Ding Li, Wen Fengmei, Zeng Liang, Li Ziwei, Xie Gangyin
Department of Breast and Thyroid Surgery, Hunan University of Medicine General Hospital, Hunan, People's Republic of China.
Teaching and Research Department, Chongqing University FuLing Hospital, Chongqing, People's Republic of China.
Sci Rep. 2025 Jul 1;15(1):20383. doi: 10.1038/s41598-025-08977-1.
Periplocin, a tumor-inhibitory compound derived from Cortex periploca, was investigated for its mechanisms of action and therapeutic potential against human papillary thyroid carcinoma cell proliferation. BCPAP/TPC-1 cells were treated with periplocin ± necrostatin-1 (a necroptosis inhibitor) or Z-Val-Ala-Asp(OMe)-fluoromethylketone (an apoptosis inhibitor). Cell proliferation was assessed using the cell counting kit 8/real-time cell analysis assay. Necrotic morphology was quantified using Hoechst 33,342/propidium iodide (PI) staining (PI⁺ rate), and apoptosis/necroptosis pathways were analyzed using Annexin V-FITC/PI flow cytometry. RNA-seq was conducted to compare the transcriptomes of periplocin-treated and untreated TPC-1 cells. The key proteins (phosphorylated mixed lineage kinase domain-like protein [p-MLKL], phosphorylated receptor-interacting protein kinase-3 [p-RIP3], IL6, IL1A, and death receptor 4 [DR4]) were validated by western blotting. In vivo, TPC-1 xenografts from BALB/c nude mice were evaluated for tumor growth, necrosis (PI staining), and expression of protein markers (proliferating cell nuclear antigen, p-MLKL, and p-RIP3) via immunohistochemistry. Periplocin suppressed the growth of BCPAP and TPC-1 thyroid carcinoma cells via concentration-dependent necroptosis. Necrostatin-1 co-treatment reduced PI⁺ cells and necrotic morphology (high Hoechst/PI staining; P < 0.05), confirming necroptosis dependence. Mechanistically, periplocin activated RIP3/MLKL signaling and damage-associated molecular patterns, whereas DR4 knockdown (si-DR4) attenuated p-MLKL expression (P < 0.01), indicating DR4-mediated pathway activation. In vivo, periplocin reduced TPC-1 xenograft volume (P < 0.01), weight, and proliferation (decreased proliferating cell nuclear antigen⁺ cells; P < 0.05), while elevating p-RIP3/p-MLKL (P < 0.01) and PI⁺ necrosis (P < 0.01). Periplocin selectively induces DR4-dependent necroptosis via RIP3/MLKL activation, providing the first evidence of necroptosis induction in papillary thyroid carcinoma. These findings suggest that periplocin is a promising therapeutic candidate, particularly for tumor necrosis factor-related apoptosis-inducing ligand-resistant papillary thyroid carcinoma that evades apoptosis.
香加皮素是一种从香加皮中提取的具有肿瘤抑制作用的化合物，本研究对其作用机制及抗人甲状腺乳头状癌细胞增殖的治疗潜力进行了探究。采用香加皮素±坏死性凋亡抑制剂Necrostatin-1或凋亡抑制剂Z-Val-Ala-Asp(OMe)-fluoromethylketone处理BCPAP/TPC-1细胞。使用细胞计数试剂盒8/实时细胞分析检测法评估细胞增殖。采用Hoechst 33342/碘化丙啶(PI)染色法(PI⁺率)对坏死形态进行定量分析，并通过Annexin V-FITC/PI流式细胞术分析凋亡/坏死性凋亡途径。进行RNA测序以比较经香加皮素处理和未处理的TPC-1细胞的转录组。通过蛋白质免疫印迹法验证关键蛋白(磷酸化混合谱系激酶样结构域蛋白[p-MLKL]、磷酸化受体相互作用蛋白激酶-3 [p-RIP3]、白细胞介素6、白细胞介素1A和死亡受体4 [DR4])。在体内，通过免疫组织化学评估BALB/c裸鼠的TPC-1异种移植瘤的肿瘤生长、坏死(PI染色)以及蛋白标志物(增殖细胞核抗原、p-MLKL和p-RIP3)的表达。香加皮素通过浓度依赖性坏死性凋亡抑制BCPAP和TPC-1甲状腺癌细胞的生长。联合使用Necrostatin-1可减少PI⁺细胞和坏死形态(高Hoechst/PI染色；P<0.05)，证实了坏死性凋亡依赖性。从机制上来说，香加皮素激活了RIP3/MLKL信号通路和损伤相关分子模式，而敲低DR4(si-DR4)可减弱p-MLKL的表达(P<0.01)，表明DR4介导的信号通路被激活。在体内，香加皮素可减小TPC-1异种移植瘤的体积(P<0.01)、重量并降低其增殖能力(增殖细胞核抗原⁺细胞减少；P<0.05)，同时提高p-RIP3/p-MLKL的表达水平(P<0.01)以及PI⁺坏死率(P<0.01)。香加皮素通过激活RIP3/MLKL选择性地诱导依赖DR4的坏死性凋亡，这为甲状腺乳头状癌中诱导坏死性凋亡提供了首个证据。这些研究结果表明，香加皮素是一种很有前景的治疗候选药物，尤其对于那些逃避凋亡的、对肿瘤坏死因子相关凋亡诱导配体耐药的甲状腺乳头状癌。
