Figure 1.
ArtinM interacts with leukemia cell lines.
A) ArtinM binding to NB4, K562, and U937 cells: cells were fixed and incubated for 30 min with biotinyl-ArtinM/streptavidin-FITC (5 µg/mL). Lectin binding to the cell surface was detected by flow cytometry and expressed as mean fluorescence intensity (MFI). B) NB4 cells adhered to Biobond-coated coverslips were incubated at 4°C for 60 min with biotinyl-ArtinM (5 µg/mL) (panel C) or PBS (panel A). For inhibition assays, biotinyl-ArtinM was pre-incubated at room temperature for 60 min with 10 µM Manα1-3[Manα1-6]Man (panel B) or 200 mM d-galactose (panel D), and then incubated with NB4 cells. After washing and incubation with streptavidin-FITC, cells were fixed and examined by fluorescence microscopy. Magnification = 40×. The result shown are representative of 3 independent experiments and are expressed as mean ± SD, * p<0.05 (Tukey's test).
Figure 2.
ArtinM inhibits cell growth inhibition and pro-apoptotic effect on NB4 cells.
A) NB4, K562, and U937 cells were cultured for 48 h in the presence or of ArtinM (3.125 to 100 µg/mL). Cell growth was evaluated by MTT assay. The curves show the ArtinM inhibition of cell growth relative to the growth of untreated cells. Each point was obtained from a triplicate assay, and the results shown are representative of 3 independent experiments. The dotted horizontal line indicates the ArtinM concentration necessary for 50% growth inhibition (IC50). B) NB4, K562, and U937 cells were cultured for 48 h in the presence or absence of ArtinM (10 µg/mL, IC50 for NB4). Cells were stained with FITC-Annexin V and propidium iodide (PI) to characterize apoptosis and to distinguish it from necrosis. Bars represent the proportion of Annexin V and Annexin V/PI-stained cells. Results represent 3 different experiments. C) Disruption of mitochondrial membrane potential (mΔΨ) of ArtinM-treated cells was assessed by flow cytometry after staining with JC-1.The bars represent the percentage of cells with mΔΨ disruption. The result shown are representative of 3 independent experiments and are expressed as mean ± SD, * p<0.05 (Tukey's test).
Figure 3.
ArtinM does not induce NB4 differentiation but promotes ROS generation.
NB4 cells were cultured for 48 h with ArtinM (10 µg/mL) or medium (CTRL), and then analyzed for 3 parameters. ATRA (1 µg/mL) or As2O3 (1 µM) were used as positive controls. A) Expression of CD11b and CD14 was evaluated by flow cytometry and expressed as mean fluorescence intensity (MFI). B) Morphology of NB4 cells was evaluated by optical microscopy of HEMA-stained cytospin preparations. Panel A: NB4 cells; Panel B: ArtinM-treated NB4 cells; Panel C: ATRA-treated NB4 cells. Bar = 10 µm. C) ROS production was evaluated by measuring the oxidative conversion of DCFH-DA to DCF in a fluorospectro-photometer. The effect of reduced glutathione (GSH) or α-tocopherol acetate (VitE) addition to ArtinM-stimulated cultures was also assayed. The results are expressed as optical density (OD) at excitation and emission wavelengths of 485 and 535 nm, respectively. The result shown are representative of 3 independent experiments and are expressed as mean ± SD, * p<0.05 (Tukey's test); # p<0.05 (ArtinM×ArtinM GSH, Tukey's test).
Figure 4.
Autophagy accounts for the caspase-independent mechanism of NB4 cell death induced by ArtinM.
NB4 cells were cultured with ArtinM (10 µg/mL) for 48 h or with Staurosporine (Stauro, 5 µM) for 4 h. A) Caspase-3 activation, manifested by cleavage of their precursor forms, was analyzed by Western blotting of RIPA cell lysates (100 µg protein) performed under reducing conditions. B) Fragmentation of genomic DNA from NB4 cells was evaluated by electrophoresis on 1% agarose gel followed by ethidium bromide visualization. St: standard markers. C) Acidic autophagic vacuoles in NB4 cells were detected through staining with 10 µg/mL acridine orange in serum-free medium. Fluorescent micrographs show that the cytoplasm and nucleus of stained cells fluoresced bright green, whereas the acidic autophagic vacuoles fluoresced bright red. Bar = 10 µm.
Figure 5.
N-Glycan recognition by ArtinM accounts for NB4 growth inhibition.
A) ArtinM binding to NB4 cells pre-treated for 24 h with tunicamycin (TM, 5 µg/mL) or swainsonine (SW, 5 µg/mL) was analyzed by flow cytometry. Fully glycosylated: untreated NB4 cells. B) Growth inhibition induced by ArtinM (10 µg/mL) in fully glycosylated cells, and in TM- or SW-treated cells. Growth rate was measured by MTT assay. Bars represent cell growth inhibition by ArtinM relative to the growth of untreated cells. As2O3 (1 µM) was used as a positive control for cell death. The results shown are representative of 3 independent experiments and are expressed as mean ± SD, * p<0.05 (Tukey's test) and # p<0.05 (Student's t-test).
Figure 6.
β1-6GlNAc branched N-glycans as targets for lectin recognition and cell growth inhibition.
A) Binding of β1-6GlNAc by lectin L-PHA in leukemia cell lines: NB4, K562, and U937 cells were incubated with L-PHA-FITC for 30 min (5 µg/mL). Blood neutrophils from healthy donors and NB4 cells pretreated with ATRA were also assayed. Lectin binding was detected by flow cytometry and expressed as mean fluorescence intensity (MFI). B) GnT-V mRNA levels in myeloid cell lines: NB4, K562, and U937 cells, as well as blood neutrophils from healthy donors (CD11b+) and NB4 pretreated with ATRA (1 µM, 48 h) were assayed. Average Ct values from triplicate samples obtained for each gene, with a standard deviation of less than 0.5 Ct units, were converted to linear values and normalized to the housekeeping gene G3PDH. C) L-PHA, but not galectin-3, competes for the ArtinM target on NB4 cells: NB4 cells were incubated with L-PHA or Galectin-3 (5 µg/mL) and then assayed for ArtinM binding (see figure 1). ArtinM binding was detected by flow cytometry and expressed as mean fluorescence intensity (MFI). D) Inhibition of leukemia cell growth by L-PHA: NB4, K562, and U937 cells were cultured for 48 h in the presence or absence of 3.125–50 µg/mL L-PHA. Cell growth was evaluated by MTT assay. The curves show the inhibition of cell growth by L-PHA relative to the growth of untreated cells. Each point was obtained from a triplicate assay. The dotted line indicates the L-PHA concentration necessary to inhibit growth by 50% (IC50). The result shown are representative of 3 independent experiments and are expressed as mean ± SD, *p<0.05 (Tukey's test).
Figure 7.
β1-6GlcNAc branched N-glycan is targeted by three different lectins.
The targeted areas by ArtinM (grey), L-PHA (brown) and galectin-3 (green) are highlighted in the figure. ArtinM binds to Manα1-3(Manα1-6)Manβ-R core and posses a sub domain that establishes additional interaction with GlcNAc in the context of α1-6Mannose branch. L-PHA binds to the sequence Galβ1-4GlcNAcβ1-2(Galβ1-4GlcNAcβ1-6)Manα-R, which partially merges with the area targeted by ArtinM. Galectin-3 binds to distal poly-N-acetyllactosamines, which does not merge with the areas targeted by ArtinM or L-PHA.