Department of Urology, The Affiliated People's Hospital of Ningbo University, Ningbo, China.
J Clin Lab Anal. 2022 Jun;36(6):e24452. doi: 10.1002/jcla.24452. Epub 2022 Apr 25.
The development of cisplatin resistance often results in cisplatin inefficacy in advanced or recurrent bladder cancer. However, effective treatment strategies for cisplatin resistance have not been well established.
Gene expression was measured by qRT-PCR and Western blotting. CCK-8 assay was performed to detect cell survival. The number of apoptotic cells was determined using the Annexin V-PI double-staining assay. The level of reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye, and the ATP level was detected using an ATP measurement kit.
The expression of receptor-interacting protein kinase 1 (RIPK1), a key regulator of necroptosis, gradually decreased during cisplatin resistance. We first used piperlongumine (PL) in combination with cisplatin to act on cisplatin-resistant BC cells and found that PL-induced activation of RIPK1 increased the sensitivity of T24 resistant cells to cisplatin treatment. Furthermore, we revealed that PL killed T24 cisplatin-resistant cells by triggering necroptosis, because cell death could be rescued by the mixed lineage kinase domain-like (MLKL) protein inhibitor necrotic sulfonamide or MLKL siRNA, but could not be suppressed by the apoptosis inhibitor z-VAD. We further explored the specific mechanism and found that PL activated RIPK1 to induce necroptosis in cisplatin-resistant cells by stimulating mitochondrial fission to produce excessive ROS.
Our results demonstrated the role of RIPK1 in cisplatin-resistant cells and the sensitization effect of the natural drug PL on bladder cancer. These may provide a new treatment strategy for overcoming cisplatin resistance in bladder cancer.
顺铂耐药的发展常常导致晚期或复发性膀胱癌对顺铂无效。然而，尚未建立有效的顺铂耐药治疗策略。
通过 qRT-PCR 和 Western blot 测定基因表达。CCK-8 测定法检测细胞存活。使用 Annexin V-PI 双重染色测定法测定凋亡细胞的数量。使用 2'，7'-二氯二氢荧光素二乙酸荧光染料测定活性氧 (ROS) 水平，并使用 ATP 测定试剂盒检测 ATP 水平。
受体相互作用蛋白激酶 1 (RIPK1) 的表达，一种坏死性凋亡的关键调节因子，在顺铂耐药过程中逐渐降低。我们首先使用胡椒碱 (PL) 与顺铂联合作用于顺铂耐药的 BC 细胞，发现 PL 诱导的 RIPK1 激活增加了 T24 耐药细胞对顺铂治疗的敏感性。此外，我们揭示 PL 通过触发坏死性凋亡杀死 T24 顺铂耐药细胞，因为细胞死亡可以被混合谱系激酶结构域样 (MLKL) 蛋白抑制剂坏死磺胺或 MLKL siRNA 挽救，但不能被凋亡抑制剂 z-VAD 抑制。我们进一步探讨了具体的机制，发现 PL 通过刺激线粒体分裂产生过多的 ROS 来激活 RIPK1，从而诱导顺铂耐药细胞发生坏死性凋亡。
我们的结果表明 RIPK1 在顺铂耐药细胞中的作用以及天然药物 PL 对膀胱癌的增敏作用。这些可能为克服膀胱癌中顺铂耐药提供新的治疗策略。
