Figure 7.

Inhibition of galectin-induced T-cell apoptosis and tumor cell growth and migration by soluble human CD6 (shCD6). (A) Relative Gal-1 and Gal-3 mRNA expression in the different tumor cell lines analyzed in this study, as assessed by qRT-PCR. (B) Splenocytes from wild-type (WT) C57BL/6J mice were activated for 72 hours with anti-CD3 (1 µg/mL) and anti-CD28 (5 µg/mL) monoclonal antibodies (mAbs) and then incubated for 30 min with 100 µg/mL of endotoxin-free glutathione S-transferase (GST), GST-Gal-1 or GST-Gal-3 in the presence of growing concentrations (10 and 20 µg/mL) of shCD6 protein (n=4/group). Percentage of apoptotic cells was determined by flow cytometry. (C) Indicated tumor cells were stained for surface ALCAM expression and mean fluorescence intensity (MFI) was determined by flow cytometry. (D) Tumor cells (2×10⁴) were cultured with increasing concentrations (5, 10 and 20 µg/mL; n=4/group) of shCD6 or HSA and their numbers counted at 24-hour intervals. (E) Wound healing assays were used to measure the migratory activity of B16.F0 cells (5×10⁵) seeded onto 12-well plates for 24 hours, followed by 2-hour exposure to mitomycin-C (50 µg/mL) and further 12-hour incubation with increasing concentrations (5 and 25 µg/mL) of shCD6 (n=4/group) or HSA control (n=3). Data represent percentage of cell-covered area. In all cases, cumulative results from two independent experiments expressed as mean±SEM are presented. *p<0.05; two-tailed Student’s t-test. ALCAM, activated leukocyte cell adhesion molecule; HSA, human serum albumin.