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目的 :探讨汉防己甲素衍生物HL-42和HL-49对三阴性乳腺癌MDAMB-231细胞增殖、克隆形成能力以及凋亡的影响,并探讨其可能的作用机制。方法 :采用不同浓度的HL-42和HL-49分别作用MDA-MB-231细胞后,应用MTT法检测细胞的增殖情况;平板克隆形成实验检测对细胞克隆形成的影响;2和10μmol/L的HL-42和HL-49分别作用于MDA-MB-231细胞24 h后,应用FCM法检测对细胞凋亡的影响;分别采用半定量RTPCR法和蛋白印迹法检测细胞中Bloom综合征解旋酶(Bloom’s syndrome helicase,BLM)、人乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)和同源重组修复的关键酶Rad51 m RNA及蛋白的表达水平。结果:1.25~10μmol/L的HL-42和HL-49处理24、48和72 h后,MDAMB-231细胞的增殖受到抑制,呈时间和浓度依赖性(P值均<0.05),HL-42作用于MDA-MB-231细胞24、48和72 h的半数抑制浓度(half maximal inhibitory concentration,IC50)值分别为(8.27±0.27)(、3.92±0.39)和(2.72±0.14)μmol/L;相应的HL-49的IC50值分别为(5.30±0.45)、(3.19±0.32)和(1.64±0.12)μmol/L;而阳性对照顺铂的IC50值则为(61.96±3.83)、(29.08±4.11)和(16.19±2.53)μmol/L。0.2、0.5、1和5μmol/L的HL-42和HL-49均可抑制MDA-MB-231细胞克隆的形成(P值均<0.001)。2和10μmol/L的HL-42和HL-49均可诱导MDA-MB-231细胞凋亡,且有浓度依赖性(P值均<0.05)。2μmol/L的HL-42处理24 h后,MDA-MB-231细胞中的Rad51 mR NA及蛋白的表达水平无明显变化(P值均>0.05);10μmol/L的HL-42处理24 h后,MDA-MB-231细胞中的Rad51 m RNA及蛋白的表达水平下调(P值均<0.05);2和10μmol/L的HL-42处理24 h后,MDA-MB-231细胞中BLM和BRCA1 m RNA及蛋白表达水平无明显变化(P值均>0.05)。2μmol/L的HL-49处理24 h后,MDA-MB-231细胞中的BRCA1 m RNA及蛋白表达水平无明显变化(P值均>0.05),10μmol/L的HL-49处理24h后,MDA-MB-231细胞中的BRCA1 m RNA及蛋白表达水平下调(P值均<0.01);2和10μmol/L的HL-49处理24 h后,MDA-MB-231细胞中的BLM、Rad51 m RNA及蛋白表达水平下调(P值均<0.01)。结论 :汉防己甲素衍生物HL-42和HL-49可明显抑制三阴性乳腺癌MDA-MB-231细胞的增殖并诱导其凋亡,作用机制可能是HL-42和HL-49部分阻断了细胞内DNA损伤修复通路。
Objective: To investigate the effects of tetrandrine derivatives HL-42 and HL-49 on the proliferation, clonality and apoptosis of triple negative breast cancer MDAMB-231 cells and to explore its possible mechanism. Methods: The proliferation of MDA-MB-231 cells was detected by MTT assay with different concentrations of HL-42 and HL-49. The effects of 2 and 10 μmol / L The effect of HL-42 and HL-49 on the apoptosis of MDA-MB-231 cells was detected by FCM 24 h after treated with HL-42 and HL-49 cells. Semiquantitative RTPCR and Western blotting were used to detect the expression of Bloom syndrome helicase (BLM), breast cancer susceptibility gene 1 (BRCA1), and Rad51 mRNA and protein levels of key enzymes involved in homologous recombination. Results: The proliferation of MDAMB-231 cells was inhibited in HL-42 and HL-49 cells treated with 1.25-10 μmol / L for 24, 48 and 72 h (P <0.05) The IC50 values of MDA-MB-231 cells at 24, 48 and 72 h were (8.27 ± 0.27), 3.92 ± 0.39 and 2.72 ± 0.14 μmol / L, respectively. The IC50 values of the corresponding HL-49 cells were (5.30 ± 0.45), (3.19 ± 0.32) and (1.64 ± 0.12) μmol / L respectively, while those of the positive control cisplatin were (61.96 ± 3.83) and (29.08 ± 4.11) and (16.19 ± 2.53) μmol / L respectively. Both HL-42 and HL-49 at 0.2, 0.5, 1 and 5 μmol / L inhibited the formation of MDA-MB-231 cell clones (all P <0.001). The apoptosis of MDA-MB-231 cells was induced by 2 and 10 μmol / L HL-42 and HL-49 in a concentration-dependent manner (P <0.05). The expression of Rad51 m RNA and protein in MDA-MB-231 cells did not change significantly after treatment with 2 μmol / L HL-42 for 24 h (P> 0.05). After treated with 10 μmol / L HL-42 for 24 h , And the expression of Rad51 m RNA and protein in MDA-MB-231 cells was down-regulated (P <0.05). After treatment with 2 and 10 μmol / L HL-42 for 24 h, the expression of BLM and BRCA1 m RNA and protein expression levels did not change significantly (P values> 0.05). The expression of BRCA1 m RNA and protein in MDA-MB-231 cells was not significantly changed after treatment with 2μmol / L HL-49 for 24 h (P> 0.05). After treatment with 10μmol / L HL-49 for 24 hours, MDA The expression of BRCA1 m RNA and protein in MDA-MB-231 cells was down-regulated (P <0.01). After treatment with 2 and 10 μmol / L HL-49 for 24 h, And protein expression (P <0.01). CONCLUSION: Tetrandrine derivatives HL-42 and HL-49 can significantly inhibit the proliferation and induce the apoptosis of triple-negative breast cancer MDA-MB-231 cells. The mechanism may be partially blocked by HL-42 and HL-49 Intracellular DNA damage repair pathway.