Nutlin-3a诱导p73α对p53突变型结肠癌细胞的作用及其机制研究

Effects of Nutlin-3a on p53 Mutant Colon Cancer Cells and the Mechanism of These Effects

目的探讨Nutlin-3a对p53突变型结肠癌细胞生长的作用及其机制。方法用不同浓度（0、2.5、5.0、10.0、20.0、40.0、60.0、80.0μmol/L）Nutlin-3a处理人体结肠癌细胞株Caco-2 72 h,MTT法检测其对癌细胞生长的抑制作用;Western blotting法检测Nutlin-3a对Caco-2细胞内p73α及p21蛋白表达的影响;细胞集落形成分析检测癌细胞在撤药后重新增殖状态。结果对照组及不同浓度Nutlin-3a组不同时间吸光度及细胞生长抑制率比较,差异均有统计学意义（P〈0.05）;Nutlin-3a浓度与处理时间存在交互作用（P〈0.05）;对照组及不同浓度Nutlin-3a组吸光度及细胞生长抑制率比较,差异有统计学意义（P〈0.05）;不同时间间差异有统计学意义（P〈0.05）。对照组及不同浓度Nutlin-3a组p73α及p21蛋白表达比较,差异有统计学意义（P〈0.05）。细胞集落形成分析结果显示,对照组及不同浓度Nutlin-3a组不同时间Caco-2细胞数量比较,差异均有统计学意义（P〈0.05）;Nutlin-3a浓度与处理时间存在交互作用（P〈0.05）;对照组及不同浓度Nutlin-3a组Caco-2细胞数量比较,差异有统计学意义（P〈0.05）;不同时间间差异有统计学意义（P〈0.05）。结论使用Nutlin-3a治疗p53突变型结肠癌时,考虑其对肿瘤细胞抑制及杀伤作用的同时,应考虑到其对残存细胞细胞周期的影响,在体外2.5～20.0μmol/L的Nutlin-3a对p53突变型结肠癌的治疗作用最为理想。

Objective To investigate the effects of Nutlin- 3a on p53 mutant colon cancer cells and to study the mechanism of these effects. Methods p53 mutant colon cancer cell line（ Caco- 2） was treated with different concentrations of Nutlin- 3a（ 0,2. 5,5. 0,10. 0,20. 0,40. 0,60. 0,80. 0 μmol / L） for 72 h. MTT assay was used to analyze the inhibitory effect of Nutlin- 3a on cellular proliferation. Colony- forming assay was utilized to detect Caco- 2 cellular re- proliferation ability after withdrawal from Nutlin- 3a. Western blotting analysis was employed to detect levels of cellular p73α and p21 protein in Caco- 2. Results According to MTT assay results,there were significant differences in absorbance value and cell inhibition rate between control group and Nutlin- 3a groups with different concentrations and among different times（ P〈0. 05）; there was significant interaction between Nutlin- 3a concentration and treatment time; there were significant differences in absorbance value and cell inhibition rate between control group and Nutlin- 3a groups with different concentrations（ P〈0. 05）; there were significant differences in absorbance value and cell inhibition rate of different groups among different times（ P〈0. 05）. According to western blotting analysis results,there were significant differences in levels of cellular p73α and p21 protein between control group and Nutlin- 3a groups with different concentrations（ P〈0. 05）. According to western blotting analysis results,there were significant differences in levels of cellular p73α and p21 protein between control group and Nutlin- 3a groups with different concentrations（ P〈0. 05）. According to colony- forming assay results,there were significant differences in Caco- 2 cell count between control group and Nutlin- 3a groups with different concentrations and among different times（ P〈0. 05）; there was significant interaction between Nutlin- 3a concentration and treatment time; there were significant differences in Caco- 2 cell count between control group and Nutlin- 3a groups with different concentrations（ P〈0. 05）; there were significant differences in Caco- 2 cell count of different groups among different times（ P〈0. 05）. Conclusion When using Nutlin- 3a to treat p53 mutant colon cancer,not only the inhibitory and killing effects of Nutlin- 3a on cells but also effect of Nutlin- 3a on cell cycle should be considered. When the concentration of Nutlin- 3a is between 2. 5 ～ 20. 0 μmol / L,Nutlin- 3a plays the best role on treating p53 mutant colon cancer in vitro.