Deng Yuan, Tao Shan-Dong, Zhang Xin, He Zheng-Mei, Chen Yue, Deng Zhi-Kui, Li Yuan-Yuan, Yu Liang
Department of Hematology, Huai'an First People's Hospital, Nanjing Medical University, Huai'an 223300, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Oct;21(5):1178-82. doi: 10.7534/j.issn.1009-2137.2013.05.018.
This study was aimed to investigate the effect of Btk inhibitor PCI-32765 and the proteasome inhibitor bortezomib on Raji and Ramos cell proliferation, apoptosis, and its mechanisms. Raji and Ramos cells were treated with PCI-32765 and bortezomib alone and/or their combination. The cell proliferation and apoptosis were detected by CCK-8 and flow cytometry respectively, the expression level of Btk, NFκB, c-IAP1, Bcl-xL and caspase-3 protein were measured by Western blot. The results indicated that: (1) after Raji and Ramos cells were treated with PCI-32765 (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 µmol/L) alone and bortezomib (10, 20, 30, 40, 50, 60, 80 nmol/L) alone and their combination for 48 h, the cell proliferation and vitality were inhibited in a dose-dependent manner and both had synergistic effect; (2) Raji and Ramos cells were treated with PCI-32765 (2.0 µmol/L) and bortezomib (20 nmol/L) alone and their combination for 8, 12, 24, 36, 48 and 72 h, the cell proliferation and vitality were inhibited in a time-dependent manner, the two drugs displayed a synergistic effects; (3) the Raji and Ramos cells were treated with PCI-32765 (2.0 µmol/L) and bortezomib (20 nmol/L) alone and their combination for 48 h, all these treatments could induce significant apoptosis of Raji and Ramos cells.In Raji cell experiment, the cell apoptosis rate in the control group, PCI-32765 group, bortezomib group and PCI-32765 and bortezomib combination group were 10.34 ± 0.53%, 24.26 ± 0.91%, 43.66 ± 1.08% and 74.06 ± 0.72% respectively, and the differences was statistically significant among the different groups (P < 0.05). In Ramos cell experiment, the cell apoptosis rate in the control group, PCI-32765 group, bortezomib group and PCI-32765 and bortezomib combination group are 15.16 ± 1.49%, 71.36 ± 0.82%, 75.32 ± 2.36% and 84.30 ± 0.91% respectively, the differences was statistically significant among the different groups (P < 0.05); (4) PCI-32765 and bortezomib could inhibit the expression level of intracellular Btk, NFκB, Bcl-xl and c-IAP1 proteins, but up-regulate the expression level of caspase-3. It is concluded that PCI-32765 and bortezomib can synergistically inhibit the proliferation and induce apoptosis of Raji and Ramos cells, the mechanism may be associated with inhibition of Btk and NFκB activity, down-regulation of anti-apoptotic proteins expression, such as Bcl-xl and c-IAP1, and increase of caspase-3 expression.
本研究旨在探讨布鲁顿酪氨酸激酶（Btk）抑制剂PCI-32765和蛋白酶体抑制剂硼替佐米对Raji和Ramos细胞增殖、凋亡的影响及其机制。将Raji和Ramos细胞分别用PCI-32765和硼替佐米单独及/或联合处理。分别采用CCK-8法和流式细胞术检测细胞增殖和凋亡情况，采用蛋白质印迹法检测Btk、核因子κB（NFκB）、细胞凋亡抑制蛋白1（c-IAP1）、Bcl-xL和半胱天冬酶-3（caspase-3）蛋白的表达水平。结果表明：（1）Raji和Ramos细胞分别用PCI-32765（0.5、1.0、2.0、3.0、4.0、5.0、6.0 μmol/L）、硼替佐米（10、20、30、40、50、60、80 nmol/L）单独及联合处理48 h后，细胞增殖和活力呈剂量依赖性受到抑制，且二者具有协同作用；（2）Raji和Ramos细胞分别用PCI-32765（2.0 μmol/L）、硼替佐米（20 nmol/L）单独及联合处理8、12、24、36、48和72 h，细胞增殖和活力呈时间依赖性受到抑制，两种药物表现出协同作用；（3）Raji和Ramos细胞分别用PCI-32765（2.0 μmol/L）、硼替佐米（20 nmol/L）单独及联合处理48 h，所有这些处理均可诱导Raji和Ramos细胞发生显著凋亡。在Raji细胞实验中，对照组、PCI-32765组、硼替佐米组及PCI-32765与硼替佐米联合组的细胞凋亡率分别为10.34±0.53%、24.26±0.91%、43.66±1.08%和74.06±0.72%，不同组间差异有统计学意义（P<0.05）。在Ramos细胞实验中，对照组、PCI-32765组、硼替佐米组及PCI-32765与硼替佐米联合组的细胞凋亡率分别为15.16±1.49%、71.36±0.82%、75.32±2.36%和84.30±0.91%，不同组间差异有统计学意义（P<0.05）；（4）PCI-32765和硼替佐米可抑制细胞内Btk、NFκB、Bcl-xL和c-IAP1蛋白的表达水平，但上调caspase-3的表达水平。结论：PCI-32765和硼替佐米可协同抑制Raji和Ramos细胞的增殖并诱导其凋亡，其机制可能与抑制Btk和NFκB活性、下调抗凋亡蛋白如Bcl-xL和c-IAP1的表达以及增加caspase-3的表达有关。
