Figure 5

Akt activation by PI3K is involved in rhEpo-mediated cisplatin resistance. a Western blot analysis demonstrating that exposure to 1 U/ml of rhEpo induces Akt activation in both UMSCC-10B and UMSCC-22B cell lines which is sustained for at least 72 h. b RhEpo-mediated Akt phosphorylation is dependent on PI3K, as determined by western blot analysis. Cells were pre-treated with either drug vehicle, 10 μM LY-294002, or 1.25 μM Akt inhibitor IV for 60 min prior to rhEpo treatment for 3 h. Total Erk was used as loading control. c MTS assays used to measure cell viability in UMSCC-10B and UMSCC-22B upon exposure to cisplatin and rhEpo show a 39.9% and 56.0% reduction in loss of cell viability when compared to cisplatin alone, respectively. Pre-treatment with Akt inhibitor IV resulted in a 69.6% and 61.2% reduced protection of rhEpo-treated UMSCC-10B and UMSCC-22B, respectively, and treatment with LY-294002 resulted in a 63.0% and 71.7% reduced protection. d TUNEL assays used to measure cell death in UMSCC-10B (top panel) and UMSCC-22B (bottom panel) reveal a significant reduction in cell death when treated with rhEpo and cisplatin (iii) when compared to cisplatin alone (ii), a 30.5% and 76.2% reduction in cell death, respectively. UMSCC-10B and UMSCC-22B cells exposed to rhEpo, cisplatin, and LY-294002 (PI3K/Akt signaling inhibitor) experienced a 69.2% and 50.9% reduced protection, respectively. Cells treated with only LY-294002 (i) experienced very little cell death (2-5%) while control cells exposed to drug vehicle, cells exposed to rhEpo alone, and cells exposed to rhEpo and LY-294002, experienced less than 1% cell death.