Objectives: To investigate the inhibitory effect of the farnesyltransferase inhibitor lonafarnib on anaplastic thyroid carcinoma (ATC) cell lines and a nude mouse xenograft model, and to elucidate the underlying mechanisms.
Methods: The anti-proliferative effect of lonafarnib on ATC cell lines (8505C, CAL62) was assessed using the CCK-8 assay. Cell migration and invasion were evaluated by Transwell assays. Apoptosis was detected by flow cytometry (Annexin V/PI double staining). Western blotting was used to measure protein expression levels of Ras, ERK, p-ERK, PARP, cleaved PARP, Caspase-3, cleaved Caspase-3, GSDME, and GSDME-N. A subcutaneous xenograft model was established in female BALB/c nude mice (4-6 weeks old, 20±1 g). 8505C cells (1×10⁶) were injected into the right flank of each mouse. When tumor volume reached 100-150 mm³, the mice were randomly divided into three groups (n=4 per group): blank control (normal saline by gavage every other day), low-dose lonafarnib (25 mg/kg by gavage every other day), and high-dose lonafarnib (50 mg/kg by gavage every other day). Treatment lasted for 15 days. Tumor volume (calculated as length×width²×0.5) and body weight were measured every two days. At the endpoint, tumors were excised and weighed.
Results: Lonafarnib significantly inhibited the proliferation of ATC cells in a concentration-dependent manner, with superior efficacy compared to cobimetinib and lenvatinib. After treatment with 5, 10, and 20 μmol/L lonafarnib, the numbers of migrated and invaded 8505C and CAL62 cells were markedly reduced (all P<0.05). Lonafarnib induced apoptosis in a concentration-dependent manner. Mechanistically, lonafarnib downregulated Ras protein expression, reduced total and phosphorylated ERK levels, decreased PARP and Caspase-3 expression, and increased cleaved PARP, cleaved Caspase-3, and GSDME-N levels in a concentration- and time-dependent manner. In vivo, the low-dose and high-dose lonafarnib groups exhibited significantly slower tumor growth compared with the blank control group. At the end of the observation period, tumor weights in the two treatment groups were significantly lower than in the control group (both P<0.05). No significant body weight loss was observed, indicating good tolerability.
Conclusions: Lonafarnib effectively suppresses the malignant progression of anaplastic thyroid carcinoma both in ATC cell lines and in a BALB/c nude mouse xenograft model. The mechanism involves inhibition of Ras farnesylation and the downstream ERK signaling pathway, leading to activation of Caspase-3/PARP-mediated apoptosis and GSDME-mediated pyroptosis.
目的: 探讨法尼基转移酶抑制剂洛那法尼对甲状腺未分化癌（ATC）细胞系及裸鼠移植瘤模型的抑制作用，并阐明其潜在机制。方法: 采用CCK-8法检测洛那法尼对8505C、CAL62等ATC细胞系增殖能力的影响;Transwell小室实验分析洛那法尼对ATC细胞迁移和侵袭能力的抑制效果;流式细胞术检测洛那法尼处理后ATC细胞（8505C，CAL62）的凋亡水平;蛋白质印迹法检测洛那法尼处理后ATC细胞中Ras蛋白、胞外信号调节激酶（ERK）、磷酸化ERK、多腺苷二磷酸核糖聚合酶（PARP）、剪切型PARP、胱天蛋白酶3（Caspase-3）、消皮素E（GSDME）和消皮素E氨基端片段（GSDME-N）等蛋白表达水平。取4~6周龄雌性BALB/c裸鼠（体重20±1 g）建立皮下移植瘤模型，于每只裸鼠右侧胁部皮下注射8505C细胞（1×10⁶个）。待肿瘤体积达到100~150 mm³时，将裸鼠随机分为三组（每组4只）:空白对照组（隔日灌胃生理盐水）、洛那法尼低剂量组（25 mg/kg，隔日灌胃）和洛那法尼高剂量组（50 mg/kg，隔日灌胃），连续给药15天。每2天测量一次肿瘤体积（按公式:长×宽²×0.5计算）和体重。实验结束时，剥离肿瘤并称重。结果: 洛那法尼能够有效抑制ATC细胞增殖，且抑制作用优于考比替尼和仑伐替尼。经5、10、20 μmol/L洛那法尼处理后8505C和CAL62细胞的迁移和侵袭数均减少（均P<0.05），细胞凋亡率均增加（均P<0.05）。机制研究表明，洛那法尼处理后ATC细胞中Ras蛋白表达水平下调，总ERK和磷酸化ERK水平降低，PARP表达呈浓度及时间依赖性减少，Caspase-3和GSDME表达水平总体呈下降趋势，同时剪切型PARP以及GSDME‑N表达呈浓度及时间依赖性增加，剪切型Caspase-3表达水平总体呈上升趋势。体内实验中，与空白对照组比较，洛那法尼低剂量组和高剂量组肿瘤生长均显著减缓。观察终点时，两个治疗组的瘤重均低于对照组（均P<0.05）。各组均未出现明显体重下降，提示药物耐受性良好。结论: 洛那法尼在ATC细胞系及BALB/c裸鼠移植瘤模型中均能有效抑制甲状腺未分化癌的恶性进展。洛那法尼通过抑制Ras蛋白法尼基化及其下游ERK信号通路，进而激活Caspase-3/PARP介导的凋亡和GSDME介导的细胞焦亡。.
Keywords: Anaplastic thyroid cancer; Cell apoptosis; Cell proliferation; Farnesylation; Lonafarnib; Nude mice; Ras protein.