亚砷酸与甲磺酸伊马替尼单用及联用对淋巴瘤细胞Raji增殖和凋亡的影响

Effects of arsenic trioxide and imatinib mesylate on proliferation and apoptosis in lymphoblastoid Raji cell line

目的研究不同浓度的亚砷酸（As2O3）、甲磺酸伊马替尼（简称伊马替尼）单用及联用对恶性淋巴瘤细胞株Raji的影响。方法采用MTT法检测两药对Raji细胞的生长抑制作用。采用流式细胞仪检测两药对Raji细胞Caspase-3的表达及G1期、S期的比例变化。采用免疫组织化学SABC法检测p16蛋白的表达。结果As2O3、伊马替尼及两药联用均使细胞抑制率升高，两药联合组与各单药组相比差异有统计学意义（P〈0．05）。As2O3上调Raji细胞Caspase-3表达呈时间、剂量依赖效应（P〈0．0001）。伊马替尼对Caspase-3表达无明显影响（P〉0．05），且两药联用无协同上调Caspase-3作用（P〉0．05）。Raji细胞随As2O3浓度升高、作用时间延长，p16蛋白平均吸光度值增加（P〈0．05）。伊马替尼在低浓度72h与高浓度48h、72h时p16蛋白表达与对照组相比差异有统计学意义（P〈0．05）。但两药联用无协同上调p16蛋白表达的作用（P〉O．05）。As2O3可使G1期细胞比例明显增高，S期比例降低，呈现剂量、时间依赖效应（P〈0．0001），并出现明显凋亡峰。伊马替尼对Raji细胞周期无影响（P〉0．05）。两药联合组与单用As2O3组差异亦无统计学意义（P〉0．05）。结论As2O3可通过激活Caspase-3表达诱导Raji细胞凋亡、上调p16蛋白、阻滞细胞周期于G1期来发挥抗肿瘤作用。伊马替尼在高浓度时可以上调Raii细胞p16蛋白的表达。As2O3和伊马替尼对恶性淋巴瘤细胞株Raji细胞无协同作用。

Objective To investigate the effect of arsenic trioxide （As203） and imatinib mesylate （imatinib） or in combination with imatinib with different concentration on Raji cells and the mechanisms. Methods The inhibition of cell proliferation was measured by MTT assay to assess the cells survival after As2O3 and imatinib or in combination with imatinib treatment with different concentration at indicated time. Caspase-3 activity changes and the relative number of cells in different phases and the percentages of cells calculated in G1 and S phase of the cell cycle were assessed by FCM. The expression of p16 protein was analyzed by SABC immunohistochemical method.. Results The results of MTF assay showed that As2O3 and imatinib or in combination with imatinib could inhibit Raji cells growth. There was clear statistical significance between the union groups and the single-drug group （P 〈0.05）. As2O3 inducing apoptosis was accompanied by up-regulation and activation of apoptosis protein Caspase-3. The mean percentage of apoptosis cells was both in time-and dosage-dependent form （P 〈0.0001）. While Raji cell lines were less sensitive to imatinib than arsenic trioxide （P 〉0.05）. Further more, Combination of imatinib with As2O3 had not a synergistic inducing apoptosis effect on Raji cells （P 〉0.05）. The optical density of p16 protein increased both in time-and dosage-dependent form with As203 by immunohistochemical method （P 〈0.05）. While at higher concentration and for a longer time, imatinib could up-regulated the optical density of p16 protein. There was no obvious statistic significance for p16 protein in Raji ceils with using As2O3 only compared with the unions group（P 〉0.05）. The cell cycle was arrested at G1 phase, the number of cells Gj period increased significantly, and S phase decreased on Raji cells after As2O3 treatment. The relationship between the cellular DNA contents and the concentration of As2O3 showed a dose-and time-dependent manner （P 〈0.0001）. But it was found that imatinib had no effect on Raji cell cycle. There was no the obvious difference （P 〉0.05） between two drugs unions group with only using As2O3 group. Conclusion The results showed that As2O3 exerted variable and definite effects on lymphoma Raji cells, and suggested that As2O3 might induce apoptosis and up-regulate the optical density of p16 protein and arrest cell cycle. As for Raji cells, imatinib might affect the expression of p16 protein at higher concentration. Two drugs unions had no effective and synergistic effect.