Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy.
Department of Health Science, Magna Græcia University, Catanzaro, Italy.
J Transl Med. 2022 Oct 22;20(1):482. doi: 10.1186/s12967-022-03705-z.
DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM.
Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells.
Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo.
Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
DNA 连接酶在 DNA 修复和细胞复制中至关重要，因为它们催化 DNA 断裂连接的最后步骤。DNA 连接酶 III（LIG3）在非同源末端连接修复（Alt-NHEJ）中发挥关键作用，这是一种易错的 DNA 修复途径，在基因组不稳定的癌症中经常上调，如多发性骨髓瘤（MM）。基于 LIG3 的三维（3D）结构，我们进行了计算筛选，以确定针对 LIG3 的天然化合物作为潜在候选物，以对抗 MM 中的 Alt-NHEJ 活性。
通过询问 Phenol Explorer 数据库进行虚拟筛选。通过饱和转移差异（STD）-核磁共振（NMR）实验验证与 LIG3 重组蛋白的结合。通过细胞毒性测定法分析细胞活力；通过 Annexin V-7AAD 染色后的流式细胞术分析评估细胞凋亡。通过质粒再连接测定法和 Cytoscan HD 评估 Alt-NHEJ 修复调节。通过全蛋白和分馏蛋白提取物的 Western blot 和免疫荧光分析分析 DNA 损伤反应蛋白水平。通过 qPCR 确定线粒体 DNA（mtDNA）拷贝数。在皮下植入 MM 细胞的 NOD-SCID 小鼠中评估体内活性。
在这里，我们提供了证据表明，一种通过计算方法选择的天然类黄酮 Rhamnetin（RHM）可拮抗 LIG3 活性并杀死依赖 Alt-NHEJ 的 MM 细胞。实际上，核磁共振（NMR）显示 RHM 与 LIG3 蛋白结合，功能实验表明 RHM 干扰 LIG3 驱动的核和线粒体 DNA 修复，导致体外和体内对 MM 的显著抗活性。
综上所述，我们的研究结果提供了概念验证，表明 RHM 靶向 MM 中的 LIG3 成瘾，因此可能代表一种新的有前途的天然抗肿瘤药物，可在早期临床环境中进行研究。
