Niu et al. Journal of Experimental & Clinical Cancer Research 2011, 30:28 http://www.jeccr.com/content/30/1/28 Page 5 of 8 Figure 5 Combination treatment of rapamycin and docetaxel decreases phosphorylation of ERK in 95D cell lines. 95D cells were treated with 1 nM and 10 nM docetaxel alone, 10 nM and 20 nM rapamycin alone and a combination with 10 nM docetaxel and 20 nM rapamycin for 24 hr. After incubation, levels of ERK1/2 and p- ERK1/2 (phosphorylated Tyr204) were examined. Con: control, Rapa: rapamycin, DTX: docetaxel. with docetaxel exhibited a marked reduction in the phosphorylation levels of ERK1/2. This suggests that there may exist positive interactions between rapamycin and docetaxel in the suppression of ERK1/2 pathway in 95D cells. Discussion The prognosis for inoperable or recurrent lung cancer patients has not been much improved despite the advent of new chemotherapeutic agents. Although early stage lung cancer is potentially curable, most lung cancer patients were already at advanced stages when diag- nosed. Moreover, most advanced lung cancer patients have a history of smoking thus suffer concurrent com- plications in both cardiovascular and pulmonary sys- tems, rendering aggressive surgery and multimodality therapy unfeasible. Docetaxel is a common second-line therapeutic agent used for advanced NSCLC. In several randomized clini- cal tries, combination cytotoxic chemotherapy regimens for second-line therapy of advanced NSCLC failed to establish patient survival benefit, although there was report of higher cytotoxic effect [23]. It has been thought that the clinical benefit of present second-line therapies for advanced NSCLC has reached its peak. More recently, combinations of molecularly targeted agents with standard chemotherapy are being investi- gated clinically with the hope to surpass the current therapeutic threshold of second-line therapies [24]. Activation of PI3K-Akt-mTOR pathway has been detected in many types of tumors including lung cancer, which is considered to be important for the survival, proliferation, angiogenesis and resistance of cancer cells to chemotherapy [25]. Consequently, this pathway has been regarded as an attractive target of molecular tar- geting therapy. Indeed, rapamycin treatment has shown some promising antitumor effect in tissue culture sys- tems [19]. However, as evidenced in clinical phase stu- dies, rapamycin analogue monotherapy exerted a modest but limited antitumor effect [26,27]. In order to achieve a greater therapeutic benefit, several combina- tion therapies of rapamycin and other cytotoxic or molecular targeting agents have been under clinical study. Encouragingly, rapamycin has clearly shown either synergistic or additive effects in these treatments [28-30]. In the present study, rapamycin treatment alone exerted modest inhibition on cell proliferation of several lung cancer cell lines in a dose-dependent manner. However, when applied together, the proliferation inhi- bition effect of docetaxel was significantly potentiated by rapamycin. This observation is in line with previous reports that regarded the mTOR pathway as a promising target of therapy in the treatment of other solid tumors refractory to conventional chemotherapies [31,32]. Apoptosis, induced by chemotherapy, radiation and cytokines, seems to be the main mechanism to kill tumor cells. We suspected that the rapamycin may also enhance the apoptosis-inducing effect of docetaxel in cancer cells. We used flow cytometry analysis to show that rapamycin and docetaxel combination indeed induced higher degree of apoptosis in lung cancer cell lines than that by either compound alone. This led us to further ponder upon the potential downstream effectors of rapamycin and docetaxel-induced signaling pathways in lung cancer cell lines. As a first step, we examined the expression and phosphorylation levels of some pro- teins known to be involved in cell proliferation and apoptosis. Interestingly, Survivin and ERK1/2 were found to be down-regulated in expression and phos- phorylation, respectively, especially by the combination treatment of rapamycin and docetaxel. In comparison, the expression of caspase-3, an apoptosis effector down- stream of mitochondrial cytochrome c release, was found to be unaffected. Survivin is a member of the inhibitor of apoptosis pro- teins (IAP) family that is typically absent in most normal adult differentiated tissues. However, its mRNA and protein are found in abundance in fetal tissue, most transformed cell lines and cancers. Survivin suppresses apoptosis and promotes angiogenesis, proliferation and metastasis in cancer cells [33-37]. Survivin can block apoptosis by inhibiting terminal apoptotic effectors cas- pase-3 and caspase-7, and by suppressing both the pro- teolytic activation and the activity of caspase-9 in the context of Survivin-IAP complexes [38-40]. Clinically, increased expression of Survivin is often associated with elevated resistance of cancer cells to apoptotic stimuli during chemotherapy and is negatively correlated with response to proapoptotic drugs and/or radiotherapy in patients with bladder cancer, breast cancer, lymphoma and multiple myeloma [41-46]. Furthermore, overex- pression of Survivin is a prognostic biomarker for decreased patient survival in multiple cancers, e.g.,