For that reason, SkE ought to be tested being a new therapeutc optocancers that exhbt consttutve actvatoof the ERK pathway.Wehave reported prevously that SkE s the two cytostatc and cytotoxc for some tumor cell lnes.The present examine was carried out to address the mechansm of actoof SkE dfferent cancer cell lnes.We frst made use of the well characterzedhumaK562 cell lne to determne irrespective of whether SkE has an effect on the prolferatoof leukemc cells.To ths end, we performed colony formatoassays soft agar usng ncreasng doses of SkE or a maxmal dose of matnb, a tyrosne knase nhbtor that targets BCR ABL, the fusooncoproteresponsble for ths dsease.As expected, matnb nhbted the clonogenc potental of K562 cells soft agar by a lot more tha90%.mportantly, SkE was ahghly potent nhbtor of K562 cell colony formatodentcal condtons, wth a maxmal effect at 500 nM.At ths dose, SkE was evemore potent thamatnb, the leadng treatment for CML.The C50 value to the SkE result was observed to get 250 nM.SkE was also an exceptionally potent nhbtor of CD34 cell growth for cells solated from two CML patents at dagnoss.
Fnally, SkE also exerted potent anteukemc results oseveral matnb resstant CML cell lnes.aattempt to dentfy the potental targets of SkE, we applied the PathScaRTK sgnalng antbody array kt from Cell Sgnalng, whch will allow the smultaneous quantfcatoof the actvty of approxmately selleck 50 knases.Among these knases, two have been sgnfcantly impacted by SkE.ndeed, SkE nhbted the actvty of ERK by 70% and c Abl by 15%.To confrm the result of SkE oBCR ABL actvty, we subsequent ncubated K562 cells for 2h wth 250 nM of SkE and analyzed the phosphorylatostatus of each BCR ABL and knowBCR ABL substrates.accordance wth the outcomes obtaned wth the RTK sgnalng array kt, we confrmed the nhbtoof c Abl by SkE as judged through the decreased phosphorylatoof c Abl as sooas 3hrs following the addtoof SkE towards the culture medum.We also noted a reduce the phosphorylatostatus of STAT5.In addition, dephosphorylatoof ERK1 two was clearly detected as sooas 30 mafter the addtoof SkE and was maxmal at 15h.
Collectvely, our outcomes confrm that SkE s an exceptionally potent nhbtor of the ERK pathway K562 cells.Furthermore, discover this info here t appears that c Abl dephosphorylatodd not precede ERK dephosphorylatobut rather followed ERK nhbton.Fgure 2C also exhibits

that SkE faed to impact autophagy K562 CML cells, as assessed through the absence of delpdatoof LC3 b cells treated wth ths drug.We upcoming applied the Raf 1ER cells, whch express anducble form in the knase Raf one, to assess the effects of SkE comparsowth U0126, a nicely knownhbtor of MEK1, the Ras Raf MEK ERK pathway.Tamoxfenduced the actvatoof the ERK pathway, as assessed from the ncreased phosphorylatoof ERK1 two.mportantly, SkE was as effcent as U0126 at abolshng tamoxfenduced ERK1 2 actvaton.