mTOR and PI3 Kinase Inhibitor Synergy Figure 3. Temsirolimus-induced Akt phosphorylation is decreased by BEZ235 and ZSTK474, but not by AZD6244. A, Ishikawa H (upper panels) and Hec50co (lower panels) cells were grown for 24 hrs and treated overnight with the indicated inhibitors. Phospho-Akt (P-Akt S473) and total Akt were assessed by Western blotting. B, Endometrial cancer cell lines were treated with the indicated inhibitors overnight. Total protein extracts were analyzed by Western blotting for P-Akt S473 and total Akt (left panels) or phospho-p70S6K T389 (P-S6K) and total p70S6K (S6K, right panels). Phospho-Akt blots for Hec1A and KLE cells were subjected to a long exposure to visualize low levels of Akt phosphorylation. doi:10.1371/journal.pone.0026343.g003 we proposed could overcome acquired resistance to mTOR inhibition. Combination treatment synergistically inhibits cell growth in endometrial cancer cell lines After demonstrating BEZ235 and ZSTK474’s effectiveness in down-regulating temsirolimus-induced Akt hyper-phosphoryla- tion, we examined the anti-proliferative properties of combined treatment of temsirolimus with BEZ235 or ZSTK474. A broad range of doses — either BEZ235 or ZSTK474 alone or in combination with temsirolimus— were tested in order to determine the optimal concentration for eliciting growth inhibitory effects. First, the panel of endometrial cancer cells were treated with single drug (BEZ235 or ZSTK474 at 1–1000 nM) and compared to vehicle (DMSO) control. BEZ235 alone reduced cell proliferation by 50% at doses as low as 1–50 nM (Fig. 4A). ZSTK474 alone was cytostatic in all the eight tested endometrial cancer cell lines; it inhibited cell growth at about 100–1000 nM (Fig. 4B). However, when either BEZ235 or ZSTK474 was combined with low dose temsirolimus (1 nM), cell proliferation was inhibited in a synergistic manner compared to BEZ235 or ZSTK474 alone. Notably, a 50% reduction in proliferation occurred with combined temsirolimus and BEZ235 at a lower concentration of BEZ235 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26343