汉防己甲素衍生物对三阴性乳腺癌MDA-MB-231细胞的抑制作用及其机制研究
发布时间：2019-04-12 10:39
【摘要】：目的:探讨汉防己甲素衍生物HL-42和HL-49对三阴性乳腺癌MDA-MB-231细胞增殖、克隆形成能力和凋亡的影响及其可能的作用机制,并初步研究HL-49联合三种化疗药物对MDA-MB-231细胞增殖的影响。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,半定量RT-PCR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 gene1,BRCA1)和同源重组修复的关键酶Rad51 mRNA的表达水平,蛋白印迹法检测细胞中BLM、BRCA1和Rad51蛋白的表达水平;1和2μmol/L的HL-49联合不同浓度的顺铂(DDP)、阿霉素(ADM)和紫杉醇(TAX)分别作用于MDA-MB-231细胞48 h后,采用MTT法检测各组细胞的增殖情况。结果:1.25~10μmol/L的HL-42和HL-49处理24、48和72 h后,MDA-MB-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(50%concentration of inhibition,IC50)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分别为(5.30±0.45)、(3.19±0.32)和(1.64±0.12)μmol/L;而阳性对照汉防己甲素的IC50分别为(23.61±2.02)、(14.85±0.56)和(12.39±0.92)μmol/L;阳性对照顺铂的IC50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 mRNA及蛋白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 mRNA及蛋白表达水平下调(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 mRNA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 mRNA及蛋白表达水平无明显变化(P值均0.05),10μmol/L的HL-49处理24 h后,MDA-MB-231细胞中的BRCA1 mRNA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、Rad51mRNA及蛋白表达水平下调(P值均0.01)。HL-49分别联合DDP、ADM和TAX对MDA-MB-231细胞的抑制作用均高于单药给药组(P值均0.05)。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;HL-49分别与DDP、ADM和TAX间存在一定的协同作用。
[Abstract]:Objective: to investigate the effects of Tetrandrine derivatives HL-42 and HL-49 on proliferation, colony forming ability and apoptosis of triple negative breast cancer MDA-MB-231 cells and its possible mechanism. The effects of HL-49 combined with three chemotherapeutic drugs on the proliferation of MDA-MB-231 cells were also studied. Methods: after MDA-MB-231 cells were treated with different concentrations of HL-42 and HL-49, the proliferation of MDA-MB-231 cells was detected by MTT assay, and the effect of plate clone formation test on cell clone formation was detected. MDA-MB-231 cells were treated with 2 and 10 渭 mol / L HL-42 and 10 渭 mol / L HL-49 for 24 h, respectively. FCM assay was used to detect the effect of apoptosis, and semi-quantitative RT-PCR method was used to detect Bloom syndrome helicase (bloom's syndrome helicase,BLM. The expression levels of human breast cancer susceptibility gene 1 (Breast cancer susceptibility gene1,BRCA1 and Rad51 mRNA, the key enzyme of homologous recombination repair, were detected by Western blot. The expression levels of BLM,BRCA1 and Rad51 proteins in cells were detected by Western blot. MDA-MB-231 cells were treated with 1 渭 mol / L and 2 渭 mol / L HL-49 combined with cisplatin, (DDP), doxorubicin (ADM) and paclitaxel (TAX) for 48 h, respectively. The proliferation of MDA-MB-231 cells was detected by MTT assay. Results: after treatment with HL-42 and HL-49 for 24 h and 48 h and 72 h, the proliferation of MDA-MB-231 cells was inhibited in a time-and concentration-dependent manner (P < 0.05). The median inhibitory concentrations (50%concentration of inhibition,IC50) of HL-42 for 24 h, 48 h and 72 h were (8.27 卤0.27), (3.92 卤0.39 and (2.72 卤0.14) 渭 mol/L;, respectively. The IC50 of the corresponding HL-49 were (5.30 卤0.45), (3.19 卤0.32) and (1.64 卤0.12) 渭 mol/L;, respectively. The IC50 of Tetrandrine in the positive control group was (23.61 卤2.02), (14.85 卤0.56) 渭 mol/L; and (12.39 卤0.92) 渭 mol/L;, respectively. The IC50 of cisplatin in positive control was (61.96 卤3.83). (29.08 卤4.11) and (16.19 卤2.53) 渭 mol/L.0.2,0.5,1 and 5 渭 mol / L HL-42 and HL-49 inhibited the clone formation of MDA-MB-231 cells (P < 0.001). 2 and 10 渭 mol / L HL-42. And HL-49 could induce apoptosis of MDA-MB-231 cells. After 24 h treatment with 2 渭 mol / L HL-42, the expression level of Rad51 mRNA and protein in MDA-MB-231 cells did not change significantly (P = 0.05). After 24 h treatment with 10 渭 mol / L HL-42, the expression level of Rad51 mRNA and protein did not change significantly (P < 0.01), but 10 渭 mol / L HL-42 for 24 h. The expression levels of Rad51 mRNA and protein in MDA-MB-231 cells were down-regulated. After treatment with 2 and 10 渭 mol / L HL-42 for 24 h, the expression levels of BLM, BRCA1 mRNA and protein in MDA-MB-231 cells did not change significantly (P < 0.05). After 24 h treatment with 2 渭 mol / L and 10 渭 mol / L HL-49, the expression levels of BLM, BRCA1 mRNA and protein were not changed significantly (P < 0.01). There was no significant change in the expression of BRCA1 mRNA and protein in MDA-MB-231 cells (P < 0.05). After treatment with 10 渭 mol / L HL-49 for 24 h, the expression levels of BRCA1 mRNA and protein in MDA-MB-231 cells were down-regulated (P = 0.01). After treatment with 2 and 10 渭 mol / L HL-49 for 24 h, the expression levels of BLM,Rad51mRNA and protein in MDA-MB-231 cells were down regulated (P < 0.01). HL-49 combined with DDP,. The inhibitory effect of ADM and TAX on MDA-MB-231 cells was higher than that of single drug group (P < 0.05). Conclusion: Tetrandrine derivatives HL-42 and HL-49 can significantly inhibit the proliferation and induce apoptosis of triple negative breast cancer MDA-MB-231 cells. The mechanism may be that HL-42 and HL-49 partially block the intracellular DNA damage repair pathway. There were some synergistic effects between HL-49 and DDP,ADM and TAX, respectively.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2456938
[Abstract]:Objective: to investigate the effects of Tetrandrine derivatives HL-42 and HL-49 on proliferation, colony forming ability and apoptosis of triple negative breast cancer MDA-MB-231 cells and its possible mechanism. The effects of HL-49 combined with three chemotherapeutic drugs on the proliferation of MDA-MB-231 cells were also studied. Methods: after MDA-MB-231 cells were treated with different concentrations of HL-42 and HL-49, the proliferation of MDA-MB-231 cells was detected by MTT assay, and the effect of plate clone formation test on cell clone formation was detected. MDA-MB-231 cells were treated with 2 and 10 渭 mol / L HL-42 and 10 渭 mol / L HL-49 for 24 h, respectively. FCM assay was used to detect the effect of apoptosis, and semi-quantitative RT-PCR method was used to detect Bloom syndrome helicase (bloom's syndrome helicase,BLM. The expression levels of human breast cancer susceptibility gene 1 (Breast cancer susceptibility gene1,BRCA1 and Rad51 mRNA, the key enzyme of homologous recombination repair, were detected by Western blot. The expression levels of BLM,BRCA1 and Rad51 proteins in cells were detected by Western blot. MDA-MB-231 cells were treated with 1 渭 mol / L and 2 渭 mol / L HL-49 combined with cisplatin, (DDP), doxorubicin (ADM) and paclitaxel (TAX) for 48 h, respectively. The proliferation of MDA-MB-231 cells was detected by MTT assay. Results: after treatment with HL-42 and HL-49 for 24 h and 48 h and 72 h, the proliferation of MDA-MB-231 cells was inhibited in a time-and concentration-dependent manner (P < 0.05). The median inhibitory concentrations (50%concentration of inhibition,IC50) of HL-42 for 24 h, 48 h and 72 h were (8.27 卤0.27), (3.92 卤0.39 and (2.72 卤0.14) 渭 mol/L;, respectively. The IC50 of the corresponding HL-49 were (5.30 卤0.45), (3.19 卤0.32) and (1.64 卤0.12) 渭 mol/L;, respectively. The IC50 of Tetrandrine in the positive control group was (23.61 卤2.02), (14.85 卤0.56) 渭 mol/L; and (12.39 卤0.92) 渭 mol/L;, respectively. The IC50 of cisplatin in positive control was (61.96 卤3.83). (29.08 卤4.11) and (16.19 卤2.53) 渭 mol/L.0.2,0.5,1 and 5 渭 mol / L HL-42 and HL-49 inhibited the clone formation of MDA-MB-231 cells (P < 0.001). 2 and 10 渭 mol / L HL-42. And HL-49 could induce apoptosis of MDA-MB-231 cells. After 24 h treatment with 2 渭 mol / L HL-42, the expression level of Rad51 mRNA and protein in MDA-MB-231 cells did not change significantly (P = 0.05). After 24 h treatment with 10 渭 mol / L HL-42, the expression level of Rad51 mRNA and protein did not change significantly (P < 0.01), but 10 渭 mol / L HL-42 for 24 h. The expression levels of Rad51 mRNA and protein in MDA-MB-231 cells were down-regulated. After treatment with 2 and 10 渭 mol / L HL-42 for 24 h, the expression levels of BLM, BRCA1 mRNA and protein in MDA-MB-231 cells did not change significantly (P < 0.05). After 24 h treatment with 2 渭 mol / L and 10 渭 mol / L HL-49, the expression levels of BLM, BRCA1 mRNA and protein were not changed significantly (P < 0.01). There was no significant change in the expression of BRCA1 mRNA and protein in MDA-MB-231 cells (P < 0.05). After treatment with 10 渭 mol / L HL-49 for 24 h, the expression levels of BRCA1 mRNA and protein in MDA-MB-231 cells were down-regulated (P = 0.01). After treatment with 2 and 10 渭 mol / L HL-49 for 24 h, the expression levels of BLM,Rad51mRNA and protein in MDA-MB-231 cells were down regulated (P < 0.01). HL-49 combined with DDP,. The inhibitory effect of ADM and TAX on MDA-MB-231 cells was higher than that of single drug group (P < 0.05). Conclusion: Tetrandrine derivatives HL-42 and HL-49 can significantly inhibit the proliferation and induce apoptosis of triple negative breast cancer MDA-MB-231 cells. The mechanism may be that HL-42 and HL-49 partially block the intracellular DNA damage repair pathway. There were some synergistic effects between HL-49 and DDP,ADM and TAX, respectively.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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