Apatinib preferentially inhibits PC9 gefitinib-resistant cancer cells by inducing cell cycle arrest and inhibiting VEGFR signaling pathway
Apatinib preferentially inhibits PC9 gefitinib-resistant cancer cells by inducing cell cycle arrest and inhibiting VEGFR signaling pathway
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DOI：
10.1186/s12935-019-0836-8
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发表时间：
2019-05-02
影响因子：
5.8
通讯作者：
Zhang, Xiao-Ju
中科院分区：
文献类型：
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作者：
Song, Yong-An;Ma, Ting;Zhang, Xiao-Ju
背景肺癌是世界上最常见和最致命的肿瘤之一。针对某些突变患者的靶向治疗，特别是通过使用靶向表皮生长因子受体（EGFR）的酪氨酸激酶抑制剂（TKI），为患者提供了显著的获益。然而，逐渐产生的对治疗的抗性成为临床实践中的主要挑战，并且需要治疗此类患者的替代方案。在此，我们报告，阿帕替尼，一种新的抗血管生成药物，有效地抑制获得的吉非替尼耐药的癌细胞，但对他们的父母敏感cells. MethodsGefitinib耐药肺癌细胞系（PC 9 GR）建立从其父母敏感株（PC 9）与传统的EGFR突变后，长期暴露于吉非替尼。采用不同浓度的阿帕替尼处理PC 9、PC 9 GR等两种肺癌细胞株，观察其抗生长作用。在阿帕替尼处理后对PC 9、PC 9 GR和两者进行RNA测序，以检测差异表达的基因和相关途径。Western blot检测细胞周期调控因子p57、p27、CDK 2、cyclin E2和pRb蛋白表达。异种移植小鼠模型被用来评估阿帕替尼在vivo.ResultsThe建立的PC 9 GR细胞的抗肿瘤活性有超过250倍增加耐吉非替尼比其敏感的亲本PC 9细胞（IC 50 5.31 10.455 M vs. 0.020 +/-0.003 M）。PC 9 GR抗性细胞获得了众所周知的T790 M突变。阿帕替尼对PC 9 GR细胞的生长抑制作用比对PC 9和其他两种肺癌细胞系A549和H460强得多（5倍）。这种抑制主要是通过将PC 9 GR细胞阻滞在G1期来实现的。RNA-seq显示，与PC 9细胞相比，PC 9 GR细胞中有多个变化的途径，阿帕替尼处理后，变化最大的途径是细胞周期和DNA复制，其中大部分基因活性受到抑制。因此，在PC 9 GR细胞中，阿帕替尼显著影响p57、CDK 2、细胞周期蛋白E2和pRb的蛋白表达。与PC 9建立的肿瘤相比，在小鼠模型中口服阿帕替尼可显著抑制PC 9 GR植入肿瘤的建立和生长。结论p id= Par 4的阿帕替尼对吉非替尼耐药的肺癌细胞具有较强的抗增殖和抗生长作用，但对吉非替尼敏感的细胞无明显影响。抗肿瘤作用主要是由于阿帕替尼诱导的细胞周期阻滞和VEGFR信号通路抑制。这些数据表明，阿帕替尼可能为EGFR-TKI治疗获得性耐药患者提供获益。
BackgroundLung cancer is one of the most common and deadly tumors around the world. Targeted therapy for patients with certain mutations, especially by use of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), has provided significant benefit to patients. However, gradually developed resistance to the therapy becomes a major challenge in clinical practice and an alternative to treat such patients is needed. Herein, we report that apatinib, a novel anti-angiogenic drug, effectively inhibits obtained gefitinib-resistant cancer cells but has no much effect on their parental sensitive cells.MethodsGefitinib-resistant lung cancer cell line (PC9GR) was established from its parental sensitive line (PC9) with a traditional EGFR mutation after long time exposure to gefitinib. Different concentrations of apatinib were used to treat PC9, PC9GR, and other two lung cancer cell lines for its anti-growth effects. RNA sequencing was performed on PC9, PC9GR, and both after apatinib treatment to detect differentially expressed genes and involved pathways. Protein expression of key cycle regulators p57, p27, CDK2, cyclin E2, and pRb was detected using Western blot. Xenograft mouse model was used to assess the anti-tumor activity of apatinib in vivo.ResultsThe established PC9GR cells had over 250-fold increased resistance to gefitinib than its sensitive parental PC9 cells (IC50 5.3110.455M vs. 0.020 +/- 0.003M). The PC9GR resistance cells obtained the well-known T790M mutation. Apatinib demonstrated much stronger (fivefold) growth inhibition on PC9GR cells than on PC9 and other two lung cancer cell lines, A549 and H460. This inhibition was mostly achieved through cell cycle arrest of PC9GR cells in G1 phase. RNA-seq revealed multiple changed pathways in PC9GR cells compared to the PC9 cells and after apatinib treatment the most changed pathways were cell cycle and DNA replication where most of gene activities were repressed. Consistently, protein expression of p57, CDK2, cyclin E2, and pRb was significantly impacted by apatinib in PC9GR cells. Oral intake of apatinib in mouse model significantly inhibited establishment and growth of PC9GR implanted tumors compared to PC9 established tumors. VEGFR2 phosphorylation in PC9GR tumors after apatinib treatment was significantly reduced along with micro-vessel formation.Conclusions p id=Par4 Apatinib demonstrated strong anti-proliferation and anti-growth effects on gefitinib resistant lung cancer cells but not its parental sensitive cells. The anti-tumor effect was mostly due to apatinib induced cell cycle arrest and VEGFR signaling pathway inhibition. These data suggested that apatinib may provide a benefit to patients with acquired resistance to EGFR-TKI treatment.