Recently, a fusion protein of echinoderm microtubule associated protein like-4 (EML4) and anaplastic lymphoma kinase (ALK) has been found in non-small cell lung cancer (NSCLC) patients. of PF-02341066 + IR. EML4-ALK and c-Met inhibition leads to activation of parallel pathways that converge on Akt signaling which abrogates any radiation-sensitizing effect. Although PF-02341066 is an effective therapy able to suppress tumor growth in tumors that exhibit positivity for either EML4-ALK or c-Met, it did not affect the intrinsic radiation response of tumor cell lines. In the present study, we demonstrated that PF-02341066 did not enhance radiation sensitivity in a panel of NSCLC cell lines. xenograft models (7). A phase I trial of PF-02341066 revealed impressive results with a 53% response rate and a disease control rate of 79% (3). PF-02341066 is currently under evaluation as a secondary agent as well as a single-drug therapy in phase III and phase II trials, respectively. While PF-02341066 has shown significant and promising results as a chemotherapeutic agent, it has not been evaluated, to date, in conjunction with radiation in NSCLC models. Tanshinone IIA sulfonic sodium IC50 In this study, we evaluated PF-02341066 as a potential radiation-sensitizing agent in 5 different established NSCLC cell Tanshinone IIA sulfonic sodium IC50 lines (H460, A549, H3122, H2228 and H1993) with varying expression levels of c-Met and EML4-ALK (8). Materials and methods Cell culture and reagents Human NSCLC cell lines H460, A549, H3122, H1993 and H2228 were kindly provided by Dr John D. Minna at the Tanshinone IIA sulfonic sodium IC50 UT Southwestern Medical Center, Dallas, TX. These cell lines were maintained in RPMI-1640 with 10% FBS and 50 units/ml penicillin and 50 g/ml streptomycin in 5% carbon dioxide at 37C. PF-02341066 (MW, 450.3) was obtained from Pfizer Inc., dissolved in DMSO to give a stock solution of 10 mM Tanshinone IIA sulfonic sodium IC50 and Rabbit Polyclonal to MBD3 stored at ?20C. Cells were irradiated using a 137Cs source (Mark 1C68 irradiator, J.L. Shepherd and Associates, San Fernando, CA) at a dose rate of 3.47 Gy/min (9). Clonogenic survival assay Exponentially growing cells were treated with PF-02341066 for 2 h and then treated with increasing doses of IR (0, 2, 4, 6 and 8 Gy). Cells were trypsinized and counted using a particle counter (Beckman Coulter, Inc.), diluted serially to appropriate concentrations and plated into a 60-mm dish in triplicate. After 7 or 14 days of incubation, the colonies were fixed and stained with 4% formaldehyde in PBS containing 0.05% crystal violet. Colonies containing >50 cells were counted. The surviving cell fraction was calculated as: (Mean colony counts)/[(cells inoculated) (plating efficiency)], in which plating efficiency was defined as (Mean colony counts)/(cells inoculated for unirradiated controls). The data are presented as the mean SD of at least 3 independent experiments. The curve S = e ?(D + D2) was fitted to the experimental data using a least square Tanshinone IIA sulfonic sodium IC50 fit algorithm using the program SigmaPlot (Systat Software, Inc.) as previously described (9). The radiation dose enhancement ratio (DER) was calculated as the dose (Gy) for radiation alone divided by the dose (Gy) for radiation plus drugs (normalized for drug toxicity) resulting in a surviving cell fraction of 0.25. Clonogenic survival assay was also performed to determine the growth inhibitory response (50%) of these NSCLC cells using increasing dosages of PF-02341066. Inhibitory dose concentrations were determined using a 4 parameter variable slope regression model. Immunoblot assay Cell lysates were prepared.