When the cell lines were classified based on phospho ERK levels ra www.selleckchem.com/products/Abiraterone.html ther than BRAF mutation Inhibitors,Modulators,Libraries status, there was no correl ation with the degree of cell growth inhibition. In contrast, high levels of pAkt in BRAF/RAS mu tant cell lines were strongly suggestive of insensitivity to E6201. Furthermore, high levels of pAkt significantly correlated with Inhibitors,Modulators,Libraries E6201 insensitivity in dependent of BRAF or PTEN status. PTEN protein was present in 20 of the melanoma cell lines tested with a lack of the tumour suppressor being sug gestive of resistance to E6201 in not only BRAF/RAS mutant lines but also if all lines are consid ered. Characterization of E6201 response in vitro MEK inhibitors have been previously shown to have a predominantly cytostatic effect on melanoma cells, although some clinically relevant inhibitors, such as CI 1040, PD0325901 and AZD6244, have been shown to induce cell death.

We sought to further Inhibitors,Modulators,Libraries evaluate the mechanism of sensitivity to E6201, as an equivocal cytocidal response in vitro may equate to the poor clinical response observed with current MEK inhibitors. Fifteen melanoma cell lines were selected such that 13 cell lines demonstrated sensitivity to E6201 and 2 cell lines were insensitive to E6201. Of these cell lines, seven were Inhibitors,Modulators,Libraries mutant for BRAF but wildtype for PTEN, five were mutant for both BRAF/NRAS and PTEN, and three were wildtype for both BRAF and PTEN. E6201 treatment induced G1 arrest in all of the sensitive cell lines and had little to no effect on cell cycle progression in the two insensitive cell lines.

E6201 treatment resulted in a greater than 2 fold increase in Annexin positive staining in eleven out of fifteen cell lines, including eleven out of thirteen Inhibitors,Modulators,Libraries sensi tive cell lines. Two sensitive cell lines, SKMEL13 and BL, did not demonstrate E6201 induced Annexin staining although both of these cell lines underwent cell cycle arrest with E6201 treatment and were hypersensitive to E6201. These experiments were repeated in duplicate to confirm this finding. E6201 induced a less than two fold increase in Annexin staining in the E6201 insensitive cell lines. Three of the five PTEN mutant cell lines exhibited a cytocidal response to E6201, demonstrating that PTEN mutation does not pre clude a cytocidal response to E6201.

E6201 also induced cell cycle arrest and cell death in cell lines with constitutively active Akt, suggesting that although high pAkt correlates http://www.selleckchem.com/products/Enzastaurin.html with E6201 insensitivity, cell lines with high pAkt can still undergo a cytocidal response to E6201. To confirm our Annexin V results we also performed an enzyme linked immunosorbent assay to de termine the degree of DNA fragmentation as an indica tor of cell death with E6201 treatment. The results from the cell death ELISA were very similar to that obtained from the Annexin studies with 10 out of 13 sensitive melanoma lines demonstrating a greater than two fold increase in DNA fragmentation with E6201.