Figure 9

Rhus coriaria stimulates the activation of the ubiquitin proteasome system and its inhibition rescue blocks autophagy and apoptosis and rescues cell viability in HT-29 cells. (A) Inactivation of mTOR through proteasome degradation precedes autophagy. Time-course analysis, by Western blotting, of phospho-mTOR, total mTOR, phospho-AKT, total AKT, mutant p53 and procaspase-3 in RCE-treated HT-29 cells. Cells were treated with 450 μg/mL RCE and proteins were extracted at the indicated time-points (3, 6, 12, 24 and 48 h) as previously described. (B) Inhibition of the proteasome rescue phospho-mTOR and block autophagy and apoptosis induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (450 µg/mL). Whole cell lysate was resolved on 6 and 15% SDS-PAGE for phospho-mTOR and LC3 and active caspase-7, respectively. (C) Inhibition of proteasome reduces cell death induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (300 and 450 µg/mL) for 48 h. Cell viability was determined as described in Material and Methods. Data are representative of three independent experiments carried out in triplicate. Statistical analysis for cell viability data on control or treated cells were performed using one-way ANOVA followed by LSD Post-Hoc test to determine the significance (***p < 0.001). (D) RCE treatment increases the cellular level of ubiquitinated proteins in HT-29 cells. Cells were treated with or without RCE (300 and 450 µg/mL) for 48 h, then whole-cell extracts were subjected to Western blot analysis for ubiquitinated proteins. (E) Time-course analysis, by Western blotting, of protein ubiquitination in RCE-treated HT-29 cells. Cells were treated with 450 μg/mL RCE and proteins were extracted at the indicated time-points (3, 6, 12, 24 and 48 h) as previously described.