-3 plays a crucial regulatory role in UVB-induced inflammation, suggesting a potential way to prevent UVB-induced skin damage by targeting galectin-3 in skin.

Galectin-1 Restores Immune Tolerance to Liver Transplantation Through Activation of Hepatic Stellate Cells

Background/Aims: Immune tolerance is considered the only way to manage liver transplantation (LT). The current study hypothesized that galectin-1 via the activation of hepatic stellate cells (HSCs) is capable of inducing immune tolerance in LT. Methods: Lentiviral-mediated gene knockdown and overexpression of galectin-1 were conducted in HSC-T6 cells. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine galectin-1 expression. LT was performed in 20 C57BL/J6 mice and 20 C3H mice. T-cells were assigned into control, Galectin-1 shRNA, Galectin-1 OE, Galectin-1 OE SB431542, Galectin-1 OE Sulforaphane, Galectin-1 OE Y27632, and Galectin-1 OE UO126 groups. CFSE, flow cytometry, and ELISA were respectively employed to detect T-cell proliferation, CD4+/ CD8+ ratio and IL-2, IL-10 and TGF-β levels. After establishing mouse models of immune tolerance and acute rejection, immunohistochemistry, TUNEL, and immunofluorescence assay were performed to determine CD3+ expression, apoptosis, α-SMA, and desmin. Mouse models of CCl4-induced liver fibrosis were established, followed by assigning the control1 and CCl4 groups. ELISA was used to determine ALT, AST, TBIL and Hyp levels. A total of 3 C57BL/J6 mice (donor) and 6 C3H mice (recipient) were grouped into the control2 and UO126 groups, followed by ELISA detection for IL-2, IL-10 and TGF-β. Results: In T-cells, galectin-1 shRNA increased cell proliferation and IL-2 levels with reduced IL-10 and TGF-β levels, while the Galectin-1 OE and Galectin-1 OE UO126 groups revealed the opposite results. Galectin-1 overexpression elevated the ratio of the CD4+ to CD8+ T-cells. The acute rejection group exhibited enhanced desmin expression and reduced α-SMA expression. Compared with the immune tolerance group, the acute rejection group displayed higher galectin-1 expression, a positive expression rate of CD3+ T-cells, and an increased apoptosis rate. Compared with the control1 group, the CCl4 group exhibited higher galectin-1 expression, ALT, AST, TBIL, and Hyp levels, α-SMA expression and CD4+/CD8+ T-cell ratio, in addition to decreased expression of desmin. Compared with the control2 group, UO126 increased galectin-1 expressions, IL-10 and TGF-β levels and reduced IL-2 levels with inactivated HSCs. Conclusions: The findings of the current study indicated that the overexpression of galectin-1 promoted the activation of HSCs, which reduced the inflammatory response by exerting immunosuppressive effects and accordingly contributed to immune tolerance in LT.

ST6GALNAC1 plays important roles in enhancing cancer stem phenotypes of colorectal cancer via the Akt pathway.

Colorectal cancer (CRC) is a mortal disease due to treatment resistance, recurrence and distant metastasis. Emerging evidence has revealed that a small sub-population of cancer cells termed cancer stem cells (CSCs)/ cancer-initiating cells (CICs) is endowed with high levels of tumor-initiating ability, self-renewal ability and differentiation ability and is responsible for treatment resistance, recurrence and distant metastasis. Eradication of CSCs/CICs is essential to improve current treatments. However, the molecular mechanisms by which CSCs/CICs are maintained are still elusive. In this study, we aimed to determine the molecular mechanisms by which colorectal (CR)-CSCs/CICs in are maintained human primary CRC cells. CR-CSCs/CICs were isolated by sphere-culture and the ALDEFLUOR assay, and transcriptome analysis revealed that the gene ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) was expressed at high levels in CR-CSCs/CICs. Overexpression of ST6GALNAC1 enhanced the expression of sialyl-Tn (STn) antigen, which is carried by the CSC marker CD44, and increased the sphere-forming ability and resistance to a chemotherapeutic reagent. The opposite phenomena were observed by gene knockdown using siRNA. Furthermore, the Akt pathway was activated in ST6GANAC1-overexpressed cells, and activation of the pathway was cancelled by gene knockdown of galectin-3. The results indicate that ST6GALNAC1 has a role in the maintenance of CR-CSCs/CICs by activating the Akt pathway in cooperation with galectin-3 and that ST6GalNAC1 (or STn antigen) might be a reasonable molecule for CSC/CIC-targeting therapy.