gain are identified in ATC as compared to well differentiated cancer, suggesting that PI3K/mTOR pathway activity is involved in the process of cancer de-differentiation [3,11,12]. For MTC, RET proto-oncogene mutations occur in almost all familiar cases (25% of MTC) and about half of sporadic MTC. This gain-of- function rearrangement enhances PI3K/mTOR signaling trans- duction [15,16]. In sporadic MTC without RET mutations, over 50% of tumor samples show activation of AKT or mTOR by immunohistochemistry [16]. BEZ235 is a dual PI3K/mTOR inhibitor that reduces PI3K and mTOR kinase activity by competitive binding to the ATP- binding cleft of these enzymes [17]. BEZ235 may treat cancers through induction of G0/G1 cell cycle arrest and apoptosis, and has recently entered phase II clinical trials [17–22]. This study was conducted to evaluate the efficacy of BEZ235 in treating thyroid cancer from four major pathological types, including PTC, FTC, ATC and MTC. We also explored combination effects of BEZ235 and currently employed chemotherapeutics against four ATC cell lines. Results Cytotoxicity of BEZ235 BEZ235 inhibited cell proliferation in all thyroid cancer lines in a dose and time dependent manner (Figure 1A). A low dose of BEZ235 at 6.25 nmol/L impeded at least 30% of cell growth in 6 of 8 cell lines on day 4. BEZ235 at 100 nmol/L arrested more than 80% cell growth in ATC lines, 78% in medullary (TT) and more than 74% in well-differentiated thyroid cancer lines. The Dm of BEZ235 on day 4 was calculated for each cell line (Figure 1B). The 4 ATC lines were the most sensitive to BEZ235 (Dm, KAT4C = 3.9, KAT18 = 6.6, 8305C = 6.7, 8505C = 9.3 nmol/L), followed by the follicular undifferentiated cancer FRO81–2 (Dm, 10.6 nmol/L). The medullary thyroid cancer (TT), the well-differentiated papillary (BHP7-13), and the follicular (WRO82–1) cancer were less sensitive (Dm 17.1, 17.2, and 43.1 nmol/L, respectively). Modulation of signaling pathways by BEZ235 The effects of BEZ235 on signaling pathways were examined in 8505C, TT and BHP7-13 cell lines (Figure 2, Figure S1). p-4E- BP1 (Thr70), p-4E-BP1 (Thr37/46) and p-S6 ribosomal protein (Ser235/236) were all consistently repressed by BEZ235 within 2 hours and the inhibition effect was significant and durable, with less than 12% of protein detected at 24 hours. p-ERK1/2 (Thr 202/Tyr204) was activated by BEZ235 with 2.6 to 8.6-fold increase at 2 to 8 hours in three cell lines. However, BEZ235 had heterogeneous effects on p-AKT (Thr308) and p-AKT (Ser473), which could be inhibited for a period in TT and BHP7-13, but were persistently increased in 8505C. As in 8505C cells, in KAT4C cells BEZ235 similarly inhibited p-S6 ribosomal protein (Ser235/236), but activated p-AKT (Thr308), p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204), (Figure S1 and S2A). The immunoblot was quantified and statistical analysis was performed for p-4E-BP1 (Thr37/46) in BHP7-13 and p-S6 ribosomal protein (Ser235/236) in 8505C and BHP7-13. BEZ235 profoundly inhibited these proteins by 2 to 4 hours, and this effect was durable for 24 hours (Figure S1). p-4E-BP1 (Thr37/46) was repressed to less than 12% from 2 to 24 hours and achieved statistical significance when compared with basal level in BHP7-13 (P,0.02, t-test). Similarly, p-S6 ribosomal protein (Ser235/236) was significantly decreased to less than 7% and 13% from 4 to 24 hours in 8505C (P,0.01, t-test) and BHP7-13 (P,0.02, t-test), respectively. These results suggest that BEZ235 has a strong ability Thyroid Cancer and NVP-BEZ235 to inhibit mTORC1 (leading to decreases of p-4E-BP1 and p-S6 ribosomal protein) and weaker effects on mTORC2 (leading to varied effects on p-AKT at Ser473). Effects of BEZ235 on cell cycle and apoptosis KAT4C, 8505C, TT, BHP7-13 and WRO82-1 were exposed to BEZ235 for 24 or 72 hours (Figure 3A). Compared with untreated control cells, BEZ235 at 6.25, 25 and 100 nmol/L induced increasing cell fractions at G0/G1phase in all cell lines. A representative cell line BHP7-13 demonstrated the effect of BEZ235 on cell cycle distribution (Figure 3B). The effects of BEZ235 (100 nmol/L) on the expression of G0/ G1 related cell cycle proteins was examined (Figure 3C, Figure S2B and S3). The transition of G0/G1 to S phase is a complex process involving cyclins, cyclin-dependent kinases (CDKs) and associated proteins, including p53, p21, p27 and cyclin D1 [31]. p53 is a tumor suppressor that may activate p21, and both p21 and p27 are inhibitors of G1 cyclin-dependent kinases. BEZ235 increased p27 by 4 to 24 hours in all cell lines, with maximal increases of 3.5 to 723.2- fold in KAT4C, TT and BHP7-13. Such a ratio of increase was not measurable in 8505C due to undetectable signal in untreated cells. Statistical analysis was performed for p27 expression in TT, and achieved significance at 8 hours as compared with basal expression (P = 0.019, t-test) (Figure S3C). This data is consistent with our finding of G0/G1 cell cycle arrest. BEZ235 had varied effects on the expression of p53, p21 and cyclin D1 in 4 cell lines, showing that there may be varied contributions of these proteins on affecting cell cycle progression. A consistent increase of p27 in BEZ235 treated cells likely exerted inhibitory effects on cell cycle progression at G0/G1. It is possible that G0/G1 cell cycle arrest is the result of a combined effect of more than one G0/G1 related protein affected by BEZ235. Alternatively, the induction of p27 alone may be enough to arrest cell cycle progression. In a p53 negative PC3M cell line, BEZ235 induced p27 expression and complete cell cycle arrest at G1 phase, without inducing p21 [17]. This study demonstrated G1 cell cycle inhibition induced by BEZ235 does not necessary require association with p53 and p21. The inhibition of the PI3K/mTOR pathway may also lead to apoptosis [6,8]. The ability of BEZ235 to cause apoptotic cell death in thyroid cancer cells was explored (Figure 3D). Compared with control, BEZ235 at 25 and 100 nmol/L significantly induced apoptosis as measured by the proportion of sub-G1 cells at 96 hours in KAT4C. Similar findings were observed in KAT18, with BEZ235 at 6.25, 25 and 100 nmol/L driving an increasing proportion of apoptotic cells. However, BEZ235 failed to show any increase of sub-G1 cells in 8505C, TT, BHP7-13, and WRO82-1. To validate the induction of apoptosis in KAT4C and KAT18 by BEZ235, caspase-3 was assessed by immunoblot after 72 hours of treatment (Figure 3E, Figure S4). In general, higher doses of BEZ235 induced more degradation of apoptotic executioner caspase-3, with less than 12% of caspase-3 detected at 100 nmol/ L in both cell lines. This data suggests that apoptotic mechanisms account for the cytotoxicity of BEZ235 in KAT4C and KAT18. These findings are consistent with previous reports of BEZ235 causing apoptosis in some, but not all, cell lines [17–22].The underlying mechanisms of the varied abilities of BEZ235 to induce apoptosis at different doses and in different cell lines remain unclear [6–8]. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46726