PI3Kδ 抑制剂CAL-101 对淋巴瘤细胞系Raji和SUDHL-10的作用及其机制研究*

目的:探讨PI 3Kδ抑制剂CAL-101 对弥漫大B 细胞淋巴瘤细胞系SUDHL- 10和Burkitt 淋巴瘤细胞系Raji 的作用及其相关机制，为这类疾病的治疗提供新的思路。方法:用不同浓度的CAL-101 处理Burkitt 淋巴瘤细胞系Raji 和弥漫大B 细胞淋巴瘤细胞系SUDHL- 10，以MTT 法检测CAL-101 对两种细胞系的增殖抑制作用；以AnnexinV/PI流式细胞术和DAPI染色法检测细胞的凋亡情况；迁移实验检测淋巴瘤细胞向淋巴瘤基质细胞系HK的迁移比例；Westernblot法检测CAL-101 处理后淋巴瘤细胞表达磷酸化ERK 的变化；MTT 法结合CalcuSynsoftware软件分析检测CAL-101 是否可协同硼替佐米抑制淋巴瘤细胞的增殖。结果:5 μmol/L 及更高浓度的CAL-101 对Raji 细胞和SUDHL- 10细胞的增殖有明显的抑制作用，且呈剂量依赖性。5、10、15、20μmol/LCAL- 101 作用于Raji 细胞48h，细胞增殖抑制率分别为（29.17± 1.23）% 、（38.15± 1.51）% 、（46.46± 1.78）% 、（55.8 ± 2.01）% ，空白对照组为（1.15± 0.02）%（P<0.05）。 作用于SUDHL- 1048h，细胞增殖抑制率也逐渐增加（P<0.05）。 CAL-101 可诱导淋巴瘤细胞的凋亡，10、20μmol/LCAL-101 作用于Raji 细胞24h，AnnexinV-FITC/PI 双标法显示其凋亡率分别为（22.69± 3.83）% 和（49.96±7.36）% ，均高于对照组（5.23± 2.04）%（P<0.05）；作用于SUDHL- 10细胞同样得到相似的结果（P<0.05）。 CAL-101 可显著降低淋巴瘤细胞向基质细胞的迁移，且对Raji 细胞和SUDHL- 10细胞的迁移率的抑制作用也呈浓度依赖性，差异均具有统计学意义（P<0.05）；Westernblot检测发现CAL-101 处理细胞后磷酸化ERK 的表达明显降低；CAL-101 与硼替佐具有协同作用，可显著抑制淋巴瘤细胞的增殖，CI<1。结论:PI 3Kδ抑制剂CAL-101 可抑制淋巴瘤细胞Raji 和SUDHL- 10的增殖，诱导凋亡，抑制其向淋巴瘤基质细胞的迁移，其机制可能通过阻断ERK 信号途径而实现；CAL-101 有望为侵袭性淋巴瘤的治疗带来希望。 

Inhibitory effects of the phosphoinostitide-3'-kinase delta inhibitor CAL-101 on Raji and SUDHL-10 lymphoma cells and its relative mechanism

Objective:To detect the inhibitory effects of CAL- 101, a selective inhibitor of phosphoinostitide-3'-kinase delta (PI 3K δ), on Burkitt's lymphoma cell line Raji and diffused large B-cell lymphoma cell line SUDHL-10and elucidate its relative mechanism. Methods:Raji and SUDHL-10cells were treated with various concentrations of CAL-101. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the inhibitory effect of CAL- 101 on lymphoma cells, and cell apoptosis was measured by Annexin V/PI and DAPI staining. Migration assays were performed with transwell to detect the migration of lymphoma cells derived from the stromal cell line HK. Western blot was used to detect the phosphorylation status of the ERK pathway. MTT and CalcuSyn software analyses were preformed to detect whether or not combining CAL-101 with bortezomib induces synergistic cytoxicity. Results:CAL-101 at con-centrations of 5, 10, 15, and 20μ mol/L inhibited cell proliferation in a dose-dependent manner. The proliferation rates of the Raji cells treated with 5, 10, 15, and 20μ mol/L for 48h were 29.17% ± 1.23%,38.15% ± 1.51%,46.46% ± 1.78%, and 55.8% ± 2.01%, respec -tively, which were significantly higher ( P<0.05) than that of the control group ( 1.15% ± 0.02% ). Similar results were found in the SUDHL- 10cells after treatment with CAL- 101 (P<0.05). CAL-101 also exerted an apoptotic effect on the lymphoma cells. The apop -totic rates of the Raji cells treated with CAL-101 for 21h were 22.69% ± 3.83% and49.96% ± 7.36%, respectively, which were signifi-cantly higher (P<0.05) than that of the control group ( 5.23% ± 2.04% ). Similar results were found in the SUDHL-10cells ( P<0.05).?Treatment with5 and 10μ mol/L CAL-101 dose-dependently inhibited the migration activity of lymphoma cells to stromal cells (P< 0.05). Western blot analysis showed that the expression level of ERK phosphorylation protein was significantly downregulated in the cells treated with CAL- 101. A synergistic effect between CAL- 101 and bortezomib was verified. That is, these two drugs can significantly inhibit the proliferation of lymphoma cells with CI values less than 1. Conclusion:The PI 3K δ-specific inhibitor CAL-101 sup- pressed the proliferation of Raji and SUDHL- 10cells, induced apoptosis, and inhibited stromal cell-derived migration. This inhibitory effect may be induced by blocking the ERK pathway. Overall, our study indicated that CAL-101 is a novel and potential agent in the therapeutic strategy against aggressive B-cell lymphoma.