The data shown are the mean from three independent experiments, each with six wells. (2S)-Octyl-α-hydroxyglutarate trypan blue staining was applied to stain dead cells (E). The data shown are the mean from three impartial experiments. *followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 14). Values of test when appropriated. CalcuSyn software (Version 2.0) was obtained from Researchsoft.com.cn (Beijing, China), and combination index (CI)<1 indicates synergism. Results RAD001 inhibits cell growth in multiple human gastric cancer cell lines We first examined the activity of RAD001 on cell growth in gastric cancer cell lines representing different genetic backgrounds, including AGS, MNK-45, HGC-27, SNU-601, MKN-28, SGC-7901 and N-87. Gastric cancer cell growth was reflected by cell viability which was detected by CCK-8 assay. RAD001 inhibited cell growth in all gastric cancer cells, as the cell viability OD decreased after different concentrations of RAD001 treatment (Physique 1A, Figure S1A and B). However, these different lines showed different sensitivity to RAD001. HGC-27 and SNU-601,were the most sensitive ones (Physique 1A, Physique S1A and B). IC-50s of RAD001 in these different lines were also presented. HGC-27 and SNU-601 had the Slc2a4 lowest IC-50, which further confirmed their highest sensitivity to RAD001 (Physique 1B,). Western blot results in Physique 1C showed the expression of PTEN and p-AKT (Ser 473) in above gastric cancer cells. Results in Physique 1C show that SNU-601 cells expressed extremely low level of (2S)-Octyl-α-hydroxyglutarate PTEN, which was also supported by paper by Byun DS et al . Results indicated that RAD001-sensitive lines were cells with no or low expression level of PTEN (HGC and SNU601). More ever, HGC-27 and AGS were both sensitive to RAD001 on mTOR (pS6) inhibition (Physique 1D and E). Open in a separate window Physique 1 RAD001 inhibits cell growth in multiple human gastric cancer cell lines.Cultured gastric cancer cell lines AGS, MNK-45, HGC-27, SNU-601, MKN-28, SGC-7901 and N-87 were treated with different concentration of RAD001 for 72 h, afterwards, cell growth was detected by CCK-8 cell viability assay (A). IC-50 of RAD001 in these cell lines was shown (B). The expression level of PTEN, pAKT (Ser 473) and -actin (equal loading) in above cell lines and GES-1 cells were also detected by western blot, PTEN and pAKT level was quantified as described (C). AGS and HGC-27 cells were treated with different concentration of RAD001 for 24 hours, phospho- and total S6 were detected by Western blot, pS6 level was quantified as described (D and E), and the number was normalized to the band labeled with 1.00. The data shown was the mean from three impartial experiments. *p<0.05. RAD001 and MK-2206 synergistically inhibits the growth of HGC-27 and SNU-601 cells The main object of this current study is usually to test the synergistic anti-gastric cancer cell ability of RAD001 and MK-2206. CCK-8 cell viability results in Physique 2A and B exhibited that either RAD001 or MK-2206 alone had a moderate effect on HGC-27 and SNU-601 cell growth, however, combination of the two at a relative lower concentration significantly inhibited the growth of both cells, as the CCK-8 OD value decreased dramatically in cells treated with both brokers (Physique 2A and B). Further, RAD001 and MK-2206 at ratio 110 showed most significant synergistic effects (Physique 2A and B). The computer software Calcusyn was used to test combination index (CI) between RAD001 and MK-2206 , CI<1.0 was considered as synergism , as seen in Physique S1C and D. Hence, the combination of RAD001 and MK-2206 showed synergistic inhibitory activity on HGC-27 and SNU-601 cell growth in vitro. Results in Physique S1E showed that RAD001 and MK-2206 synergistically induced HGC-27 cell death, as the number of trypan blue cells (dead cells) increased significantly after the co-administration, comparable results were also obtained in SNU-601 cells (data not shown). We repeated the same (2S)-Octyl-α-hydroxyglutarate treatment (RAD001 plus MK-2206 combination) in SGC-7901, GES-1 cells (high PTEN expression) and MKN-28 cells(middle PTEN expression). Results clearly showed that this synergistic effects was most significant in low-PTEN expression cells.