背景:奥希替尼(AZD9291)是第三代表皮生长因子受体(EGFR)酪氨酸激酶抑制剂,在EGFR致敏突变或EGFRT790M突变患者中显示出显著的临床获益。同源重组(HR)途径对于修复DNA双链断裂(DSB)至关重要。Rad51在人力资源中起着核心作用,促进同源性搜索和促进同源DNA分子之间的DNA链交换。Rad51在许多类型的癌细胞中过表达。Rad51的特异性小分子抑制剂B02抑制Rad51的DNA链交换活性。先前的研究表明,B02破坏了Rad51病灶的形成,以响应DNA损伤,并抑制了人细胞中的DSBs修复,并使其在体外和体内对化疗药物敏感。然而,奥希替尼与Rad51抑制剂联合使用的潜在治疗效果尚不清楚.这项研究的目的是阐明Rad51表达和活性的下调是否可以增强奥希替尼诱导的非小细胞肺癌(NSCLC)细胞的细胞毒性。
方法:我们使用了MTS,锥虫蓝染料排除和集落形成能力测定以确定奥希替尼单独或与B02联合使用是否对NSCLC细胞系具有细胞毒性作用.进行实时PCR以测量Rad51mRNA的量。通过蛋白质印迹分析确定磷酸化AKT和Rad51的蛋白质水平。
结果:我们发现奥希替尼通过使AKT活性失活而降低Rad51表达。通过磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002或si-AKTRNA转染使用siRNA或AKT灭活的Rad51敲除增强了奥希替尼的细胞毒性和生长抑制作用。相比之下,AKT-CA(AKT的组成型活性形式)载体增强表达可以减轻奥希替尼的细胞毒性和细胞生长抑制作用。此外,B02显著增强奥希替尼在NSCLC细胞中的细胞毒性和细胞生长抑制作用。与亲代细胞相比,奥希替尼治疗不能显著抑制奥希替尼耐药细胞中AKT活化和Rad51表达.此外,Rad51表达增加与奥希替尼耐药H1975和A549细胞的耐药机制相关.
结论:总的来说,Rad51表达和活性的下调增强了奥希替尼在人NSCLC细胞中的细胞毒作用.
BACKGROUND: Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has shown significant clinical benefits in patients with EGFR-sensitizing mutations or the EGFR T790M mutation. The homologous recombination (HR) pathway is crucial for repairing DNA double-strand breaks (DSBs). Rad51 plays a central role in HR, facilitating the search for homology and promoting DNA strand exchange between homologous DNA molecules.
Rad51 is overexpressed in numerous types of cancer cells. B02, a specific small molecule inhibitor of
Rad51, inhibits the DNA strand exchange activity of
Rad51. Previous studies have indicated that B02 disrupted
Rad51 foci formation in response to DNA damage and inhibited DSBs repair in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. However, the potential therapeutic effects of combining osimertinib with a Rad51 inhibitor are not well understood. The aim of this study was to elucidate whether the downregulation of Rad51 expression and activity can enhance the osimertinib-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells.
METHODS: We used the MTS, trypan blue dye exclusion and colony-formation ability assay to determine whether osimertinib alone or in combination with B02 had cytotoxic effects on NSCLC cell lines. Real-time polymerase chain reaction was conducted to measure the amounts of
Rad51 mRNA. The protein levels of phosphorylated AKT and
Rad51 were determined by Western blot analysis.
RESULTS: We found that osimertinib reduced Rad51 expression by inactivating AKT activity. Rad51 knockdown using small interfering RNA or AKT inactivation through the phosphatidylinositol 3-kinase inhibitor LY294002 or si-AKT RNA transfection enhanced the cytotoxic and growth inhibitory effects of osimertinib. In contrast, AKT-CA (a constitutively active form of AKT) vector-enforced expression could mitigate the cytotoxic and cell growth inhibitory effects of osimertinib. Furthermore, B02 significantly enhanced the cytotoxic and cell growth inhibitory effects of osimertinib in NSCLC cells. Compared to parental cells, the activation of AKT and Rad51 expression in osimertinib-resistant cells could not be significantly inhibited by osimertinib treatment. Moreover, the increased expression of Rad51 is associated with the resistance mechanism in osimertinib-resistant H1975 and A549 cells.
CONCLUSIONS: Collectively, the downregulation of Rad51 expression and activity enhances the cytotoxic effect of osimertinib in human NSCLC cells.