Fig. 6: CD44 fucosylation is responsible for the enhanced acquisition of sorafenib resistance and antitumor effect of oncolytic adenoviruses expressing various shRNAs.
A SNU449 (left) or SNU398 (right) cells were pretreated with 2DGal (10 mM, 12 h) before sorafenib treatment (10 μM, 5 μM for 2 h, respectively). GRP78 and CD44 at the cell surface membrane, as well as their total cell lysates, were subsequently examined. Phosphorylated IGF1R, IGF1R, and FUT1 proteins in cell lysates were also examined. B SNU449 (left) or SNU398 (right) cells were pretreated with 2DGal (10 mM, 12 h) before sorafenib treatment (10 μM, 5 μM for 2 h, respectively). Then, the cells were lysed and subjected to immunoprecipitation with an anti-GRP78 antibody to detect the interaction between GRP78 and CD44 in total cell lysates. C After CD44-overexpressing SNU398 cells were selected via retroviral infection with pBabe-puro-CD44, the selected cells were treated with sorafenib (5 μM, 2 h). Then, the cells were lysed and subjected to immunoprecipitation with an anti-CD44 antibody to detect the interaction between CD44 and fucosylation-related factors and H antigen from total cell lysate (right). (D) Sorafenib-resistant SNU398 (SNU398-SR) tumors were grown in male BALB/c nude mice. Tumors were established via the subcutaneous injection of 2 × 106 cells and were allowed to grow to an average size of 70–120 mm3. PBS and adenoviruses were intratumorally injected twice every other day. Oncolytic adenoviruses coexpressing shHSP27-shTGF-β1 or shTGF-β1-shGRP78, shHSP27-shTGF-β1-shHIF1α or shTGF-β1-shGRP78-shHIF1α were intratumorally injected into SNU398-SR tumors. Tumor growth was measured every 2 days for more than 14 days via calipers. The arrows indicate when the oncolytic adenovirus was administered. The statistical significance was determined by two-way analysis of variance (ANOVA). Data are shown as mean values ± SD. **p < 0.01; ****p < 0.0001; ns not significant.
