RITA对TP53突变人套细胞淋巴瘤细胞株的作用及调控机制研究

Effect of RITA on TP53 Mutant Human Mantle Cell Lymphoma Cell Line and Its Mechanism

目的:研究RITA对TP53突变的人套细胞淋巴瘤细胞株Mino的作用及其调控机制。方法:体外培养Mino细胞,不同浓度(0-16μmol/L) RITA作用于Mino细胞24、48、72 h后,采用CCK-8法检测RITA对Mino细胞的增殖抑制作用;0-8μmol/L浓度RITA作用于Mino细胞48 h,采用Annexin V/PI流式细胞术检测RITA对Mino细胞的凋亡诱导作用,采用Western blot法检测蛋白BCL-2、Caspase-3、Cleaved Caspase-3、PARP、MDM2、P53的表达。结果:RITA能显著抑制TP53突变细胞株Mino增殖,经0.5、1、2、4、8、16μmol/L的RITA作用Mino细胞48 h后,细胞的增殖抑制率分别为(1.2±5.6)%、(14.9±4.9)%、(41.7±5.0)%、(61.8±2.4)%、(70.2±2.8)%和(70.8±2.4)%,随着RITA作用浓度的增加,抑制率也增加(r=0.767)。Mino细胞经4μmol/L的RITA作用24、48和72 h后的增殖抑制率分别为(25.2±3.8)%、(61.8±2.4)%和(87.0±0.7)%,随着RITA作用时间增加,抑制率亦增加(r=0.978)。细胞凋亡检测结果显示,Mino细胞经0、2、4、8μmol/L的RITA作用48 h后的凋亡率分别为(5.4±0.4)%、(15.3±0.6)%、(38.7±1.7)%和(50.8±1.1)%,随着RITA作用浓度增加,诱导Mino细胞凋亡的作用增强(r=0.961)。Western blot检测结果显示,随着RITA浓度的增加,BCL-2蛋白表达下降(r=0.932),出现PARP切割和Caspase-3激活,而MDM2、P53蛋白表达无变化。结论:P53小分子RITA对套细胞淋巴瘤TP53突变细胞株Mino有显著的增殖抑制和诱导凋亡的作用,其机制可能依赖于Caspase途径,而与P53通路无关。

Objective: To investigate the effect of RITA on TP53 mutant human mantle cell lymphoma( MCL) cell line Mino and its possible mechanism. Methods: Mino cells were cultured in RPMI-1640 and treated with RITA at a concentration of 0-16 μmol/L for 24, 48, 72 hours. Cell viability was assessed by CCK-8 assay. The cells were treated by RITA( 0-8 μmol/L) for 48 h,the cell apoptosis induced by RITA was detected by annexin V/PI flow cytometry.Western blot was performed to evaluate the expression of protein BCL-2,Caspase-3,Cleaved Caspase-3,PARP,MDM2,and P53 in Mino cells. Results: After treatment with 0.5,1,2,4,8,and 16 μmol/L RITA for 48 h,the proliferation inhibition rate of Mino cells was( 1.2±5.6) %,( 14.9±4.9) %,( 41.7±5.0) %,( 61.8±2. 4) %,( 70.2±2. 8) %,and( 70.8±2.4) %,respectively. RITA could inhibit the proliferation of Mino cells significantly,and statistical analysis showed that the inhibition rate was increased with the increasing of RITA concentration( r = 0. 767). After the cells were treated by 4 μmol/L RITA for 24,48,and 72 h,the proliferation inhibition rate was( 25. 2±3.8) %,( 61.8±2. 4) %,and( 87.0±0.7) %,respectively. Satistical analysis showed that the inhibition rate was also increased with the increasing of treatment time( r = 0.978). The apoptosis rate of Mino cells treated by 0,2,4,and 8 μmol/L RITA for 48 h was( 5.4±0.4) %,( 15.3±0.6) %,( 38.7±1.7) %,and( 50.8±1.1) %,respectively,and it showed dose-dependent manner( r = 0.961). Western blot showed that with the increasing of RITA concentration,the BCL-2 protein expression was decreased in a dose-dependent manner( r = 0. 932),moreover,PARP cleavage and Caspase-3 activation were found,while the protein expression of MDM2 and P53 showed no change. Conclusion: RITA can inhibit the proliferation and induce the apoptosis of Mino cells significantly. The mechanism may be dependent on the Caspase pathway,but independent on the P53 pathway.