姜黄素衍生物C085抑制K562/G01细胞及Bcr-Abl激酶的体外研究

Curcumin derivative C085 inhibits proliferation of K562/G01 cells and activity of Bcr-Abl kinase in vitro

目的研究姜黄素衍生物C085对K562/G01细胞及野生型和伊马替尼耐药型Bcr-Abl激酶的作用,以期能找到一种对IM耐药点突变激酶有效的酪氨酸激酶抑制剂。方法分别采用MTT法及流式细胞仪检测及观察C085对细胞增殖及凋亡的影响;应用Kinase-Glo~激酶发光检测试剂盒检测C085对4种Abl激酶(WT、T135I、Y253F、Q252H)活性的影响;蛋白免疫印迹检测C085对Bcr-Abl和下游信号转导通路蛋白的磷酸化水平及其他凋亡相关蛋白表达的影响;JC-1荧光染色法检测C085对细胞线粒体膜电位的影响;集落形成法观察C085对CML病人骨髓CD34+细胞增殖的影响。结果 C085低浓度下即可抑制K562/G01的增殖,且可非ATP竞争性地抑制野生型及IM耐药点突变Abl激酶的活性;C085可抑制Bcr-Abl的自磷酸化,下调下游信号转通路蛋白的磷酸化水平和凋亡相关蛋白的表达,作用强于IM,具有明显的量效关系;C085通过直接作用于线粒体的PT孔使其开放,激活凋亡相关蛋白,诱导细胞凋亡,促凋亡作用强于IM;镜下观察及集落数量统计发现,C085可对已经产生IM耐药的CD34^+祖/干细胞集落有明显的抑制作用。结论C085可抑制K562/G01细胞的生长可能与其抑制Bcr-Abl蛋白激酶活性,下调相关信号通路;直接作用于线粒体的PT孔使其开放,激活凋亡相关蛋白,诱导细胞凋亡有关。C085对IM敏感及耐药的白血病细胞均有杀伤作用,有望克服IM的耐药而成为新的TKI。

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＆nbsp;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 .