Niu et al. Journal of Experimental & Clinical Cancer Research 2011, 30:28 http://www.jeccr.com/content/30/1/28 5% nonfat milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween-20) at room temperature for 2 h and incubated with the indicated primary antibodies at 4° C overnight with gentle rocking. After washing with TBST, the membranes were reacted with appropriate horseradish peroxidase (HRP)-conjugated secondary anti- bodies for 1 h at room temperature. After extensive wash- ing with TBST, the presence of proteins was visualized by the enhanced chemiluminescence (ECL) detection kit in accordance with the manufacture’s recommendation. Statistical analysis Each experiment involving tissue culture was performed in triplicates. All analyses were performed using the SPSS 13.0 software. Results are expressed as mean ± SD. The one-way analysis of variance (ANOVA) was used to com- pare the difference between treatment groups. Differences were considered significant if the p value is less than 0.05. Results Growth inhibitory effect of rapamycin on lung cancer cells We first set out to examine whether and at what levels rapamycin inhibits the growth of four different lung cancer cell lines (NCI-H446, A549, SPC-A-1 and 95D). As shown in Figure 1, rapamycin treatment exerted modest inhibitory effect on lung cancer cell proliferation in a dose-dependent manner in all cell lines tested. In addition, the effect of rapamycin seems to level off with its increasing concentration, achieving about 30 - 40% reduction in cell proliferation at 100 nM vs. ~ 10% reduction at 12.5 nM. Finally, the inhibitory effect and its saturating trend towards higher doses of rapamycin are the same for all four cancer cell lines, suggesting rapamycin may act on some targets/pathways common in all of them. Figure 1 Rapamycin exerts growth inhibitory effects in four lung cancer cell lines in a dose-dependent fashion. Cells were treated with increasing levels of rapamycin for 24 hours before cell viability was examined by MTT assay. Control group received treatment of DMSO solution of the same volume and concentration used to dissolve rapamycin. Page 3 of 8 Growth inhibitory effect of rapamycin with docetaxel on lung cancer cells Next we checked the effect of rapamycin on docetaxel- induced growth inhibition in lung cancer cells. It was found that 20 nM rapamycin can potentiate the growth inhibition activity of docetaxel in all four cancer cell lines (Figure 2). This enhancing effect of rapamycin is espe- cially pronounced at low docetaxel concentration (1 nM), having led to an additional 20 - 40% of reduction in cell growth. Although rapamycin does not change the maxi- mum level of cell growth inhibition elicited by docetaxel (e.g., at 100 nM), the co-treatment of rapamycin with docetaxel effectively lowered the EC50 (concentration needed to achieve 50% of maximal effect) of the latter. Rapamycin induces apoptosis in synergy with docetaxel To further investigate whether the enhancing effect that rapamycin showed in docetaxel-co-treated cancer cells is associated with an increased level of apoptosis, we performed flow cytomety analysis using Annexin V/pro- pidium iodide-stained cells. As shown in Figure 3, rapa- mycin enhances the effects of docetaxel in promoting cancer cell death. Discounting the basal apoptosis level as shown in the control sample, the level of apoptosis in the Rapa+DTX group is close to the sum of those in the two monotreaments using either compound alone. These findings indicate that rapamycin may further enhance the efficacy of docetaxel by inducing a higher degree of apoptosis. Combination treatment of rapamycin with docetaxel decreases the expression of Survivin As we wondered whether the enhancing effect of rapa- mycin might come from its ability to block cellular pathways that can counteract the cytotoxic activity of docetaxel, the effect of rapamycin on the expression of Survivin was next examined. Treatment of 95D cells with either rapamycin or docetaxel alone resulted in moderate but significant reduction on the level of Survi- vin expression compared with that of the untreated cells. Moreover, the co-treatment resulted in an even bigger reduction in the Survivin protein level than those of the two single drug treatments added together (Figure 4). In contrast, the expression of a key marker in the apoptotic pathway, caspase-3, is largely unaffected by these treatments. Combination treatment of rapamycin with docetaxel decreases the phosphorylation level of ERK1/2 in 95D cell lines To further clarify the cell growth inhibitory mechanism of rapamycin with docetaxel, we examined the changes in the expression levels of the enzymes involved in cell growth signal transduction pathways. 95D cells were