复发性喉鳞状细胞癌中 TRAIL 耐药性的形成与 DR4 CpG 岛的辐射诱导高甲基化有关。

Development of TRAIL resistance by radiation-induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma.

机构信息

Department of Otorhinolaryngology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.

Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea.

出版信息

Int J Radiat Oncol Biol Phys. 2014 Apr 1;88(5):1203-11. doi: 10.1016/j.ijrobp.2013.12.016.

DOI:10.1016/j.ijrobp.2013.12.016

PMID:24661673

Abstract

PURPOSE

There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis.

METHODS AND MATERIALS

Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line.

RESULTS

HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine.

CONCLUSION

Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

摘要

目的

对于放射治疗失败后复发性头颈部癌症患者，治疗选择有限。为了评估肿瘤坏死因子相关凋亡诱导配体（TRAIL）作为放射失败后复发性癌症的挽救化疗药物的作用，我们研究了临床上相关的累积照射对 TRAIL 诱导凋亡的影响。

方法和材料

使用先前从喉癌患者中建立的 HN3 细胞系，我们生成了一个慢性辐照的 HN3R 同源细胞系。用 MTS 和 PI/annexin V-FITC 测定法分析 HN3 和 HN3R 细胞在 TRAIL 处理下的活力和凋亡。Western blot 和流式细胞术用于确定 TRAIL 耐药的潜在机制。用 107 个喉癌标本的组织微阵列对 DR4 表达进行半定量评分。对每个细胞系的基因组 DNA 进行甲基化特异性聚合酶链反应和 bisulfite 测序以检测 DR4。

结果

HN3R 细胞比 HN3 细胞对 TRAIL 诱导的凋亡更具抗性，因为 DR4 受体水平明显降低。在 70 例未接受放射治疗的标本中，37 例放射治疗失败后的挽救手术标本中的 DR4 染色评分较低（分别为 3.03±2.75 与 5.46±3.30；P<.001）。HN3R 细胞具有一个甲基化的 DR4 CpG 岛，该岛被 DNA 去甲基化剂 5-aza-2'-脱氧胞苷部分去甲基化。