PO.CL08.01 · 临床研究
抑制PLK4通过基因组不稳定性和凋亡性细胞死亡克服局部晚期直肠癌的放射抵抗
Inhibition of PLK4 overcomes radioresistance in locally advanced rectal cancer through genomic instability and apoptotic cell death
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：Polo样激酶4（PLK4）是中心体复制的关键调节因子，已被证实与癌症进展和治疗抵抗相关。尽管在多种恶性肿瘤中经常观察到PLK4过表达，但其对结直肠癌（CRC）放疗（RT）反应的功能性作用仍知之甚少。
方法：为研究PLK4抑制是否增强放射敏感性，将CRC细胞系HCT116（p53野生型）和HT29（p53突变型）用选择性PLK4抑制剂CFI-400945、RT或二者联合处理。进行细胞活力和克隆形成实验以评估细胞毒性。通过gammaH2AX免疫荧光以及对p-ATM、p-CHK2和DNA-PKcs的蛋白质印迹分析评估DNA损伤应答。分别通过流式细胞术和gamma-tubulin染色检测细胞周期分布和中心体异常。通过cleaved PARP-1和caspase-3表达水平测定凋亡。
结果：CFI-400945处理以剂量和时间依赖的方式降低细胞活力，并显著增强了RT在两株CRC细胞系中的细胞毒性作用。联合治疗显著增加gammaH2AX病灶形成并上调DNA损伤应答蛋白，包括p-ATM、p-CHK2和DNA-PKcs。PLK4抑制还诱导中心体扩增、微核形成和多核化，提示导致基因组不稳定性的有丝分裂缺陷。此外，联合治疗增强了G2/M阻滞和凋亡，表现为cleaved PARP-1和caspase-3水平升高。
结论：PLK4抑制通过促进DNA损伤累积、有丝分裂灾变和凋亡性细胞死亡，使结直肠癌细胞对放疗敏感。这些发现提示，靶向PLK4可能成为克服CRC放射抵抗的一种潜在治疗策略。
查看英文原文 English abstract
Background: Polo-like kinase 4 (PLK4), a key regulator of centrosome duplication, has been implicated in cancer progression and therapeutic resistance. Although PLK4 overexpression is frequently observed in various malignancies, its functional contribution to radiotherapy (RT) response in colorectal cancer (CRC) remains poorly understood.
Methods: To investigate whether PLK4 inhibition enhances radiosensitivity, CRC cell lines HCT116 (p53 wild-type) and HT29 (p53 mutant) were treated with the selective PLK4 inhibitor CFI-400945, RT or their combination. Cell viability and clonogenic assays were performed to evaluate cytotoxicity. DNA damage responses were assessed by gammaH2AX immunofluorescence and Western blot analysis of p-ATM, p-CHK2, and DNA-PKcs. Cell-cycle profiles and centrosomal abnormalities were examined by flow cytometry and gamma-tubulin staining, respectively. Apoptosis was determined through cleaved PARP-1 and caspase-3 expression levels.
Results: CFI-400945 treatment reduced cell viability in a dose- and time-dependent manner and significantly enhanced the cytotoxic effects of RT in both CRC cell lines. Combined treatment markedly increased gammaH2AX foci formation and upregulated DNA damage response proteins, including p-ATM, p-CHK2, and DNA-PKcs. PLK4 inhibition also induced centrosome amplification, micronuclei formation, and multinucleation, indicating mitotic defects leading to genomic instability. Furthermore, the combination therapy enhanced G2/M arrest and apoptosis, as evidenced by elevated levels of cleaved PARP-1 and caspase-3.
Conclusions: PLK4 inhibition sensitizes colorectal cancer cells to radiation by promoting DNA damage accumulation, mitotic catastrophe, and apoptotic cell death. These findings suggest that targeting PLK4 may serve as a potential therapeutic strategy to overcome radioresistance in CRC.
利益披露 Disclosure
S. Bae, None..
J. Hwang, None.