姜黄素调控p53蛋白入核诱导骨髓瘤细胞凋亡

Curcumin regulates P53 protein entry into nucleus and induces apoptosis of P3X63Ag8

为探讨姜黄素对小鼠骨髓瘤细胞核内p53蛋白含量的影响及其凋亡作用,体外培养P3X63Ag8细胞,给予不同浓度姜黄素处理(0、10、20、30、40、50μmol/L)24 h,通过还原性辅酶Ⅰ氧化法测定乳酸脱氢酶(LDH)活性、Caspase3荧光分析试剂盒检测Caspase3活性的变化,采用2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)法检测细胞活性氧(ROS)量的变化,利用Western-Blot技术检测细胞质与细胞核中p53蛋白的分布情况.结果显示,姜黄素浓度为20μmol/L时,P3X63Ag8细胞LDH活性极显著升高(P <0.01),并在40μmol/L组达到顶峰.30μmol/L姜黄素处理后,Caspase-3活性极显著提高(P <0.01),其余姜黄素处理组与未处理组相比未呈现显著变化.与阴性对照组相比,当姜黄素浓度为30μmol/L时细胞内活性氧水平显著上升(P <0.01),而随着姜黄素浓度提高到40和50μmol/L,细胞内活性氧水平均开始极显著下降(P <0.01),且高浓度组下降程度高于中浓度组.从细胞质和细胞核蛋白分离的试验结果可以看出,在细胞质蛋白中,p53蛋白含量随着姜黄素浓度的增加而降低,与此相反,细胞核蛋白中p53蛋白含量则呈现了上升趋势.因此,姜黄素通过调控LDH活性、Caspase-3活性以及活性氧水平来促进细胞损伤,从而抑制骨髓瘤细胞P3X63Ag8的增殖,促进其凋亡;在蛋白水平上促进p53蛋白入核,从而进一步刺激细胞凋亡程序的启动.

The aim of this study was to investigate the effect of curcumin on the protein level of p53 in the nucleus of mouse myeloma cells and curcumin’s ability to induce apoptosis. P3 X63 Ag8 cells were cultured with different concentrations of curcumin(0, 10, 20, 30, 40, 50 μmol/L) for 24 h. Lactate dehydrogenase(LDH) activity was determined by reducing the coenzyme I oxidation method. Caspase-3 was detected by Caspase-3 fluorescence assay kit. 2’,7’-dichlorofluorescein diacetate(DCFH-DA) method was used to detect changes in cellular reactive oxygen species(ROS). To detect the protein level of P53 in the cytoplasm and nucleus, the cytoplasmic proteins and nuclear protein were separated by western blotting. When the concentration of curcumin was 20 μmol/L, the activity of LDH in P3 X63 Ag8 cells was significantly increased(P < 0.01) and reached a peak under 40 μmol/L. The activity of Caspase-3 was significantly increased(P < 0.01) in the 30 μmol/L curcumin treatment. The other curcumin treatment groups showed no significant difference compared with the control group. Compared with the negative control group, the level of ROS was significantly increased when the concentration of curcumin was 30 μmol/L(P < 0.01). However, the level of ROS showed a significant decrease(P < 0.01) when the concentration of curcumin was 40 and 50 μmol/L. Western blot results showed that the cytoplasmic protein and the protein level of p53 decreased with the increasing concentration of curcumin. In contrast, the protein level of nuclear p53 had an upward trend. In summary, curcumin can regulate the activity of LDH, Caspase-3, and ROS to promote cell damage, thereby inhibiting the proliferation of myeloma cells P3 X63 Ag8 and leading to its apoptosis. In addition, it promotes p53 protein entry into the nucleus and regulates apoptosisrelated proteins at the protein level.