We determined the overall tyrosine twice phosphorylation pat tern in cell lysates prepared from oxaliplatin and/or doviti nib treated cells. Oxaliplatin showed insignificant changes in the phosphorylation pattern while treatment with dovitinib inhibited the phosphorylation of several pro teins in all three cell lines. Next, we checked the expres sion levels of receptors in the presence of oxaliplatin and/or dovitinib in all three CRC cell lines by western blot analyses. Only HCT 116 and HT 29 were found to have expression of both receptors. These two cell lines showed insignificant changes in the phosphorylation of FGFR and VEGFR after treatment with either oxaliplatin or dovitinib while the combination of the drugs showed a significant down regulation of phosphorylation of both the receptors.

Since SW480 cells showed ab sence or very low expression most of these receptors, we checked the effect of the combination on downstream sig nalling pathway proteins. RAS proteins are small GTPases known for their in volvement in oncogenesis. Around 25% of human tu mors present mutations in a member of this family. Many studies have cleared that activating mutations in members of the RAS family of genes are among the most common genetic lesions in human tumors and these mutations lock RAS proteins into a constitutively acti vated state in which they signal to downstream effectors even in the absence of extracellular stimuli. Involvement of RAS signaling in cancer is accentuated by the inci dence not only of RAS mutations but also the deregula tion of many of its regulators or effectors pathways.

The first RAS effector pathway to be identified was the RAF MEK ERK pathway. Next, if RAS or RAF mutations play any role in the response to the treatment with oxaliplatin and dovitinib, we determined the expression levels of these proteins in the presence/ absence of these drugs. Irrespective of RAS RAF muta tions, dovitinib inhibited the expression of both RAS and RAF in all three cell lines tested. The combination of the two drugs showed an even more pronounced in hibition of both RAS and RAF proteins. In addition, dovitinib inhibited phosphorylation of ERK, a down stream signaling molecule of RTKs. These data indicate that dovitinib inhibits the activity of its target receptors in tumor cells and results in down modulation of the signaling pathway and .

The second best characterized RAS effector family is phosphoinositide AV-951 3 kinases, which play important roles as mediators of RAS mediated cell survival and prolif eration. When active, PI3K converts phosphatidylinositol bisphosphate into phosphatidylinositol trisphosphate. PIP3, in turn, binds the pleckstrin homology inhibitor Erlotinib domain of Akt/PKB, stimulating its kinase activity, resulting in the phosphorylation of a host of other proteins that affect cell growth, cell cycle entry, and cell survival. Next, we determined the phosphorylation of AKT in response to treatment with oxaliplatin and/or dovitinib.