姜黄素衍生物C1209抑制慢性粒细胞白血病细胞增殖与阻断Hsp90伴侣功能的关系

Inhibition of curcumin derivative C1209 on proliferation of CML cells by disrupting Hsp90 chaperon function

目的研究姜黄素衍生物C1209抑制慢性粒细胞白血病细胞增殖与阻断Hsp90伴侣功能的关系。方法采用荧光光谱法,研究不同浓度C1209与Hsp90、其N端片段(NHsp90)、M端片段(MHsp90)、C端片段(CHsp90)的相互作用,以及不同温度(293 K、303 K、310 K)下,C1209与Hsp90的相互作用;实验选取280 nm为激发波长,290~510 nm的波长范围内进行荧光光谱扫描。采用孔雀绿磷钼酸铵-无机磷检测法,研究C1209对Hsp90-ATPase活性的抑制。四甲基偶氮唑蓝(MTT)法和羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)染色法体外检测C1209对白血病细胞K562及耐伊马替尼(IM)的白血病细胞K562/G01细胞的增殖抑制作用;应用蛋白免疫印迹法检测Hsp90客户蛋白及其下游蛋白、Hsp90分子伴侣的表达。结果C1209解离常数为(14.733±0.713)μmol·L^(-1);C1209与Hsp90之间的主要作用力为静电作用力;C1209与Hsp90的C端片段结合能力最强。当ATP为1 mmol·L^(-1)时,C1209作用于Hsp90的IC50值为11.4μmol·L^(-1)。C1209呈剂量依赖性地抑制K562和K562/G01细胞的增殖,作用24 h的IC50为1.14μmol·L^(-1)和0.56μmol·L^(-1);C1209影响Hsp90的分子伴侣功能,且下调K562和K562/G01细胞中Bcr-Abl、Akt、MEK、ERK、c-Raf及其相应磷酸化蛋白p-Akt、p-MEK、p-ERK等Hsp90客户蛋白及其下游蛋白的表达。结论姜黄素衍生物C1209是Hsp90抑制剂,对K562和耐IM的白血病细胞K562/G01具有明显的增殖抑制作用,可能与其抑制Hsp90的分子伴侣功能、下调Hsp90客户蛋白有关。

Aim To estimate the inhibition of curcumin derivative C1209 on the proliferation of chronic myeloid leukemia（ CML） cells involving the disruption Hsp90 chaperon function. Methods The fluorescence spectrum experiment was applied to examine the affinity between different C1209 concentrations and Hsp90,NHsp90,MHsp90,CHsp90; fluorescence intensities 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 C1209 on the activity of Hsp90 ATPase. MTT assay and CFSE were used for K562 and K562/G01 cell proliferation determination in vitro by C1209. Western blot was used to detect the client proteins and the molecular chaperone of Hsp90 level. Results The dissociation constant KDvalues of C1209 was（ 14. 733 ±0. 713） μmol · L-1. The interaction between C1209 and Hsp90 was driven mainly by electrostatic interaction. C1209 showed the strongest affinity with CHsp90. When the concentration of ATP was 1 mmol·L-1,the inhibition of Hsp90 ATPase activity of C1209 with the IC50 value was 11. 4 μmol · L-1; C1209 showed inhibition of K562 and K562/G01 cells in dosedependent proliferation and the IC50 value was 1. 14μmol · L-1 and 0. 56 μmol · L-1,respectively after24 h incubation. C1209 affected the molecular chaperone functions of Hsp90 and down-regulated Bcr-Abl,Akt,MEK,ERK,c-Raf,p-Akt,p-MEK and p-ERK protein levels which were client proteins of Hsp90 in K562 and K562/G01 cells. Conclusions Curcumin derivative C1209 is Hsp90 inhibitor; C1209 has significant inhibitory effects on proliferation of K562 and K562/G01,which may be related to C1209 affecting the molecular chaperone functions of Hsp90 and downregulating client proteins of Hsp90 level.