ABSTRACT
Aim To find new kinase inhibitors that o-vercome the imatinib resistance in the treatment of chronic myeloid leukemia ( CML ) by synthesizing C085, a novel derivative of curcumin , and testing its activities against wild-type( WT) and imatinib-resistant mutant Abl kinases , as well as in imatinib-resistant CML cells in vitro.Methods Cell proliferation and apoptosis were examined using MTT assay and flow cy-tometry, respectively;Kinase activity was measured u-sing Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant ( Q252H, Y253F, and T315I) Abl kinases.The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting .Colony forming units ( CFU ) growth was used to test the effects of C 085 on human leukemia progenitor/stem cells.Results C085 suppressed the growth of imatinib-resistant K562/G01 cells and po-tently inhibited both WT and mutant ( Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP com-petitive manner with the values of IC 50 at low nanomole levels.C085 dose-dependently down-regulated Bcr-Abl kinase activity in K562/G01 cells as judged by auto-phosphorylation and Stat 5 , Crkl phosphorylation , and inhibited the phosphorylation of downstream targets Raf,Mek and Erk with protein content reducing .C085 could directly impact mitochondrial PT hole and make it open, which prevents the activation of caspase cas-cade reaction and induces the apoptosis .Furthermore, C085 significantly suppressed CFU growth , implicating that C085 could inhibit human leukemia progenitor/stem cells.Conclusion C085 may inhibit K562/G01 cells through inhibiting Bcr-Abl kinase activity and down-regulating the downstream signal proteins .Di-rectly acting on mitochondrial PT hole and then activa-ting apoptosis-associated proteins are also involved in the pro-apoptotic effect of C085.C085 is a promising compound for the treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib re-sistance .
ABSTRACT
Aim To explore the anti-proliferation and apoptotic effects of C085, a curcumin derivative, on K562 cells and its mechanism. Methods MTT assay and flow cytometry were used to examine cell prolifera-tion and apoptosis, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were ana-lyzed using Western blot. Results The results showed that C085 suppressed the growth of K562 cells and the IC50 value was about 5-fold lower than that of Cur. C085 also induced significant apoptosis on K562 cells in 24 hours when compared with imatinib. Western blot results demonstrated that C085 down-regulated the phosphorylation of Bcr-Abl in K562 cells in a dose-de-pendent manner. The phosphorylation of Stat 5 and Crkl, which were downstream signaling proteins of Bcr-Abl kinase, was also inhibited by C085. C085 caused the opening of mitochondrial PT holes as detected by JC-1 fluorescent, which inhibited Bcl-2 and enhanced Bax , then induced apoptosis. Conclusion C085 in-hibited BCR-ABL+ K562 cells through inhibiting BCR-ABL kinase activity and down-regulating its down-stream signal proteins. Directly acting on mitochondrial PT hole and then activating apoptosis- associated pro-teins are also involved in the pro-apoptotic effect of C085 .
ABSTRACT
Aim To estimate the affinity between C085 and Hsp90 and the inhibitory effects of C085 on the activity of Hsp90 ATPase. Methods The fluores-cence spectrum experiment was applied to examine the affinity between different C085 concentrations and Hsp90 , NHsp90 , MHsp90 , CHsp90; fluorescence in-tensities were recorded in the range of 290-510 nm at 293 K, 303 K and 310 K, respectively;a colorimetric assay for inorganic phosphate based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green was used to examine the inhibitory effects of C085 on the activity of Hsp90 ATPase. Re-sults The dissociation constant KD value of C085 was (11. 163 ± 0. 316 ) μmol · L-1 . The interaction be-tween C085 and Hsp90 was driven mainly by electro-static interaction. C085 showed strongest affinity with CHsp90. When the concentration of ATP was 1 mmol· L-1 ,the inhibition of Hsp90 ATPase activity of C085 with the IC50 value was 6. 04μmol·L-1 . Conclusions The interaction mechanism between C085 and Hsp90 can be analyzed by fluorescence spectrum. C085 shows strong inhibition ATPase activity of Hsp90 .