汉防己甲素衍生物HL-49协同顺铂或多柔比星促进三阴性乳腺癌MDA-MB-231细胞的凋亡

目的:研究汉防己甲素衍生物HL-49协同顺铂(cisplatin,DDP)或多柔比星(adriamycin,ADM)对三阴性乳腺癌MDA-MB-231细胞增殖及凋亡的影响,并探讨其可能的作用机制。方法:用不同浓度的HL-49、DDP、ADM单药以及HL-49联合DDP或ADM处理MDA-MB-231细胞;采用MTT法检测细胞的增殖抑制率并计算Q值,平板克隆实验检测细胞的集落形成能力,FCM法检测细胞的凋亡率并计算Q值;蛋白质印迹法检测细胞中Bloom综合征解旋酶(Bloom’s symdrome helicase,BLM)、乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)和DNA修复关键酶Rad51以及凋亡相关蛋白caspase 3、caspase 8、 caspase 10、Bax、p53和Bcl-2蛋白的表达水平。结果:与HL-49、DDP和ADM单药组相比,HL-49(1或2μmol/L)联合DDP(5或10μmol/L)或ADM(2或5μmol/L)组MDA-MB-231细胞的增殖抑制率均明显提高(P值均<0.05),其中HL-49联合DDP(单药浓度两两组合)4个浓度组的Q值分别为2.04、1.88、1.87和1.83,HL-49联合ADM的Q值分别为1.37、1.21、1.36和1.23,两药联用均具有协同效应。在平板克隆形成实验中,联合用药组细胞的集落形成数均较单药组明显减少(P值均<0.05)。HL-49(1或2μmol/L)联合DDP(5或10μmol/L)或ADM(2或5μmol/L)组MDA-MB-231细胞的凋亡率均高于单药组(P值均<0.05),其中HL-49联合DDP的Q值分别为1.20、1.35、1.70和2.06,HL-49联合ADM的Q值分别为1.48、1.17、1.99和2.13,提示两药联用有协同作用。HL-49联合DDP或ADM用药组MDA-MB-231细胞中的caspase 3、caspase 8、caspase 10和Bax蛋白的表达水平均较单药组明显上调(P值均<0.05),Rad51、BRCA1、BLM、p53和Bcl-2蛋白表达水平均明显下调(P值均<0.05)。结论:汉防己甲素衍生物HL-49与DDP或ADM联合用药均具有协同作用,且其联用抗肿瘤活性可能与诱导三阴性乳腺癌细胞凋亡相关。

Tetrandrine derivative HL-49 combined with cisplatin or adriamycin promotes apoptosis of triple-negative breast cancer MDA-MB-231 cells

Objective: To investigate the effects of tetrandrine derivative HL-49 combined with cisplatin(DDP) or adriamycin(ADM) on the proliferation and apoptosis of triple-negative breast cancer MDAMB-231 cells, and to explore the possible mechanism.Methods: MDA-MB-231 cells were treated with different concentrations of HL-49, DDP or ADM alone and HL-49 combined with DDP or ADM, respectively. The proliferation inhibitory rate of MDA-MB-231 cells was detected by MTT assay and the Q value was calculated. The colony forming ability of MDA-MB-231 cells was detected by plate cloning test. The apoptosis rate was detected by FCM method and the Q value was calculated. The expression levels of Bloom’s symdrome helicase(BLM), breast cancer susceptibility gene1(BRCA1), DNA repair key enzyme Rad51, apoptosis-related protein caspase 3, caspase 8, caspase 10, Bax, p53 and Bcl-2 were detected by Western blotting.Results: The proliferation inhibitory rate of MDA-MB-231 cells in HL-49 combined with DDP or ADM group was significantly higher than that in HL-49, DDP or ADM alone group(all P < 0 05);the Q values of HL-49(1 or 2 μmol/L) combined with DDP(5 or 10 μmol/L)were 2.04, 1.88, 1.87 and 1.83, respectively;while the Q values of HL-49(1 or 2 μmol/L) combined with ADM(2 or 5 μmol/L) were 1.37,1.21, 1.36 and 1.23, respectively. The colony formation number of MDA-MB-231 cells in HL-49 combined with DDP or ADM group was significantly lower than that in HL-49, DDP or ADM alone group(all P < 0 05). The apoptosis rate of MDA-MB-231 cells in HL-49 combined with DDP or ADM group was higher than that in HL-49,DDP or ADM alone group(all P < 0 05);the Q values of HL-49(1 or 2 μmol/L) combined with DDP(5 or 10 μmol/L) were 1.20, 1.35,1.70 and 2.06, respectively;the Q values of HL-49(1 or 2 μmol/L)combined with ADM(2 or 5 μmol/L) were 1.48, 1.17, 1.99 and 2.13,respectively. The above results suggested that HL-49 and DDP(or ADM) had synergistic effects. As compared with HL-49, DDP or ADM alone group, the expression levels of caspase 3, caspase 8, caspase10 and Bax proteins in HL-49 combined with DDP or ADM group were up-regulated, and the expression levels of Rad51, BRCA1, BLM, p53 and Bcl-2 proteins were down-regulated(all P < 0. 05).Conclusion: Tetrandrine derivative HL-49 combined with DDP or ADM has synergetic effect.The anti-tumor activity of the combination treatment is related to the induction of apoptosis in triple-negative breast cancer MDA-MB-231 cells.