Next, we determined whether HB cells are sensitive to mTOR inhibitors. For this purpose, we chose MLN0128, a second generation mTOR inhibitor . Unlike allosteric mTOR inhibitors (such as Rapamaycin), which partially inhibit mTORC1, MLN0128 has been shown to complete suppress mTORC1 activity as indicated by the strong inhibition of p-4EBP1 . Importantly, both Hep293TT and HepG2 cells were found to be highly sensitive to MLN0128, with an IC50 around 50nM (Figure 2A). At the molecular level, administration of MLN0128 led to an increased inhibition of mTORC1 targets, including p-mTOR, p-RPS6 and p-4EBP1, in a dose-dependent manner (Figure 2B). In the time course study, sustained inhibition of p-mTOR, p-S6K, p-RPS6 and p-4EBP1 was detected when Hep293TT and HepG2 cells were treated with MLN0128 at 50nM (Figure 2C). Furthermore, MLN0128 dramatically decreased cell proliferation and increased cell apoptosis in a dose-dependent manner (Figure 2D and 2E). Consistently, pro-survival proteins, including Survivin and MCL-1, were found to be decreased, whereas the pro-apoptotic protein Bak was upregulated in MLN0128 treated HB cell lines. Consistent with increased apoptosis, cleaved caspase 3 levels were highest in Hep293TT and HepG2 cells treated with MLN0128 (Figure 2C).