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. 2012 Mar;19(3):523-33.
doi: 10.1038/cdd.2011.123. Epub 2011 Sep 23.

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

N Mazurek  1 , J C Byrd, Y Sun, M Hafley, K Ramirez, J Burks, R S Bresalier
Affiliations

"V体育2025版" Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

"VSports app下载" N Mazurek et al. Cell Death Differ. 2012 Mar.

"VSports手机版" Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance VSports手机版. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal. .

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Figure 1
Figure 1
Generation of TRAIL-resistant LiM6-TR colon cancer cells. The TRAIL-resistant LiM6-TR stable cell line was obtained as described under ‘Materials and Methods'. The parental LS-LiM6 and resistant LiM6-TR were periodically tested for TRAIL response. (a) TRAIL-dependent loss of viable cells, measured by the MTT assay. Each data point represents the mean and standard deviation of experiments performed in triplicate. (b) Apoptotic cell death was confirmed by the accumulation of cells at sub-diploid fraction following exposure to 100 ng/ml TRAIL for 4 h and 24 h. Bars denote percentage of sub-diploid cells. (c) Immunoblots of PARP show the full length 116- and 89-kDa apoptosis related cleaved fragments only in the parental LS-LiM6 exposed to 100 μg/ml TRAIL, confirming apoptotic cell death induced by TRAIL
Figure 2
Figure 2
Differential regulation of pro-survival signals is not responsible for the acquisition of TRAIL resistance by LiM6-TR. (a) Parental LS-LiM6 and TRAIL-resistant LiM6-TR were treated with 100 ng/ml TRAIL for 24 h and modulation of pro-survival signaling was compared by western blot analysis. (b) Effects of inhibition of apoptosis by the caspase-3 inhibitor Ac-DEVD-FMK (10 and 100 μ) on the activation status of JNK, PTEN, and beta-catenin in parental LS-LiM6 cells. (c) JNK inhibitor SP600125 (25 μ) failed to confer resistance to TRAIL as determined by western blots of PARP cleavage. (d) TRAIL-dependent accumulation of apoptotic cells in sub-diploid faction is not prevented by JNK inhibitor. Bars denote percentage of sub-diploid cells
Figure 3
Figure 3
TRAIL fails to activate caspase-8 and the subsequent apoptosis cascade in TRAIL-resistant colon cancer cells. (a) Western blot analysis of apoptosis cascade in parental LS-LiM6 cells and TRAIL-resistant LiM6-TR cells with or without TRAIL treatment. (b) Secretion of cytochrome C from the mitochondria (M) to cytosol (C) after TRAIL treatment of LS-LiM6, but not LiM6-TR cells. COX4 is the mitochondrial marker
Figure 4
Figure 4
The expression of the DISC components is not changed in LiM6-TR. (a) Total protein expression of members of the DISC was compared between LS-LiM6 and LiM6-TR by western blot analysis. (b) Comparison of cell surface expression of the death receptor DR4 between LS-LiM6 (blue) and LiM6-TR (red) was performed by FACS analysis, using Alexa-Fluor 647 fluorophore and Fortessa FACS cytometer. (c) Comparison of cell surface expression of the death receptor DR5 between LS-LiM6 (blue) and LiM6-TR (red) was performed by FACS analysis, using Alexa-Fluor 488 fluorophore and XL-MCL FACS cytometer. Control staining (gray) differs between DR4 and DR5 labeling. (d) Cells were treated with a complex of Flag-tagged TRAIL and anti-Flag antibody, and cell lysates were subjected to DISC immunoprecipitation. Associated proteins were detected by immunoblotting with the indicated antibodies
Figure 5
Figure 5
Cell surface galectin-3 confers TRAIL resistance. (a) Comparison of total galectin-3 protein expression between LS-LiM6 and LiM6-TR by western blot analysis. (b) Comparison of surface expression of galectin-3 between parental LS-LiM6 and TRAIL-resistant LiM6-TR cells. Cells were fixed and processed for confocal microscopy using anti-galectin-3 (mAb TIB166, red) and visualized using Alexa Fluor-conjugated secondary antibody. (c) Comparison of cell surface expression of galectin-3 between LS-LiM6 (blue) and LiM6-TR (red) was performed by FACS analysis. Control staining, gray. (d) LiM6-TR cells were treated with galectin-3 sh-RNA (TR-SH-G) or non-targeting control sh-RNA (TR-C-SH) lentiviral particles. Depletion of galectin-3 expression was confirmed by western blot analysis. (e) Cell surface expression of galectin-3 was monitored by FACS analysis of LiM6-TR-G-SH (red) and LiM6-TR-C-SH (blue). Control staining, gray. (f) LiM6-TR,TR-G-SH, and TR-C-SH cells were treated with 100 ng/ml TRAIL for 5 h and loss of viable cells was determined by the MTT assay. Data are reported as mean±S.E.M. (g) Apoptotic cell death was quantified by flow cytometry analysis of the PI-stained cells. Bars denote percentage of sub-diploid cells
Figure 6
Figure 6
Surface galectin-3 anchors DR4 and DR5 in nano-clusters and impedes endocytosis. (a) Cells were labeled with anti-DR4 (green), anti-DR5 (gray), anti-galectin-3 (red) and nuclear staining by DAPI (blue), and surface labeling was visualized with Alexa Fluor-conjugated secondary antibodies by confocal microscopy. The white boxes of merged images are magnified to show detailed morphology (detail). Morphometric analysis performed by the 3I's Slide book 5.0, computing Pearson's coefficient for degree of overlapping. (b) Lysates from LS-LIM6 or LIM6-TR cells were immunoprecipitated with either anti-DR4, anti-DR5, or control IgG antibodies. The precipitates were immunoblotted with anti-galectin-3 antibody (TIB 166). (c) TRAIL-dependent internalization of DR4 and DR5. Cells were pre-labeled with anti-DR4 (green) and anti-DR5 (gray) antibodies, and exposed to 100 ng/ml TRAIL on ice for 1 h. The temperature was raised to 37°C for 30 min to allow receptor internalization or kept at 0°C for controls. Surface labeling was removed by treatment with 2 m acetic acid, then the cells were fixed and permeabilized. Internalization of death receptor immunocomplexes was visualized with Alexa Fluor-conjugated secondary antibodies by confocal microscopy. (d) TRAIL-dependent loss of cell surface DR4 and DR5. Cells were treated with 100 ng/ml TRAIL for 30 min at 37°C followed by labeling with either anti-DR4 or anti-DR-5 antibodies and cell surface death receptor expression was determined by flow cytometry analysis
Figure 7
Figure 7
Inhibitors of glycosylation and galectin-3 binding re-sensitize LiM6-TR to TRAIL. (a) LiM6-TR cells were pretreated with either 5 m LacNAc, 2 μg/ml tunicamycin, 2 μg/ml swainsonine, or 1 m benzyl-alpha-GalNAc for 24 h followed by exposure to 100 ng/ml TRAIL for 5 h. Apoptosis was determined by the accumulation of PI-labeled cells in the sub-diploid fractions by flow cytometry analysis. (b) LiM6-TR cells were treated with inhibitors of glycosylation and galectin-3 binding and analyzed for TRAIL-dependent loss of surface DR5 receptor by flow cytometry. In control experiments (red), cells were pre-treated but were not stimulated with TRAIL. Control staining, gray. Only data for DR5 is shown; similar results showed TRAIL-dependent loss of surface DR4 in tunicamycin-treated LiM6-TR
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