Combined inhibition shows increased efficiency in a variety of cancer genotypes in pre clinical studies and numerous early phase clinical studies have been in progress. Clinical studies demonstrate the simultaneous inhibition of multiple pathways Hedgehog antagonist to stay all likelihood more toxic than inhibition of one process, and no dose has been established. PI3K mTOR inhibitors could be split into twin PI3K?mTOR inhibitors, PI3K inhibitors and mTOR inhibitors. Rapalog mTOR inhibitors are known to induce IRS 1 mediated, upstream feedback activation of PI3K AKT, that is considered to be important for the limited scientific effectiveness of the treatment for most cancers, including NSCLC. PI3K/mTOR and pi3k inhibitors must lack such feedback service and theoretically become more active. Numerous early stage clinical Lymph node trials are currently testing both single PI3K and twin PI3K/mTOR inhibitors, but it is unknown whether either is more efficient, although it is likely that a drug which hits multiple targets is going to be more harmful in a clinical setting. Recent oncological treatments have moderate disease changing effects in cases of non-small cell lung cancer, although some disease subgroups attentive to specific therapy have been identified recently. These include EGFR mutant and ALK translocated, where patients are highly responsive to EGFR or ALK tyrosine kinase inhibitors. Furthermore, other major oncogenic condition sub-groups are the K Ras mutant, that is regarded as undruggable with presently available pharmacological agents. We set out here to investigate combined inhibition with PI3K and MEK in non-small cell lung cancer cell lines of numerous genotypes. Dual inhibition is proved to be an even more effective type of treatment in some cell lines. This study also addresses administration times for the inhibitors Gemcitabine ic50 that might prove less-toxic in a clinical setting. Methods Cell lines The cell lines used here involved NSCLC lines with EGFR mutation, a K Ras mutation, ALK translocation and the triple bad genotype, a basal like breast cancer line MDA MB231 and HCT116, a K Ras mutant colorectal cell line. The NSCLC cell lines were kind gifts from Dr. Pasi J?nne, and the breast and colorectal lines from Dr. Peppi Koivunen. The cell lines were cultured in RPMI 1640 supplemented with 5 or ten percent fetal bovine serum and 100 IU/ml penicillin and streptomycin. Each of the cell culture reagents were purchased from HyClone. Inhibitors These inhibitors were used: CI 1040, PI 103, ZSTK474, and TAE684. Most of the inhibitors were dissolved in DMSO to a final concentration of 10mM and stored at 20 C. The drug options for the tests were prepared from a 10mM stock solution instantly before use.