Figure 4. PEITC inhibits the activation of EGFR in ovarian cancer cells. Representative blots showing the concentration depen- dent effect of PEITC on p-EGFR (Tyr-1068) and EGFR in (A) SKOV-3 and (B) OVCAR-3 ovarian cancer cells. Actin was used as loading control. Each *p,0.05 when compared to control. doi:10.1371/journal.pone.0043577.g004 PEITC Induces Apoptosis in Ovarian Cancer Cells In another experiment, we tested whether growth inhibitory effects of PEITC were concurrent with its apoptosis inducing ability. Treatment of SKOV-3 cells with various concentrations of PEITC resulted in approximately 2 to 5 fold apoptosis over control (Fig. 3A i). To rule out the cell specific effects of PEITC, we evaluated PEITC-induced apoptosis in two other ovarian cancer cell lines. Our results showed that apoptosis induced by PEITC was nearly 4 to 6 fold in OVCAR-3 and 2 to 10 fold in TOV-21G cells (Fig. 3 B–C). These observations clearly establish PEITC Targets EGFR to Suppress Ovarian Cancer that PEITC reduces the proliferation of ovarian cancer cells by inducing apoptosis. Apoptosis inducing effects of PEITC were further confirmed by western blot analysis that revealed cleavage of Caspase-3 and PARP in PEITC- treated ovarian cancer cells (Figure 3 A–C). PEITC Inhibits the Activation of EGFR in Ovarian Cancer Cells To extend the molecular observations made in vivo, we treated SKOV-3 and OVCAR-3 cells with PEITC for 24 hours. As shown in Figure 4, PEITC treatment significantly blocked the phosphor- ylation of EGFR at Tyr-1068 in a concentration dependent manner in all the three cell lines tested. Approximately, 50% decrease in the phosphorylation of EGFR at Tyr-1068 was observed in SKOV-3 cells when treated with 30 mM PEITC (Figure 4A), whereas 65% inhibition was observed in OVCAR-3 cells exposed to 10 mM PEITC for 24 hours (Figure 4B). Interestingly, our results also revealed a dramatic reduction in the protein expression of EGFR in ovarian cancer cells treated with PEITC (Figure 4 A–B). Taken together, these results demonstrate that PEITC blocks the phosphorylation as well as reduce protein levels of EGFR in ovarian cancer cells. PEITC Treatment Blocks AKT Activation EGFR regulates various cellular processes by directly acting on downstream molecules such as AKT. Activation of EGFR leads to the phosphorylation of Akt at Ser 473. Because we observed a significant blockade in EGFR activation by PEITC treatment, we sought to determine the effect of PEITC on both activation and constitutive expression of AKT. Exposure of SKOV-3, OVCAR-3 or TOV-21G cells to different concentrations of PEITC for 24 h resulted in the significant inhibition of the phosphorylation as well as constitutive expression of AKT (Fig. 5 A–C). More than 50% reduction was observed in the phosphorylation of AKT at Ser 473 in SKOV-3 cells (Figure 5A). AKT phosphorylation was reduced by70% and 90% in OVCAR-3 and TOV-21G cells respectively (Figure 5 B–C). PEITC had similar effects on constitutive expression of AKT in all the three ovarian cancer cell lines. These results indicate that PEITC modulates the downstream molecules of EGFR pathway. AKT Kinase Activity Since substantially reduced phosphorylation of AKT was observed by PEITC treatment; we wanted to confirm these observations by AKT kinase activity, which measures the functionality of AKT. AKT activity was determined by evaluating the phosphorylation of its downstream substrate GSK. As shown in Figure 5D, a significant reduction in the kinase activity of AKT was observed in OVCAR-3 and TOV-21G cells. For example, treatment of OVCAR-3 cells with 10 mM PEITC for 24 hours resulted in the inhibition of approximately 80% AKT kinase activity as compared with control cells (Figure 5D). Early Inhibition of EGFR by PEITC To evaluate whether induction of apoptosis by PEITC was associated with EGFR inhibition, SKOV-3 cells were treated with 15 mM PEITC at different time intervals. The reduction in the phosphorylation of EGFR and AKT was observed just after 2 hours of PEITC treatment and this effect increased at later time points (Figure 6A). However, cleavage of PARP was observed only after 8 hours PEITC treatment suggesting that inhibition of EGFR/AKT lead to apoptosis in our model (Figure 6A). PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43577