2(nd) Medical Clinic, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Str. 22 81675 Munich, Germany.
2(nd) Medical Clinic, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Str. 22 81675 Munich, Germany.
Life Sci. 2023 Oct 1;330:122000. doi: 10.1016/j.lfs.2023.122000. Epub 2023 Aug 3.
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.
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.
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.
The combination of VdU and PINK represents a novel and promising DNA-templated "click" approach for cancer treatment via selective induction of DNA damage.
点击化学为生物医学研究提供了有价值的工具，但它在治疗中的直接应用几乎未被探索。对于癌症治疗，核苷类似物（NA），如 5-乙烯基-2'-脱氧尿苷（VdU），可以被代谢掺入癌细胞 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 Diels-Alder（IEDDA）反应以前曾用于标记培养细胞的细胞核。在这里，我们报告了 VdU 和 PINK 的串联使用以诱导细胞毒性。
随后用 VdU 和 PINK 处理细胞系，并通过细胞密度和 3D 肿瘤球体测定法测量细胞活力。使用 Western Blotting 和流式细胞术的细胞周期分析评估 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。通过荧光检测细胞外的磷脂酰丝氨酸残基来评估细胞凋亡。
我们报告 VdU 和 PINK 的组合协同诱导培养的人类细胞的细胞毒性。VdU 和 PINK 的组合强烈降低了 2D 和 3D 培养的癌细胞中的细胞活力。从机制上讲，这些化合物通过 DSB 的形成诱导 DNA 损伤，从而导致 S 期积累和细胞凋亡。
VdU 和 PINK 的组合代表了一种新颖且有前途的基于 DNA 模板的“点击”方法，可通过选择性诱导 DNA 损伤来治疗癌症。
