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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (02) : 90 -98. doi: 10.3877/cma.j.issn.2095-1221.2021.02.004

所属专题: 文献

论著

阿帕替尼与白蛋白结合型紫杉醇在MDA- MB-231乳腺癌细胞系中的协同抗癌作用
李静1,(), 王志芬1, 张晓慧2, 杨庚武1, 刘峥1, 牛广旭3   
  1. 1. 056000 邯郸,河北邯郸市中心医院肿瘤三科,
    2. 056000 邯郸,河北邯郸市中心医院血液内科,
    3. 056000 邯郸,河北邯郸市中心医院病理科
  • 收稿日期:2019-09-08 出版日期:2021-04-01
  • 通信作者: 李静

Synergistic anticancer effects of apatinib and nab-paclitaxel in MDA-MB-231 breast cancer cell line

Jing Li1,(), Zhifen Wang1, Xiaohui Zhang2, Gengwu Yang1, Zheng Liu1, Guangxu Niu3   

  1. 1. Third Department of Oncology, Handan Central Hospital, Handan 056000, China
    2. Department of Hematology, Handan Central Hospital, Handan 056000, China
    3. Department of Pathology, Handan Central Hospital, Handan 056000, China
  • Received:2019-09-08 Published:2021-04-01
  • Corresponding author: Jing Li
引用本文:

李静, 王志芬, 张晓慧, 杨庚武, 刘峥, 牛广旭. 阿帕替尼与白蛋白结合型紫杉醇在MDA- MB-231乳腺癌细胞系中的协同抗癌作用[J/OL]. 中华细胞与干细胞杂志(电子版), 2021, 11(02): 90-98.

Jing Li, Zhifen Wang, Xiaohui Zhang, Gengwu Yang, Zheng Liu, Guangxu Niu. Synergistic anticancer effects of apatinib and nab-paclitaxel in MDA-MB-231 breast cancer cell line[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(02): 90-98.

目的

阐明阿帕替尼(apatinib)和白蛋白结合型紫杉醇(nab-Paclitaxel)诱导MDA-MB-231乳腺癌细胞凋亡的分子机制。

方法

本研究以MDA-MB-231乳腺癌细胞为研究对象,并以apatinib和nab-Paclitaxel处理细胞后分组:0.1 ﹪DMSO处理为阴性对照组;10 μmol/L apatinib处理组(APA组);经5、10、15、20 nmol/L nab-Paclitaxel处理组(Nab-p 5组、Nab-p 10组、Nab-p 15组和Nab-p 20组);以及10 μmol/L apatinib分别与5、10、15、20 nmol/L nab-Paclitaxel联合处理组(APA+Nab-p 5组、APA+Nab-p 10组、APA+Nab-p 15组和APA+Nab-p 20组)。使用乳酸脱氢酶释放测定法测定apatinib和nab-Paclitaxel对MDA-MB-231细胞诱导的细胞毒活性,结合流式细胞术分析不同处理组细胞凋亡情况,通过JC-1染色法测定不同干预方式对MDA-MB-231细胞线粒体膜电位变化(ΔΨm)的影响,借助FAM-FLICA荧光成像检测caspase-8和caspase-9活性,通过彗星试验评估apatinib和nab-Paclitaxel对非致瘤上皮细胞株MCF-10A细胞DNA损伤的影响。两组之间独立样本t检验或单向ANOVA进行比较,多组之间使用Tukey事后检验。

结果

细胞毒性检测结果显示,与阴性对照组相比,Nab-p 20组MDA-MB-231细胞杀伤率在24 h时接近90 ﹪;与单药处理组(Nab-p 5组和Nab-p 10组)相比,APA+Nab-p 5组和APA+Nab-p 10组联合处理24 h和48 h后,分别检测到约85 ﹪和95 ﹪细胞死亡,差异均具有统计学意义(P均< 0.001)。流式细胞术统计结果显示,与Nab-p5组和Nab-p10组相比,APA+Nab-p 5组和APA+Nab-p 10组24 h时MDA-MB-231细胞凋亡率(31.8 ﹪ ± 1.48 ﹪、33.25 ﹪±1.77 ﹪比76.11 ﹪±1.14 ﹪、89.4 ﹪± 1.07﹪)升高(P均< 0.05)。线粒体膜电位检测结果表明,与对照组相比,在单药处理组中,仅APA组和Nab-p 10组24 h时去极化细胞(12.35 ﹪ ± 1.05﹪比78.33﹪± 1.11﹪、46.74﹪± 1.75﹪)增多;在联用处理组中,APA+Nab-p 5组和APA+Nab-p 10组24 h时去极化细胞(68.47﹪± 1.94﹪比90.03﹪± 1.79﹪)增多,差异均有统计学意义(P均< 0.05)。FAM-FLICA荧光成像结果显示,相较于单一处理组,apatinib和nab-Paclitaxel联用处理组的caspase-8和caspase-9蛋白高度活化。结合彗星试验分析,apatinib和nab-Paclitaxel干预对MCF-10A非致瘤上皮细胞株DNA完整性没有显著影响。

结论

apatinib/nab-Paclitaxel联用通过内源性的线粒体功能扰动和外源性的caspase激活诱导三阴性乳腺癌细胞MDA-MB-231凋亡,发挥协同抗癌作用。

Objective

To elucidate the molecular mechanisms of apatinib and nab-Paclitaxel in inducing apoptosis of MDA-MB-231 breast cancer cells.

Methods

MDA-MB-231 cells were treated with apatinib and nab-Paclitaxel alone or together and named: control group (0.1 ﹪ DMSO treatment) ; 10 μmol/L apatinib treatment group (APA group) ; 5, 10, 15, 20 nmol/L nab-Paclitaxel treatment group (Nab-p 5 groups, Nab-p 10 groups, Nab-p 15 groups and Nab-p 20 groups) and 10 μmol/L apatinib combined with 5, 10, 15, 20 nmol/L nab-Paclitaxel treatment group (APA Nab-p 5 groups, APA Nab-p 10 groups, APA Nab-p 15 groups and APA Nab-p 20 groups) . The cytotoxic activity induced by apatinib and albumin-bound paclitaxel on MDA-MB-231 cells was determined by a lactate dehydrogenase release assay. Flow cytometry was used to quantify apoptosis in different treatment groups. Effects of different intervention methods on mitochondrial membrane potential change (ΔΨm) of MDA-MB-231 cells were examined by JC-1 staining. Caspase-8 and Caspase-9 activities were detected by FAM-FLICA fluorescence imaging. The effects of apatinib and albumin-bound paclitaxel on DNA damage in non-tumorigenic epithelial cell line MCF-10A were evaluated by comet assay. Student's t test or one-way ANOVA was used for comparison between two groups, and Tukey post-test was used for comparison within multiple groups.

Results

Cytotoxicity test results showed that the MDA-MB-231 cell death rate in the Nab-p 20 group was close to 90 ﹪ at 24 h, and the difference was extremely significant compared with the control group (P < 0.001) . After treatment for 24 h and 48 h, about 85 ﹪ and 95 ﹪ of cell deaths were detected in the APA+Nab-p 5 group and APA+Nab-p 10 group respectively, which showed a significantly difference compared with the single-drug treatment group (Nab-p 5 group and Nab-p 10 group) (P < 0.001) . The results of flow cytometry showed that the apoptotic rate of APA+Nab-p5 (76.11±1.14) and APA+Nab-p10 (89.4±1.07) groups were significantly increased (P < 0.05) compared with the Nab-p5 (31.8±1.48) and Nab-p10 (33.25±1.77) groups. The results of mitochondrial membrane potential test showed that only depolarized cells in APA group (78.33±1.11) and Nab-p 10 group (46.74±1.75) was increased significantly at 24 h (all P < 0.01) compared with the control group (12.35±1.05) , in the single-drug treatment group, while in the combined treatment group, the depolarised cells in the APA+Nab-p 5 (68.47±1.94) and APA+Nab-p 10 group (90.03±1.79) also increased significantly (P < 0.05) . FAM-FLICA fluorescence imaging results showed that compared with the single treatment group, the caspase-8 and caspase-9 proteins in the combination group of apatinib and nab-Paclitaxel were highly activated. Comet assay analysis showed that the intervention of apatinib and nab-Paclitaxel did not significantly affect the DNA integrity of MCF-10A non-tumorigenic epithelial cell line.

Conclusion

Combined apatinib with nab-paclitaxel induces apoptosis through intrinsic mitochondrial function perturbation and extrinsic caspase activation in triple-negative breast cancer cells MDA-MB-231, and may exhibit a synergistic anticancer effect.

表1 apatinib和nab-Paclitaxel诱导MDA-MB-231细胞杀伤率比较(﹪,±s
表2 流式细胞术检测统计结果(﹪,±s
图1 不同干预组MDA-MB-231 24 h处理的典型流式细胞术结果
表3 线粒体膜去极化检测结果比较(﹪,±s
图2 不同干预组MDA-MB-231细胞24 h处理典型流式细胞术结果
图3 倒置荧光显微镜观察MDA-MB-231细胞中caspase-8和caspase-9的激活情况(×100)
表4 apatinib和nab-Paclitaxel诱导MCF-10A细胞杀伤率比较(﹪,±s
图4 倒置荧光显微镜观察MCF-10A细胞DNA损伤情况(×200)
1
Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells[J]. Proc Natl Acad Sci U S A, 1980, 77(3):1561-1565.
2
于海涛. 抗癌药物-紫杉醇合成和应用研究进展[J]. 黑龙江科技信息, 2017(5):129-129.
3
Verweij J, Clavel M, Chevalier B. Paclitaxel (taxoltm) and docetaxel (taxoteretm): Not simply two of a kind[J]. Ann Oncol, 1994, 5(6):495-505.
4
潘力敏. 白蛋白结合型紫杉醇与多西他赛在HER-2阴性早期乳腺癌新辅助治疗中的临床对比[D]. 郑州:郑州大学, 2019.
5
Desai N, Trieu V, Yao Z, et al. Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin-bound paclitaxel, abi-007, compared with cremophor-based paclitaxel[J]. Clin Cancer Res, 2006, 12(4):1317-24.
6
Desai NP, Trieu V, Hwang LY et al. Improved effectiveness of nanoparticle albumin-bound (nab) paclitaxel versus polysorbate-based docetaxel in multiple xenografts as a function of her2 and sparc status[J]. Anticancer Drugs, 2008, 19(9):899-909.
7
Gradishar WJ, Tjulandin S, Davidson N, et al. Phase iii trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer[J]. J Clin Oncol, 2005, 23(31):7794-7803.
8
杨芳. 阿帕替尼在晚期转移性三阴性乳腺癌中的临床疗效及预后分析[D].开封:河南大学, 2019.
9
王明婷. 贝伐珠单抗联合阿帕替尼对肺癌抗肿瘤作用实验研究[D]. 泸州:西南医科大学, 2020.
10
董世奇,张爱杰,樊慧蓉. 阿帕替尼临床应用的研究进展[J]. 药物评价研究, 2020, 43(07):1446-1451.
11
Zhang H. Apatinib for molecular targeted therapy in tumor[J]. Drug Des Devel Ther, 2015, 9:6075-6081.
12
张香梅,何明,陈新, 等. 阿帕替尼在进展期恶性肿瘤治疗中的研究进展[J]. 广东医学, 2017, 38(S2):168-170+173.
13
李蓉. 阿帕替尼联合白蛋白紫杉醇在耐药性卵巢癌中的作用[D]. 太原:山西医科大学, 2020.
14
Kundranda MN, Niu J. Albumin-bound paclitaxel in solid tumors: Clinical development and future directions[J]. Drug Des Devel Ther, 2015, 9:3767-3777.
15
Zong Y, Wu J, Shen K. Nanoparticle albumin-bound paclitaxel as neoadjuvant chemotherapy of breast cancer: A systematic review and meta-analysis[J]. Oncotarget, 2017, 8(10):17360-17372.
16
Park CH, Han SE, Nam-Goong IS, et al. Combined effects of baicalein and docetaxel on apoptosis in 8505c anaplastic thyroid cancer cells via downregulation of the erk and akt/mtor pathways[J]. Endocrinol Metab (Seoul), 2018, 33(1):121-132.
17
Speyer CL, Bukhsh MA, Jafry WS, et al. Riluzole synergizes with paclitaxel to inhibit cell growth and induce apoptosis in triple-negative breast cancer[J]. Breast Cancer Res Treat, 2017, 166(2):407-419.
18
Ireland L, Santos A, Campbell F, et al. Blockade of insulin-like growth factors increases efficacy of paclitaxel in metastatic breast cancer[J]. Oncogene, 2018, 37(15):2022-2036.
19
Johnson LV, Walsh ML, Bockus BJ, et al. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy[J]. J Cell Biol, 1981, 88(3):526-535.
20
Elmore S. Apoptosis: A review of programmed cell death[J]. Toxicol Pathol, 2007, 35(4):495-516.
21
Debatin KM, Poncet D, Kroemer G. Chemotherapy: Targeting the mitochondrial cell death pathway[J]. Oncogene, 2002, 21(57):8786-8803.
22
Heiskanen KM, Bhat MB, Wang HW, et al. Mitochondrial depolarization accompanies cytochrome c release during apoptosis in pc6 cells[J]. J Biol Chem, 1999, 274(9):5654-5658.
23
Salvesen GS, Dixit VM. Caspases: Intracellular signaling by proteolysis[J]. Cell, 1997, 91(4):443-446.
24
Rossi D, Gaidano G. Messengers of cell death: Apoptotic signaling in health and disease[J]. Haematologica, 2003, 88(2):212-218.
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