Figure 6. TGF treatment of AKT overexpression overrides the effects of PEITC. (A) Representative blot of time dependent effect of 15 mM PEITC on p-EGFR (Tyr 1068), p-AKT (Ser-473), Cl-PARP in SKOV-3 ovarian cancer cells. Actin was used as loading control. (B) OVCAR-3 cells were stimulated with 50 ng/mL TGF for 1 hour after treatment with varying concentrations of PEITC for 24 hours. Whole-cell lysates were resolved on 10% SDS-PAGE for the analysis of phosphorylation of EGFR at Tyr-1068, phosphorylation of AKT at Ser-473, and cleavage of caspase-3 and PARP. Actin was used as a control for loading. Effect of AKT overexpression on PEITC induced apoptosis was also determined in SKOV-3 cells. (C) Representative blots showing p-AKT (Ser 473), AKT and Cl-PARP. Actin was used as a loading control. doi:10.1371/journal.pone.0043577.g006 Activation of EGFR-AKT by TGF is Inhibited by PEITC EGFR can be activated by growth factors and ligands such as TGF and EGF. When TGF binds, EGFR is phosphorylated which in-turn phosphorylates its downstream molecules such as AKT. Treatment OVCAR-3 cells with 50 ng/ml TGF resulted in the 3 fold activation (phosphorylation) of EGFR and AKT (Fig. 6B). However, PEITC treatment was able to reduce the activation of EGFR and AKT induced by TGF (Figure 6B). The effect of TGF and PEITC on cleavage of caspase-3 and PARP was also evaluated. From Figure 6B, it is clear that PEITC treatment was able to induce cleavage of caspase-3 and PARP in OVCAR-3 cells. PEITC Targets EGFR to Suppress Ovarian Cancer Figure 7. PEITC treatment inhibits mTOR, Raptor and Rictor of mTORC1 and mTORC2 complexes. (A) Representative blots of the concentration dependent effect of PEITC (0–7.5 mM) on p-mTOR (Ser- 2481), Rictor and Raptor on SKOV-3 ovarian cancer cells. Actin was used as loading control. (B) SKOV3 cells treated with 5 mM PEITC for 24 h were immunoprecipitated with mTOR antibody. The blots were probed with antibodies for Rictor and Raptor. doi:10.1371/journal.pone.0043577.g007 AKT Overexpression Abrogates the Effects of PEITC To further strengthen our observation that PEITC-induced apoptosis was mediated by AKT inhibition in ovarian cancer cells, we transiently transfected SKOV-3 cells with AKT encoding plasmid for 24 hours, resulting in almost 7 fold increase in the expression of AKT. Correspondingly, phosphorylation of AKT at Ser-473 also increased substantially. AKT overexpression com- pletely prevented PEITC induced suppression of AKT phosphor- ylation. Furthermore, AKT overexpression abrogated PEITC induced apoptosis as evaluated by cleavage of PARP (Figure 6C). Taken together, our results clearly indicate that apoptosis induced by PEITC was almost completely blocked in the cells overex- pressing AKT, indicating AKT as a target of PEITC in ovarian cancer cells. PEITC Inhibits mTORC1 and mTORC2 Complexes Recent literature suggests that EGFR-AKT activation regulates mTOR activity which is important for cell proliferation [28,29]. mTOR is regulated by two complexes- mTORC1 and mTORC2. The mTORC1complex consists of regulatory associated protein of mTOR (Raptor), which binds to mTOR and facilitates mTOR signaling in translation and cell growth [30,31]. On the other hand, mTORC2 complex consists of Rapamycin insensitive companion of mTOR (Rictor) bound to mTOR. The Rictor- mTOR (mTORC2) complex regulates the phosphorylation of AKT at Ser 473 [32]. Since, we observed that PEITC suppressed the phosphorylation of AKT, we hypothesized PEITC treatment would disturb mTOR signaling. As expected, our results demonstrated that PEITC treatment disrupted mTOR signaling PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43577