Galectin-8 promotes migration and proliferation and prevents apoptosis in U87 glioblastoma cells

Abstract

Background: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model.

Methods: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS.

Results: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels.

Conclusions: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.

Keywords: Apoptosis; Cancer; Cell cycle; Galectin-8; Glioblastoma; Migration; Proliferation.

Fig. 1

Gal-8 silencing in U87 glioma cells. U87 cells were transduced with lentiviral particles containing Gal-8-silencing shRNAs (shGal-8#4 or shGal-8#5) or irrelevant shRNA (shC). Expression of Gal-8 isoforms (Gal-8S and Gal-8L) was determined 3 days post-infection by semi-quantitative RT-PCR (a) qRT-PCR corrected by β-actin mRNA level, (b) and western blot in cell extracts (Cells) and 72 h conditioned medium (Medium) (c)

Fig. 2

Soluble Gal-8 as a chemoattractant induces migration of U87 cells, while partial silencing of Gal-8 does not inhibit migration. a U87 cells (50,000 per transwell chamber) were seeded in medium without FBS in the upper compartment of transwell chambers and the lower chamber was filled with medium in the following conditions: without serum (control), 10 % FBS or 50 μg/ml Gal-8 in the absence or presence of 20 mM lactose for 16 h. The bottom sides of the filters were stained with crystal violet to count the cells that migrated across the filter. The graph represents cells per field (five different fields in duplicate for each condition) (*p < 0.0001). b Cell migration of U87 cells transduced by control shC or Gal-8-silencing sh-Gal-8#5, using 10 % FBS as a chemoattractant, show no significant differences

Fig. 3

Soluble Gal-8S promotes cell growth and its partial silencing reduces proliferation of U87 cells. a Cell growth under Gal-8 treatment. U87 cells were seeded on plastic plates, treated with soluble Gal-8S (50 μg/ml) for 24 h in the presence or absence of 20 mM lactose and a crystal violet assay was performed in triplicate. Gal-8 increased cell growth (*p < 0.0001) except when co-incubated with lactose, indicating a glycan interaction-mediated effect. b Effect of Gal-8 silencing on cell growth. U87 cells transduced with shGal-8#5 or irrelevant shRNA (shC) lentiviral particles were cultivated for 1 week, seeded (100,000 viable cells per well) for 48 h and viable cells were counted after trypan blue staining. Graph represents the percentage of cell number increment in two independent experiments in triplicate (*p < 0.01). c Effect of Gal-8 silencing on proliferation. Transduced U87 cells were stained with CFSE and fluorescence dilution, reflecting cell proliferation, was analyzed after 24 h by flow cytometry. A representative histogram shows a lower decrease of CFSE fluorescence in shGal-8#5 versus control shC-transduced U87 cells. The graph represents the quantification of CFSE median fluorescence index of three independent experiments. (*p < 0.05)

Fig. 4

Gal-8 partial silencing increases apoptosis in U87 cells. a Flow cytometry analysis of cell cycle and fragmented DNA. The histogram shows the percentage of U87 cells (transduced with shC or shGal-8#5) in different phases of the cell cycle and the sub-G1 phase containing fragmented DNA, indicative of apoptotic cells. The proportion of sh-Gal-8#5-transduced cells compared with shC cells decreased in G0/G1 and G2/M, while increased in S and more significantly (sixfold) in Sub-G1. b Apoptosis assessed by caspase-3 activation. U87 cells transduced with shC or shGal-8#5 and treated for cleaved caspase-3 indirect immunofluorescence were analyzed by flow cytometry (b) a representative dot blot of two experiments with the corresponding graph and an histogram of four experiments with an inset graph representing normalized mean fluorescence (MF) intensity of cleaved caspase-3 reveals increased apoptosis in shGal-8-#5-transduced cells. (*p < 0.05). c Apoptotic cells detected by indirect immunofluorescence against cleaved caspase-3 and Hoechst stained nucleus