by down-regulating p- mTOR (Ser 2481) and expression of Raptor and Rictor, which are involved in mTORC1 and mTORC2 complexes (Fig. 7A). To confirm the dissociation of the complex, mTOR was immunoprecipitated from control and PEITC treated cells and immunoblotted for Rictor and Raptor. Our results clearly showed that mTOR complex partners Rictor and Raptor were suppressed by PEITC treatment in SKOV3 cells (Fig. 7B). The expression of mTOR-associated Rictor was reduced more than Raptor by PEITC treatment (Fig. 7B). These observations suggest that the mTORC2 complex was affected more by PEITC treatment than mTORC1 complex. Nonetheless, further studies are required to delineate the exact mechanism by which PEITC disrupts mTORC1 and mTORC2 complexes. Discussion In the present study, we showed the mechanism by which PEITC suppressed the growth of ovarian cancer cells in culture as well as in animal model. Our results show that oral administration of 12 mmol PEITC to athymic nude mice significantly suppressed the growth of SKOV-3 ovarian tumor xenografts. Tumor growth suppression by PEITC treatment was associated with increased apoptosis in the tumor cells, which in turn was linked with the inhibition of EGFR and AKT activation. Inhibition of phosphor- ylation of EGFR and AKT were confirmed in SKOV-3 and OVCAR-3 ovarian cancer cells by western blotting. PEITC also blocked TGF-mediated activation of both EGFR and AKT. Overexpression of AKT by transient transfection substantially blocked the apoptosis inducing effects of PEITC. Our results showed that PEITC inhibits ovarian tumor growth in vivo by suppressing EGFR-AKT pathway and identifies EGFR-Akt axis as a target of PEITC. Constitutive activation of EGFR has been reported in various cancers including breast, prostate and ovarian [33–35]. EGFR family of proteins is vital for cell growth. Activated EGFR is diversely involved in transducing growth signal and inhibiting apoptosis. In addition, it is also involved in angiogenesis, EMT and metastasis [9,10]. A study conducted by Alper and colleagues have shown that approximately 70% of ovarian tumors express high levels of EGFR [36]. Inhibition of EGFR activation has been shown to suppress the growth of human cancer cells [37,38]. Moreover, most of the cancers acquire drug resistance due to the activation of EGFR pathway [3,39]. Therefore, inhibiting EGFR could be one potential approach to treat ovarian cancer. Our results showed that PEITC treatment substantially suppressed the phosphorylation of EGFR at Tyr-1068 as well as constitutive protein expression of EGFR in different ovarian cancer cell lines. In agreement with these results, tumors from PEITC treated mice also showed drastic suppression of both active and constitutive expression of EGFR. Our results indicate that PEITC suppress the growth of ovarian cancer cells by disrupting the phosphorylation of EGFR. These results are in agreement with several studies which showed that chemopreventive agents such as diindolyl- methane, resveratrol, capsaicin and silibilin suppress the growth of prostate, breast and lung cancer cells by targeting EGFR [40–43]. Studies by Trachootham et al., and Satyan et al. demonstrated the anti-cancer effects of PEITC in ovarian cancer cells through ROS generation and MAPK activation [44,45]. Our current study indicates an additional mechanism of action of PEITC in ovarian cancer cells. AKT is pivotal to EGFR activation. Activated EGFR further activates AKT by phosphorylating it at Ser 473 [46]. AKT is a potent survival pathway that may mediate resistance to the apoptosis inducing effects of chemotherapy and radiation therapy PEITC Targets EGFR to Suppress Ovarian Cancer in a variety of cancer types including ovarian cancer [47]. Several studies have shown the involvement of AKT signaling in apoptosis [48]. AKT is frequently overexpressed in ovarian cancer and plays a major role in ovarian carcinogenesis [14,15]. Overexpression of AKT is frequently associated with aggressive phenotype and poor prognosis of ovarian cancer. Blockade of AKT has been shown to cause apoptosis in breast and pancreatic cancers [16,49,50]. Our study reveals that PEITC blocks both the activation and protein expression of AKT in all the three ovarian cancer cells. PEITC mediated inhibition of AKT was further verified by kinase activity of AKT by determining the phosphorylation of GSK. Several studies demonstrated the association of AKT activity with EGFR activation [51,52]. Our results also showed that PEITC treatment reduced mTOR, Raptor and Rictor indicating that PEITC targets mTORC1 and mTORC2 complexes. Curcumin and fisetin also were shown to modulate the expression of mTOR, Rictor and Raptor in colorectal and prostate cancer cells respectively [53,54]. The immunoprecipitation studies demonstrated that the expres- sion of mTOR-associated Rictor was reduced more than Raptor by PEITC treatment. These observations suggest that the mTORC2 complex was affected more by PEITC treatment than mTORC1 complex. Since mTORC2 complex regulate the activation of AKT in cancer cells, our results suggest that reduced phosphorylation of AKT by PEITC treatment was primarily associated with the disruption of mTORC2 complex. Our results are in agreement with the studies published by Toschi et al. demonstrating the dissociation of Rictor from mTORC2 complex to enhance cell death by Rapamycin [55]. Furthermore, exposure of cells to TGF, a ligand of EGFR substantially increased the activation (phosphorylation) of AKT but suppressed by PEITC. These observations indicate that EGFR is upstream and pivotal to the activation of AKT in our model. Our results also showed that ovarian tumor growth suppression by PEITC was associated with the inhibition of AKT phosphorylation and expression. Our results thus established AKT as a target of PEITC in ovarian cancer cells. It is important to note that oral administration of PEITC has reasonable bioavailability. Oral administration of 10 mmol PEITC in rats resulted in approximately 9.260.6 mM PEITC in the plasma after 0.44 hour of treatment [56]. In another study performed in healthy human volunteers, consumption of 100 g watercress by human volunteers resulted in approximately 9286250 nM PEITC in plasma [57]. In yet another study, consumption of a single hydrolyzed extract of 3-day old broccoli sprouts (containing approximately 200 mM total isothiocyanate) resulted in peak concentration of 0.94 to 2.27 mM isothiocyanates in plasma and serum within 1 hour of broccoli consumption in humans [58]. These results indicate that the therapeutic concen- tration of PEITC can be achieved. It is important to note that PEITC is currently under clinical trials for lung cancer. Outcome of the clinical trial would further validate the use of PEITC as a therapeutic agent against various cancers including ovarian cancer. Several EGFR targeted therapies such as monoclonal antibodies, small molecule inhibitors or RTK inhibitors failed to pass phase II clinical trials of ovarian cancer [59], giving rationale to develop newer therapies. In conclusion, our results established that PEITC suppresses the growth of ovarian cancer in vitro and in vivo by inhibiting EGFR signaling. Our results also provide evidence that PEITC suppress the phosphorylation of AKT, which is regulated by EGFR. Taken together, our study provides support for the use of PEITC in pre- clinical and clinical settings for the management of ovarian cancer. PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43577