Sunitinib Synergizes with Cisplatin by Suppressing DNA Repair Pathways in High-Grade Serous Ovarian Carcinoma Cells

Abstract Background: High-grade serous ovarian carcinoma (HGSOC) frequently develops resistance to platinum-based chemotherapy such as cisplatin, leading to high mortality. Sunitinib, a multi-target Receptor tyrosine kinase inhibitor, has shown potential in the treatment of ovarian carcinoma (OC). However, the synergistic effects between cisplatin and sunitinib, and the sensitization mechanism of sunitinib in HGSOC are not yet understood. Methods: The anti-tumor effects of sunitinib on HGSOC cells were assessed using CCK-8, EdU, colony formation, wound healing, Transwell, and flow cytometry assays. RNA sequencing was performed on sunitinib-treated cells, followed by differential expression, enrichment, and protein-protein interaction network (PPI) analyses. Genes on cisplatin sensitivity were predicted using the CellMiner and GEPIA3 databases. Synergy between sunitinib and cisplatin was evaluated using SynergyFinder 3.0, and DNA damage was assessed by ?-H2AX expression. Results: Sunitinib significantly inhibited proliferation, migration, and invasion in HGSOC cells, while further inducing cell cycle arrest, promoting necrosis. Besides, sunitinib downregulated critical DNA damage repair pathways, including homologous recombination, fanconi anemia, and base excision repair. Furthermore, sunitinib synergizes with cisplatin in HGSOC cells, enhancing DNA damage compared to monotherapy. Additionally, we screened out 25 sunitinib-downregulated core genes (SDCs). Drug-sensitivity analyses showed that higher SDCs expression was significantly associated with cisplatin resistance in OC. Notably, in cisplatin-resistant HEY-A8/DDP cells, sunitinib displayed stronger cytotoxicity than cisplatin. Conclusion: Sunitinib induces cell-cycle arrest and necrosis. In addition, sunitinib synergized with cisplatin and enhanced cisplatin sensitivity by impairing DNA repair pathways. Drug-sensitivity analyses showed that SDCs are associated with cisplatin resistance in OC, suggesting that sunitinib may help overcome cisplatin chemoresistance. Notably, sunitinib retains substantial cytotoxic activity in cisplatin-resistant cells. Together, these findings suggest that the sunitinib–cisplatin combination is a promising strategy to overcome cisplatin resistance in HGSOC. Overall design: In this study, HEYA8 cells were treated with either sunitinib (treatment group) or an equivalent volume of DMSO (control group), with three biological replicates established for each condition. Total RNA was extracted from these samples and subjected to RNA sequencing. Differential expression analysis was subsequently performed to compare the transcriptomic profiles, thereby evaluating the impact of sunitinib on HEYA8 cells and high-grade serous ovarian cancer (HGSOC). Additionally, we included RNA-seq data from HEYA8 cells treated with Compound 32 for 24 hours to evaluate its regulatory effects on the HGSOC transcriptome. Total RNA was extracted from these samples and subjected to RNA sequencing. Differential expression analysis was subsequently performed to compare the transcriptomic profiles, thereby assessing the impact of both sunitinib and Compound 32 on HGSOC.

研究背景：高级别浆液性卵巢癌（High-grade serous ovarian carcinoma, HGSOC）常对顺铂等铂类化疗药物产生耐药性，导致患者死亡率居高不下。舒尼替尼（Sunitinib）作为一种多靶点受体酪氨酸激酶抑制剂，在卵巢癌（Ovarian carcinoma, OC）治疗中展现出应用潜力。然而，顺铂与舒尼替尼的协同作用，以及舒尼替尼在HGSOC中的增敏机制尚未明确。
研究方法：本研究采用CCK-8实验、EdU实验、集落形成实验、划痕愈合实验、Transwell实验及流式细胞术，评估舒尼替尼对HGSOC细胞的抗肿瘤活性。对经舒尼替尼处理的细胞进行RNA测序，随后开展差异表达分析、富集分析及蛋白质相互作用网络（Protein-protein interaction network, PPI）分析。通过CellMiner与GEPIA3数据库预测与顺铂敏感性相关的基因。采用SynergyFinder 3.0评估舒尼替尼与顺铂的协同作用，并通过γ-H2AX的表达水平检测DNA损伤程度。
研究结果：舒尼替尼可显著抑制HGSOC细胞的增殖、迁移与侵袭能力，同时进一步诱导细胞周期阻滞，促进细胞坏死。此外，舒尼替尼可下调关键的DNA损伤修复通路，包括同源重组（homologous recombination）、范可尼贫血通路及碱基切除修复通路。进一步研究发现，舒尼替尼与顺铂在HGSOC细胞中具有协同作用，相较于单一用药可加重DNA损伤。本研究共筛选出25个舒尼替尼下调核心基因（Sunitinib-downregulated core genes, SDCs）。药物敏感性分析显示，SDCs的高表达与OC患者的顺铂耐药性显著相关。值得注意的是，在顺铂耐药的HEY-A8/DDP细胞中，舒尼替尼的细胞毒性作用强于顺铂。
研究结论：舒尼替尼可诱导细胞周期阻滞与细胞坏死。此外，舒尼替尼可通过损伤DNA修复通路增强顺铂的敏感性，二者具有协同抗肿瘤作用。药物敏感性分析结果表明，SDCs与OC患者的顺铂耐药性相关，提示舒尼替尼有望克服顺铂化疗耐药。值得注意的是，舒尼替尼在顺铂耐药细胞中仍保留较强的细胞毒性活性。综上，本研究结果表明，舒尼替尼与顺铂联合用药有望解决HGSOC的顺铂耐药问题。
整体实验设计：本研究将HEYA8细胞分为两组，分别用舒尼替尼（处理组）或等体积二甲基亚砜（Dimethyl sulfoxide, DMSO，对照组）处理，每组设置3个生物学重复。提取各组样本的总RNA并进行RNA测序，随后开展差异表达分析以比较两组的转录组特征，以此评估舒尼替尼对HEYA8细胞及HGSOC的影响。此外，本研究还纳入了经Compound 32处理24小时的HEYA8细胞的RNA测序数据，以评估Compound 32对HGSOC转录组的调控作用。同样提取该组样本的总RNA并进行RNA测序，随后开展差异表达分析以比较转录组特征，进而评估舒尼替尼与Compound 32对HGSOC的共同影响。