Department of Cardiothoracic Surgery, the 1st Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, China.
Eur Rev Med Pharmacol Sci. 2017 Dec;21(23):5370-5377. doi: 10.26355/eurrev_201712_13922.
To observe the reversal effect of apatinib on the resistance to cisplatin (DDP) of A549/cisplatin (A549/DDP) cells and its relevant mechanism.
A549/DDP cells were treated with the control method, apatinib alone, DDP alone and DDP combined with apatinib. The cell proliferation was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the cell clone formation assay. The cell apoptosis was detected by Hoechst 33258 staining and annexin V and propidium iodide (PI) double labeling. The changes in apoptotic proteins, multidrug resistance protein 1 (MDR1) and extracellular signal-regulated kinase (ERK) signaling pathway proteins in each group after treatment were detected by Western blotting.
MTT assay results showed that compared with A549 cells, A549/DDP cells had obvious resistance to DDP. MTT assay and cell clone formation assay revealed that the tumor inhibition rate of the sub-lethal dose of apatinib (10 μM) combined with DDP was higher than that of DDP alone. The apoptosis detection results indicated that the proportion of apoptotic cells in the apatinib (10 μM) combined with DDP group was significantly increased. Western blotting results revealed that compared with that in parental A549 cells, the expression level of MDR1 in A549/DDP cells was significantly increased, and the ERK signaling pathway was activated. In the apatinib combined with DDP group, the levels of cleaved caspase-3, cleaved caspase-9 and B-cell lymphoma-2 (Bcl-2)-associated X (BAX) proteins were significantly upregulated, while the level of Bcl-2 proteins was downregulated. Apatinib could inhibit the expression of MDR1 and the activity of the ERK signaling pathway in a dose-dependent manner.
Apatinib can restore the sensitivity of A549/DDP cells to DDP by down-regulating the expression level of MDR1 and inhibiting the activity of the ERK signaling pathway.
观察阿帕替尼对 A549/顺铂（A549/DDP）细胞顺铂耐药的逆转作用及其相关机制。
采用对照方法、阿帕替尼单药、顺铂单药和顺铂联合阿帕替尼处理 A549/DDP 细胞。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和细胞克隆形成实验检测细胞增殖。通过 Hoechst 33258 染色和 Annexin V 和碘化丙啶（PI）双标记检测细胞凋亡。通过 Western blot 检测各组处理后凋亡蛋白、多药耐药蛋白 1（MDR1）和细胞外信号调节激酶（ERK）信号通路蛋白的变化。
MTT 检测结果显示，与 A549 细胞相比，A549/DDP 细胞对顺铂具有明显的耐药性。MTT 检测和细胞克隆形成实验表明，亚致死剂量的阿帕替尼（10 μM）联合顺铂的肿瘤抑制率高于顺铂单药组。凋亡检测结果表明，阿帕替尼（10 μM）联合顺铂组中凋亡细胞的比例明显增加。Western blot 结果显示，与亲本 A549 细胞相比，A549/DDP 细胞中 MDR1 的表达水平明显升高，ERK 信号通路被激活。在阿帕替尼联合顺铂组中，裂解 caspase-3、裂解 caspase-9 和 B 细胞淋巴瘤-2（Bcl-2）相关 X（BAX）蛋白的水平显著上调，而 Bcl-2 蛋白的水平下调。阿帕替尼可以呈剂量依赖性抑制 MDR1 的表达和 ERK 信号通路的活性。
阿帕替尼可以通过下调 MDR1 的表达水平和抑制 ERK 信号通路的活性，恢复 A549/DDP 细胞对顺铂的敏感性。
