Hsieh Kun-Lin, Huang Kuan-Hua, Chang Ching-Ping, Tsai Hung-Wen, Chang Yu-Hao, Zheng Yi-Ru, Huang Huei-Sheng
Division of Urology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan.
Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan.
PLoS One. 2025 Sep 2;20(9):e0331289. doi: 10.1371/journal.pone.0331289. eCollection 2025.
Gemcitabine is commonly used in the standard first-line treatment of urothelial carcinoma (UC); however, the emergence of drug resistance significantly limits its clinical benefit. The present study aims to investigate the role of CUB domain-containing protein 1 (CDCP1) in mediating resistance to gemcitabine in UC cells. Gemcitabine-resistant T24 (T24-GR) cells exhibited downregulation of human equilibrative nucleoside transporter 1 and upregulation of cytidine deaminase, key regulators of gemcitabine metabolism, as well as increased CDCP1 expression. Notably, silencing CDCP1 reversed these resistance-associated expression patterns. Mechanistically, T24-GR cells displayed elevated expression of CDCP1 and increased phosphorylation of c-Src and PKCδ, indicating activation of downstream survival signaling. Overexpression of CDCP1 in T24-CD cells activated similar pathways and modulated regulators of gemcitabine metabolism. In contrast, CRISPR/Cas9-mediated knockout of CDCP1 in T24-CDKO cells suppressed c-Src/PKCδ signaling and increased sensitivity to gemcitabine-induced cytotoxicity. Using flow cytometry, we observed that treatment with gemcitabine induced apoptosis in parental T24 cells, as indicated by an increase in the sub-G1 population. In contrast, T24-GR and T24-CD cells showed minimal sub-G1 accumulation, suggesting resistance to gemcitabine-induced apoptosis. Western blot analysis revealed decreased levels of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in T24-GR and T24-CD cells following gemcitabine exposure, whereas these markers were upregulated in parental T24 and T24-CDKO cells. Furthermore, the knockdown of CDCP1 and the utilization of c-Src/PKCδ signaling inhibitors in T24-GR cells led to the restoration of sensitivity to gemcitabine. By suppressing apoptosis and altering drug metabolism pathways, highlighting CDCP1 as a potential therapeutic target for overcoming gemcitabine resistance in UC.
吉西他滨常用于尿路上皮癌（UC）的标准一线治疗；然而，耐药性的出现显著限制了其临床疗效。本研究旨在探讨含CUB结构域蛋白1（CDCP1）在介导UC细胞对吉西他滨耐药中的作用。吉西他滨耐药的T24（T24-GR）细胞表现出人类平衡核苷转运体1的下调和胞苷脱氨酶的上调，胞苷脱氨酶是吉西他滨代谢的关键调节因子，同时CDCP1表达增加。值得注意的是，沉默CDCP1可逆转这些与耐药相关的表达模式。从机制上讲，T24-GR细胞显示出CDCP1表达升高以及c-Src和PKCδ磷酸化增加，表明下游生存信号通路被激活。T24-CD细胞中CDCP1的过表达激活了类似的信号通路并调节了吉西他滨代谢的调节因子。相反，CRISPR/Cas9介导的T24-CDKO细胞中CDCP1的敲除抑制了c-Src/PKCδ信号通路，并增加了对吉西他滨诱导的细胞毒性的敏感性。使用流式细胞术，我们观察到用吉西他滨处理可诱导亲本T24细胞凋亡，表现为亚G1期细胞群体增加。相比之下，T24-GR和T24-CD细胞显示出最小的亚G1期积累，表明对吉西他滨诱导的凋亡具有抗性。蛋白质印迹分析显示，吉西他滨处理后，T24-GR和T24-CD细胞中裂解的caspase-3和裂解的聚（ADP-核糖）聚合酶水平降低，而这些标志物在亲本T24和T24-CDKO细胞中上调。此外，T24-GR细胞中CDCP1的敲低以及c-Src/PKCδ信号通路抑制剂的使用导致对吉西他滨的敏感性恢复。通过抑制凋亡和改变药物代谢途径，突出了CDCP1作为克服UC中吉西他滨耐药性的潜在治疗靶点。
