Aims

Click Chemistry is providing valuable tools to biomedical research, but its direct use in therapies remains nearly unexplored. For cancer treatment, nucleoside analogues (NA) such as 5-vinyl-2′-deoxyuridine (VdU) can be metabolically incorporated into cancer cell DNA and subsequently “clicked” to form a toxic product. The inverse electron-demand Diels-Alder (IEDDA) reaction between VdU and an acridine-tetrazine conjugate (PINK) has previously been used to label cell nuclei of cultured cells. Here, we report tandem usage of VdU and PINK to induce cytotoxicity.

Main methods

Cell lines were subsequently treated with VdU and PINK, and cell viability was measured via well confluency and 3D tumor spheroid assays. DNA damage and apoptosis were evaluated using Western Blotting and cell cycle analysis by flow cytometry. Double stranded DNA break (DSB) formation was measured using the comet assay. Apoptosis was assessed by fluorescent detection of externalized phosphatidylserine residues.

Key findings

We report that the combination of VdU and PINK synergistically induces cytotoxicity in cultured human cells. The combination of VdU and PINK strongly reduced cell viability in 2D and 3D cultured cancer cells. Mechanistically, the compounds induced DNA damage through DSB formation, which leads to S-phase accumulation and apoptosis.

Significance

The combination of VdU and PINK represents a novel and promising DNA-templated “click” approach for cancer treatment via selective induction of DNA damage.

中文翻译：

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DNA 模板点击化学通过 5-乙烯基-2′-脱氧尿苷和吖啶-四嗪缀合物诱导癌细胞 DNA 损伤和细胞凋亡

目标

点击化学为生物医学研究提供了有价值的工具，但其在治疗中的直接应用几乎尚未被探索。对于癌症治疗，核苷类似物 (NA) 如 5-乙烯基-2'-脱氧尿苷 (VdU) 可以通过代谢方式掺入癌细胞 DNA 中，随后“点击”形成有毒产物。VdU 和吖啶-四嗪缀合物 (PINK) 之间的逆电子需求狄尔斯-阿尔德 (IEDDA) 反应先前已用于标记培养细胞的细胞核。在这里，我们报告了 VdU 和 PINK 的串联使用来诱导细胞毒性。

主要方法

随后用 VdU 和 PINK 处理细胞系，并通过孔汇合和 3D 肿瘤球体测定来测量细胞活力。使用蛋白质印迹法和流式细胞术进行细胞周期分析来评估DNA损伤和细胞凋亡。使用彗星测定法测量双链 DNA 断裂 (DSB) 的形成。通过荧光检测外化磷脂酰丝氨酸残基来评估细胞凋亡。

主要发现

我们报告说，VdU 和 PINK 的组合可协同诱导培养的人类细胞的细胞毒性。VdU 和 PINK 的组合大大降低了 2D 和 3D 培养癌细胞的细胞活力。从机制上讲，这些化合物通过 DSB 形成诱导 DNA 损伤，从而导致 S 期积累和细胞凋亡。

意义

VdU 和 PINK 的组合代表了一种新颖且有前途的 DNA 模板“点击”方法，通过选择性诱导 DNA 损伤来治疗癌症。