mTOR and PI3 Kinase Inhibitor Synergy Figure 4. Combination treatment of BEZ235 or ZSTK474 with temsirolimus synergistically inhibits cell proliferation. A, B, Cell viability was determined in the indicated endometrial cancer cell lines after treatment with increasing concentrations of (A) BEZ235 or (B) ZSTK474 alone or in the presence of 1 nM temsirolimus for 72 hrs. doi:10.1371/journal.pone.0026343.g004 than with BEZ235 alone (Fig. 4A). Co-treatment of ZSTK474 with 1 nM temsirolimus resulted in a dose-dependent synergistic effect on cell proliferation at concentrations similar to BEZ235 with temsirolimus (Fig. 4B). The synergistic effect was seen in all cell lines except KLE, in which the drug combinations exhibited an additive effect. Combination indices were calculated and are shown in Supporting Table S1. These data signify that inhibiting both the upstream and downstream components of PI3K/Akt/ mTOR signaling is an effective approach to synergistically decrease cell proliferation. Co-treatment of BEZ235 or ZSTK474 with temsirolimus induces G1 cell cycle arrest and up-regulates p27 Temsirolimus and BEZ235 have been reported to inhibit cancer cell growth by inducing G0/G1 cell cycle arrest [26,27]. Since we have two cell populations whose proliferation is differentially affected by temsirolimus treatment (sensitive vs. resistant), we chose two representative cell lines (AN3CA-sensitive; Hec50co- resistant) as models to test the combination approach on cell cycle progression. Cell cycle content analysis of AN3CA cells demon- strated that temsirolimus treatment increased the percent of cells PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26343