Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences and Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona (UB), 08028 Barcelona, Spain.
Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences and Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona (UB), 08028 Barcelona, Spain.
Biochem Pharmacol. 2020 May;175:113911. doi: 10.1016/j.bcp.2020.113911. Epub 2020 Mar 13.
In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.
针对 DNA 损伤，细胞周期检查点会产生细胞周期停滞，以修复和维持基因组完整性。由于复制率高和遗传异常，癌细胞依赖于复制应激反应 (RSR)，并且该途径的抑制剂正被作为一种抗癌方法进行研究。在这个方向上，我们使用多嘧啶反向 Hoogsteen 发夹 (PPRHs) 作为基因沉默工具，研究了抑制 RSR 的关键组成部分 CHK1 和 WEE1。针对 WEE1 或 CHK1 设计的 PPRHs 降低了不同癌细胞系的活力，并在 HeLa 细胞中显示出凋亡增加。PPRHs 对 HeLa 细胞活力的影响具有剂量和时间依赖性。在用 PPRHs 处理后，每种基因的 mRNA 和蛋白水平都降低。在分析两种 CHK1 mRNA 剪接变体 CHK1 和 CHK1-S 的水平时，两种形式呈比例下降，从而保持相同的表达比例。针对 WEE1 和 CHK1 的 PPRHs 也被证明在处理 15 小时后会破坏细胞周期。此外，PPRHs 与 DNA 损伤剂（如甲氨蝶呤或 5-氟尿嘧啶）联合使用时显示出协同效应，这些药物广泛用于临床实践。这项工作在体外验证了 PPRHs 作为针对 RSR 基因 WEE1 和 CHK1 的沉默工具的使用，并证实了抑制这些靶点作为单一药物治疗或与其他化疗药物联合用于癌症研究的潜力。
