维莫非尼与 Navitoclax 联合治疗 BRAF 突变型甲状腺癌。

Combination Treatment with the BRAF Inhibitor Vemurafenib and the BH3 Mimetic Navitoclax for BRAF-Mutant Thyroid Carcinoma.

机构信息

1 Department of Nuclear medicine, Kyungpook National University Chilgok Hospital, Daegu, Republic of Korea.

2 Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.

出版信息

Thyroid. 2019 Apr;29(4):540-548. doi: 10.1089/thy.2018.0511.

DOI:10.1089/thy.2018.0511

PMID:30869573

Abstract

BACKGROUND

Vemurafenib is a selective BRAF inhibitor (BRAFi) that has shown promising activity in BRAF-positive papillary thyroid cancer (PTC). However, adverse events and resistance to a single-agent BRAFi often require discontinuation of the targeted therapy in BRAF-positive PTC. Thus, this study investigated the expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) family members, which are frequently overexpressed in many human cancers to inhibit apoptosis, in PTC harboring the BRAF mutation after BRAFi treatment, and then evaluated the cytotoxic effects of a homology 3 domain (BH3)-mimetic in combination with a BRAFi.

METHODS

K1 cells (BRAF-positive human PTC) were treated with various concentrations of vemurafenib to investigate the effect of the BRAFi. In addition, the study analyzed the protein expression profiles of phosphorylated ERK1/2 (p-ERK 1/2) and anti-apoptotic BCL-2 family after vemurafenib treatment and selected the target anti-apoptotic protein. Antitumor effects were measured by cell counting, and effects on apoptosis were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and Western blot analysis.

RESULTS

At a concentration of 10 μM, vemurafenib inhibited the growth of K1 cells by 49.4%. Western blot analysis following exposure to 10 μM vemurafenib revealed that p-ERK1/2 gradually decreased over 24 hours, but the expression of B-cell lymphoma-extralarge (BCL-XL) and BCL-2 increased after 12 hours of treatment. Based on this result, the K1 cells were treated with navitoclax (BCL-2/BCL-XL inhibitor) for 24 hours up to a concentration of 4 μM, which resulted in negligible effects on cell survival. However, a combination treatment of 0.5 μM navitoclax with 1 μM vemurafenib resulted in significantly enhanced cell growth inhibition and increased apoptosis.

CONCLUSIONS

The results of the present study show that vemurafenib increased the expression of anti-apoptotic proteins of the BCL-2 family. Thus, the combination of vemurafenib with navitoclax may be effective in BRAF-positive PTC treatment.

摘要

背景

维莫非尼是一种选择性 BRAF 抑制剂（BRAFi），在 BRAF 阳性甲状腺乳头状癌（PTC）中显示出有前景的活性。然而，不良事件和对单一靶向 BRAFi 的耐药性常常需要停止 BRAF 阳性 PTC 的靶向治疗。因此，本研究调查了在 BRAFi 治疗后携带 BRAF 突变的 PTC 中抗凋亡 B 细胞淋巴瘤 2（BCL-2）家族成员的表达情况，这些成员在许多人类癌症中经常过表达以抑制凋亡，并随后评估同源结构域 3（BH3）模拟物与 BRAFi 联合的细胞毒性作用。

方法

用不同浓度的维莫非尼处理 K1 细胞（BRAF 阳性人 PTC），以研究 BRAFi 的作用。此外，该研究还分析了维莫非尼治疗后磷酸化 ERK1/2（p-ERK 1/2）和抗凋亡 BCL-2 家族的蛋白表达谱，并选择了靶抗凋亡蛋白。通过细胞计数测量抗肿瘤作用，通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记测定和 Western blot 分析测定对凋亡的影响。

结果

浓度为 10 μM 时，维莫非尼抑制 K1 细胞生长 49.4%。暴露于 10 μM 维莫非尼 24 小时后，Western blot 分析显示 p-ERK1/2 逐渐减少，但 B 细胞淋巴瘤特大（BCL-XL）和 BCL-2 的表达在治疗 12 小时后增加。基于这一结果，用 navitoclax（BCL-2/BCL-XL 抑制剂）处理 K1 细胞 24 小时，浓度高达 4 μM，对细胞存活几乎没有影响。然而，0.5 μM navitoclax 与 1 μM 维莫非尼联合治疗可显著增强细胞生长抑制和增加凋亡。