Lin Shengbin, Sun Binghe, Zhu Yin, Huang Yi, Qin Yu, Yao Nan, Liu Yongzhu, Chen Guo
School of Biopharmacy, China Pharmaceutical University, Nanjing, P.R. China.
Department of Clinical Laboratory, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, China.
J Pharm Pharmacol. 2025 Apr 3;77(4):511-523. doi: 10.1093/jpp/rgaf006.
As a strategy in the development of effective cancer therapeutics, synthetic lethality has been used in clinical practice. Poly adenosine diphosphate (ADP)-ribose polymerase inhibitors are the first approved drug utilized synthetic lethality and achieved promising therapeutic efficacy in cancer cells with BRCA1/2 mutation. Nonetheless, most cancer patients with wild-type BRCA1/2 gene are not qualified for PARPi therapy. To induce BRCAness phenotype in cancer cells with normal BRCA1/2 status, we identified Pulsatilla Saponin D (SB365), which efficiently inhibited recruitment of BRCA1 at DNA double-strand breaks, leading to homologous recombination repair deficiency.
We utilized the HR repair reporter system. The reporter cells were treated with a natural compounds library to identify the agent that significantly decreased HR activity. Then, we detected the expression of HR related proteins using immunofluorescence and western blot. Colony formation and CCK8 was used to detect the inhibitory effect of Pulsatilla Saponin D on cell proliferation. Apoptosis was measured using Annexin V/PI staining. Comet assay kits were used to carry out the comet assay. Ovarian cancer xenograft model, immunohistochemical staining and Hematoxylin-Eosin staining was used to detect the antitumor efficacy and toxicity of Pulsatilla Saponin D.
Pulsatilla Saponin D greatly increased PARPi-induced DNA DSBs, growth inhibition and apoptosis in ovarian cancer cells. Combined administration of PARPi and Pulsatilla Saponin D induced synergistic anti-tumor effects in ovarian cancer cells and xenograft mouse model without obvious toxicity.
In summary, our study found Pulsatilla Saponin D is a novel HR repair inhibitor and would optimize clinical application of PARP inhibitors on cancer patients with WT BRCA1/2.
作为开发有效癌症治疗方法的一种策略，合成致死性已应用于临床实践。聚腺苷二磷酸（ADP）-核糖聚合酶抑制剂是首个获批的利用合成致死性的药物，在具有BRCA1/2突变的癌细胞中取得了有前景的治疗效果。然而，大多数野生型BRCA1/2基因的癌症患者不符合PARPi治疗条件。为了在BRCA1/2状态正常的癌细胞中诱导BRCA缺陷表型，我们鉴定出白头翁皂苷D（SB365），它能有效抑制BRCA1在DNA双链断裂处的募集，导致同源重组修复缺陷。
我们利用了HR修复报告系统。用天然化合物文库处理报告细胞，以鉴定能显著降低HR活性的试剂。然后，我们通过免疫荧光和蛋白质印迹检测HR相关蛋白的表达。使用集落形成和CCK8检测白头翁皂苷D对细胞增殖的抑制作用。使用Annexin V/PI染色测量细胞凋亡。使用彗星试验试剂盒进行彗星试验。利用卵巢癌异种移植模型、免疫组织化学染色和苏木精-伊红染色检测白头翁皂苷D的抗肿瘤疗效和毒性。
白头翁皂苷D显著增加PARPi诱导的卵巢癌细胞中的DNA双链断裂、生长抑制和细胞凋亡。PARPi与白头翁皂苷D联合给药在卵巢癌细胞和异种移植小鼠模型中诱导了协同抗肿瘤作用，且无明显毒性。
总之，我们的研究发现白头翁皂苷D是一种新型的HR修复抑制剂，将优化PARP抑制剂在野生型BRCA1/2癌症患者中的临床应用。
