Niu et al. Journal of Experimental & Clinical Cancer Research 2011, 30:28 http://www.jeccr.com/content/30/1/28 Page 2 of 8 of the PI3K/Akt/mTOR signalling pathway can cause ovarian cancer cells to develop resistance to taxane dur- ing the course of the therapy [12]. However, a combina- tion treatment using specific PI3K inhibitor and paclitaxel seemed more effective than using paclitaxel alone not only in the reduction of tumor growth, but also in minimizing side effects [12]. Rapamycin and related compounds are molecular tar- geting agents that specifically inhibit the mammalian target of rapamycin (mTOR). Originally intended for use in transplantation procedures to prevent organ or graft rejection, rapamycin has recently become of signifi- cant interest as a potential anti-cancer drug. It has been reported that rapamycin can exert antitumor activity with cytostatic activities such as G1 phase arrest and that it can exhibit anti-angiogenesis properties [13,14]. Rapamycin was also demonstrated to have synergistic cytotoxic effect in conjunction with other chemothera- peutic agents on several cancer cell types [15-19]. Sev- eral rapamycin analogues have been synthesized and put under evaluation in phase Ⅰ/Ⅱ clinical trials, showing a promising antitumor effect in several types of refractory or advanced tumors. This evidence prompted us to examine whether the administration of rapamycin will result in some beneficial modulation of the cancer kill- ing properties of docetaxel in lung cancer cells [20,21]. To the best of our knowledge, the effect of including rapamycin in combination therapies intended to treat advanced stage lung cancer has not been reported in the literature. This prompted us to examine whether juxta- posed administration of rapamycin will result in some beneficial modulation of the cancer killing properties of docetaxel in lung cancer cells. Our results showed that rapamycin can sensitize lung cancer cells for more effec- tive killing by docetaxel and suggested that such enhancement may involve down-regulation of the expression of Survivin and the inactivation of ERK signalling. Materials and methods Therapeutic compounds and reagents Lung cancer cell lines A549, SPC-A-1, 95D and NCI- H446 were purchased from Shanghai Institue of Bio- chemistry and Cell Biology, Chinese Academy of Sciences. Rapamycin, DMSO and MTT were purchased from Sigma (St Louis, MO, USA). Docetaxel was pur- chased from Shanghai Sanwei Pharmaceutical Company (Shanghai, China). Annexin V-FITC apoptosis detection kit was from Jingmei Biotech (Shenzhen, China). RPMI tissue culture medium and fetal bovine serum (FBS) were purchased from GIBCO (USA). Anti-Survivin, anti-caspase-3, anti-ERK1/2, anti-p-ERK1/2, anti- GAPDH and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Chemiluminescence (ECL) reagent kit was purchased from Pierce Biotechnology (Rockford, IL, USA). Cell culture A549, SPC-A-1, 95D and NCI-H446 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomy- cin. The cells were grown in a humidified incubator at 37°C and in an atmosphere of 5% CO2 in air. Cells were grown on sterile tissue culture petri dishes and passaged once every 2 to 3 days. MTT cell viability assay Cell were seeded in a 96-well plate at a density of 1 × 106/ml and cultured in medium for 24 h. Cell viability was determined using the conversion of MTT to forma- zan via mitochondrial oxidation. Various treatments of cells included the addition of rapamycin (12.5 nM, 25 nM, 50 nM, 100 nM), docetaxel (1 nM, 10 nM, 50 nM, 100 nM) and the combination of docetaxel and 20 nM rapamycin for 24 h. Cells in the control group were treated with only the DMSO solution used to dilute rapamycin. MTT solution was then added to each well at a final concentration of 1 mg/ml per well and the plates were incubated at 37°C for another 4 h. After incubation, 150 μl DMSO was added to each well to dis- solve the formazan formed and the absorbance was read at 490 nm using a spectrophotometer. Flow cytometry apoptosis assay Cellular apoptosis was determined using the Annexin V- FITC and propidium iodide (PI) double staining kit according to the manufacturer’s protocol. Briefly, 95D cells were seeded in six-well plates and allowed to attach overnight; they were then treated with 20 nM rapamycin (Rapa), 10 nM docetaxel (DTX) alone or a combination (20 nM Rapa + 10 nM DTX). After 48 h, cells were har- vested, washed twice with cold PBS, resuspended in 250 μl of binding buffer, and stained with staining solu- tion containing Annexin V/FITC and PI. After incuba- tion in the dark for 30 min, cells were analyzed by FACSCalibur flow cytometry (BD Biosciences). Western blot Western Blotting was performed using standard techni- ques as previously described [22]. Briefly, cells were washed twice with PBS buffer and lysed in RIPA lysis buf- fer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 2 μg/ml aprotinin) on ice. 50 μg total proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked with