METHODS: Prolonged exposure to gefitinib resulted in the emergence of a resistant lung cancer cell line (PC9GR) from the parental sensitive cell line (PC9), which exhibited a traditional EGFR mutation. The CCK-8 assay was employed to assess the impact of various concentrations of pitavastatin on cellular proliferation. RNA sequencing was conducted to detect differentially expressed genes and their correlated pathways. For the detection of protein expression, Western blot was performed. The antitumor activity of pitavastatin was evaluated in vivo via a xenograft mouse model.
RESULTS: PC9 gefitinib resistant strains were induced by low-dose maintenance. Cell culture and animal-related studies validated that the application of pitavastatin inhibited the proliferation of lung cancer cells, promoted cell apoptosis, and restrained the acquired resistance to EGFR-TKIs. KEGG pathway analysis showed that the hippo/YAP signaling pathway was activated in PC9GR cells relative to PC9 cells, and the YAP expression was inhibited by pitavastatin administration. With YAP RNA interference, pAKT, pBAD and BCL-2 expression was decreased, while BAX expression as increased. Accordingly, YAP down-regulated significantly increased apoptosis and decreased the survival rate of gefitinib-resistant lung cancer cells. After pAKT was increased by SC79, apoptosis of YAP down-regulated cells induced by gefitinib was decreased, and the cell survival rate was increased. Mechanistically, these effects of pitavastatin are associated with the YAP pathway, thereby inhibiting the downstream AKT/BAD-BCL-2 signaling pathway.
CONCLUSIONS: Our study provides a molecular basis for the clinical application of the lipid-lowering drug pitavastatin enhances the susceptibility of lung cancer to EGFR-TKI drugs and alleviates drug resistance.
方法:长时间接触吉非替尼导致从亲本敏感细胞系(PC9)中出现耐药肺癌细胞系(PC9GR),表现出传统的EGFR突变。CCK-8测定用于评估各种浓度的匹伐他汀对细胞增殖的影响。进行RNA测序以检测差异表达的基因及其相关途径。为了检测蛋白质的表达,进行蛋白质印迹。通过异种移植小鼠模型在体内评估匹伐他汀的抗肿瘤活性。
结果:低剂量维持诱导PC9吉非替尼耐药菌株。细胞培养和动物相关研究验证了匹伐他汀对肺癌细胞增殖的抑制作用,促进细胞凋亡,抑制了EGFR-TKIs的获得性耐药。KEGG通路分析表明,相对于PC9细胞,在PC9GR细胞中hippo/YAP信号通路被激活,匹伐他汀对YAP的表达有抑制作用。用YAPRNA干扰,pAKT,pBAD和BCL-2表达降低,而BAX表达则增加。因此,YAP下调显著增加吉非替尼耐药肺癌细胞的凋亡并降低其存活率。SC79增加pAKT后,吉非替尼诱导的YAP下调细胞凋亡减少,细胞存活率提高。机械上,匹伐他汀的这些作用与YAP途径有关,从而抑制下游AKT/BAD-BCL-2信号通路。
结论:本研究为临床应用降脂药物匹伐他汀增强肺癌患者对EGFR-TKI药物的敏感性、缓解耐药提供了分子依据。