漢防己甲素衍生物對三陰性乳腺癌MDA-MB-231細胞的抑制作用及其機制研究
發(fā)布時間：2019-04-12 10:39
【摘要】：目的:探討漢防己甲素衍生物HL-42和HL-49對三陰性乳腺癌MDA-MB-231細胞增殖、克隆形成能力和凋亡的影響及其可能的作用機制,并初步研究HL-49聯(lián)合三種化療藥物對MDA-MB-231細胞增殖的影響。方法:采用不同濃度的HL-42和HL-49分別作用MDA-MB-231細胞后,應(yīng)用MTT法檢測細胞的增殖情況;平板克隆形成實驗檢測對細胞克隆形成的影響;2和10μmol/L的HL-42和HL-49分別作用于MDA-MB-231細胞24 h后,應(yīng)用FCM法檢測對細胞凋亡的影響,半定量RT-PCR法檢測細胞中Bloom綜合征解旋酶(bloom’s syndrome helicase,BLM)、人乳腺癌易感基因1(Breast cancer susceptibility gene1,BRCA1)和同源重組修復(fù)的關(guān)鍵酶Rad51 mRNA的表達水平,蛋白印跡法檢測細胞中BLM、BRCA1和Rad51蛋白的表達水平;1和2μmol/L的HL-49聯(lián)合不同濃度的順鉑(DDP)、阿霉素(ADM)和紫杉醇(TAX)分別作用于MDA-MB-231細胞48 h后,采用MTT法檢測各組細胞的增殖情況。結(jié)果:1.25~10μmol/L的HL-42和HL-49處理24、48和72 h后,MDA-MB-231細胞的增殖受到抑制,呈時間和濃度依賴性(P值均0.05),HL-42作用于MDA-MB-231細胞24、48和72 h的半數(shù)抑制濃度(50%concentration of inhibition,IC50)分別為(8.27±0.27)、(3.92±0.39)和(2.72±0.14)μmol/L;相應(yīng)的HL-49的IC50分別為(5.30±0.45)、(3.19±0.32)和(1.64±0.12)μmol/L;而陽性對照漢防己甲素的IC50分別為(23.61±2.02)、(14.85±0.56)和(12.39±0.92)μmol/L;陽性對照順鉑的IC50則為(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細胞克隆的形成(P值均0.001)。2和10μmol/L的HL-42和HL-49均可誘導(dǎo)MDA-MB-231細胞凋亡,且有濃度依賴性(P值均0.05)。2μmol/L的HL-42處理24 h后,MDA-MB-231細胞中的Rad51 mRNA及蛋白表達水平無明顯變化(P值均0.05),10μmol/L的HL-42處理24 h后,MDA-MB-231細胞中的Rad51 mRNA及蛋白表達水平下調(diào)(P值均0.05);2和10μmol/L的HL-42處理24 h后,MDA-MB-231細胞中BLM和BRCA1 mRNA及蛋白表達水平無明顯變化(P值均0.05)。2μmol/L的HL-49處理24 h后,MDA-MB-231細胞中的BRCA1 mRNA及蛋白表達水平無明顯變化(P值均0.05),10μmol/L的HL-49處理24 h后,MDA-MB-231細胞中的BRCA1 mRNA及蛋白表達水平下調(diào)(P值均0.01);2和10μmol/L的HL-49處理24 h后,MDA-MB-231細胞中的BLM、Rad51mRNA及蛋白表達水平下調(diào)(P值均0.01)。HL-49分別聯(lián)合DDP、ADM和TAX對MDA-MB-231細胞的抑制作用均高于單藥給藥組(P值均0.05)。結(jié)論:漢防己甲素衍生物HL-42和HL-49可明顯抑制三陰性乳腺癌MDA-MB-231細胞的增殖并誘導(dǎo)其凋亡,作用機制可能是HL-42和HL-49部分阻斷了細胞內(nèi)DNA損傷修復(fù)通路;HL-49分別與DDP、ADM和TAX間存在一定的協(xié)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 鹵0.27), (3.92 鹵0.39 and (2.72 鹵0.14) 渭 mol/L;, respectively. The IC50 of the corresponding HL-49 were (5.30 鹵0.45), (3.19 鹵0.32) and (1.64 鹵0.12) 渭 mol/L;, respectively. The IC50 of Tetrandrine in the positive control group was (23.61 鹵2.02), (14.85 鹵0.56) 渭 mol/L; and (12.39 鹵0.92) 渭 mol/L;, respectively. The IC50 of cisplatin in positive control was (61.96 鹵3.83). (29.08 鹵4.11) and (16.19 鹵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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
本文編號：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 鹵0.27), (3.92 鹵0.39 and (2.72 鹵0.14) 渭 mol/L;, respectively. The IC50 of the corresponding HL-49 were (5.30 鹵0.45), (3.19 鹵0.32) and (1.64 鹵0.12) 渭 mol/L;, respectively. The IC50 of Tetrandrine in the positive control group was (23.61 鹵2.02), (14.85 鹵0.56) 渭 mol/L; and (12.39 鹵0.92) 渭 mol/L;, respectively. The IC50 of cisplatin in positive control was (61.96 鹵3.83). (29.08 鹵4.11) and (16.19 鹵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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
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