Erlotinib (ERL) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of pancreatic cancer (PC). However, the clinical efficacy of ERL is limited due to the activation of alternative pathways that bypass the EGFR signaling. Kaempferol (KAE), a natural flavonoid compound, has been reported to possess potent anti-tumor and anti-inflammatory properties, and in this study, we aimed at identifying the sensitization effect of KAE on ERL monotherapy in PC cells and mouse models. Briefly, the CCK-8, colony formation, and flow cytometry were used to assess the proliferation and apoptosis of two PC cell lines in response to a treatment combination of KAE and ERL. Additionally, the drug–disease targets and related anti-PC mechanisms of KAE and ERL were predicted with a network pharmacology method. The survival outcome for PC patients with EGFR differential expression was evaluated through survival analysis. The molecular docking technique predicted the affinity between KAE and EGFR. Moreover, western blot (WB) and immunohistochemistry (IHC) analyses were applied to verify the expression levels of related proteins. As a result, in vitro results showed that the combination of KAE and ERL significantly inhibited cell proliferation and promoted cell apoptosis compared to that with ERL alone. Network pharmacology results demonstrated that KAE sensitized PC to ERL treatment may likely be related to the PI3K/AKT signaling pathway and EGFR TKI resistance. Survival analysis illustrated that PC patients with high expression of EGFR had a relative lower survival rate. Molecular docking results further suggested that KAE had a high binding affinity of – 8.9 kcal/mol with EGFR. WB results indicated that the combination of KAE and ERL dramatically downregulated the expression levels of p-EGFR, p-AKT, p-ERK1/2, and Bcl-2, and upregulated the expression levels of cleaved caspase-9, cleaved PARP, and Bax. The in vivo results revealed that treatment combination of KAE and ERL further reduced the volume and weight of subcutaneous grafted tumors. IHC results confirmed the WB results. These data imply that KAE may be a valid therapeutic candidate to potentiate PC cell sensitivity to ERL via inhibiting PI3K/AKT and EGFR signaling.

厄洛替尼 (ERL) 是胰腺癌 (PC) 的表皮生长因子受体 (EGFR) 酪氨酸激酶抑制剂 (TKI)。然而，由于激活了绕过 EGFR 信号传导的替代途径，ERL 的临床疗效受到限制。据报道，山奈酚 (KAE) 是一种天然类黄酮化合物，具有有效的抗肿瘤和抗炎特性，在本研究中，我们旨在确定 KAE 对 PC 细胞和小鼠模型中 ERL 单一疗法的致敏作用。简而言之，CCK-8、集落形成和流式细胞术用于评估两种 PC 细胞系响应 KAE 和 ERL 治疗组合的增殖和凋亡。此外，通过网络药理学方法预测了 KAE 和 ERL 的药物-疾病靶点和相关的抗 PC 机制。通过生存分析评估具有EGFR差异表达的PC患者的生存结果。分子对接技术预测了 KAE 和 EGFR 之间的亲和力。此外，应用蛋白质印迹（WB）和免疫组织化学（IHC）分析来验证相关蛋白的表达水平。结果，体外结果表明，与单独使用 ERL 相比，KAE 和 ERL 的组合显着抑制细胞增殖并促进细胞凋亡。网络药理学结果表明，KAE 使 PC 对 ERL 治疗敏感，可能与 PI3K/AKT 信号通路和 EGFR TKI 耐药有关。生存分析表明，EGFR高表达的PC患者生存率相对较低。分子对接结果进一步表明，KAE 与 EGFR 具有 - 8.9 kcal/mol 的高结合亲和力。WB 结果表明，KAE 和 ERL 的组合显着下调 p-EGFR、p-AKT、p-ERK1/2 和 Bcl-2 的表达水平，并上调 cleaved caspase-9、cleaved PARP 和巴克斯。体内结果显示，KAE 和 ERL 的治疗组合进一步减少了皮下移植肿瘤的体积和重量。IHC 结果证实了 WB 结果。这些数据表明，KAE 可能是一种有效的治疗候选药物，可通过抑制 PI3K/AKT 和 EGFR 信号传导来增强 PC 细胞对 ERL 的敏感性。并上调 cleaved caspase-9、cleaved PARP 和 Bax 的表达水平。体内结果显示，KAE 和 ERL 的治疗组合进一步减少了皮下移植肿瘤的体积和重量。IHC 结果证实了 WB 结果。这些数据表明，KAE 可能是一种有效的治疗候选药物，可通过抑制 PI3K/AKT 和 EGFR 信号传导来增强 PC 细胞对 ERL 的敏感性。并上调 cleaved caspase-9、cleaved PARP 和 Bax 的表达水平。体内结果显示，KAE 和 ERL 的治疗组合进一步减少了皮下移植肿瘤的体积和重量。IHC 结果证实了 WB 结果。这些数据表明，KAE 可能是一种有效的治疗候选药物，可通过抑制 PI3K/AKT 和 EGFR 信号传导来增强 PC 细胞对 ERL 的敏感性。