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 spec-trum experiment was applied to examine the affinity be-tween different C1209 concentrations and Hsp90, NH-sp90,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 in-organic phosphate based on the formation of a phospho-molybdate complex and subsequent reaction with mala-chite 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/G01cell pro-liferation determination in vitro by C1209. Western blot was used to detect the client proteins and the mo-lecular chaperone of Hsp90 level. Results The disso-ciation constant KDvalues of C1209 was (14.733 ± 0.713) μmol·L-1. The interaction between C1209 and Hsp90 was driven mainly by electrostatic interac-tion. C1209 showed the strongest affinity with CHsp90. When the concentration of ATP was 1mmol· L-1,the inhibition of Hsp90 ATPase activity of C1209 with the IC50value was 11.4 μmol·L-1; C1209 showed inhibition of K562 and K562/G01cells in dose-dependent proliferation and the IC50value was 1.14 μmol·L-1and 0.56 μmol·L-1, respectively after 24 h incubation. C1209 affected the molecular chaper-one 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/G01cells. Conclusions Curcumin derivative C1209 is Hsp90 inhibitor;C1209 has signif-icant inhibitory effects on proliferation of K562 and K562 / G01, which may be related to C1209 affecting the molecular chaperone functions of Hsp90 and down-regulating client proteins of Hsp90 level.%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(NH-sp90)、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11.4 μmol·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1.14 μmol·L-1和0.56 μmol·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.
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