| 徐霄鹏,戴军,高昳,等.三磷酸鸟苷环化水解酶1去磷酸化突变体对食管癌放射敏感性的影响及机制研究[J].中华放射医学与防护杂志,2024,44(10):819-826.Xu Xiaopeng,Dai Jun,Gao Yi,et al.Exploring the impacts and mechanisms of GCH1 dephosphorylated mutants on the radiosensitivity of esophageal cancers[J].Chin J Radiol Med Prot,2024,44(10):819-826 |
| 三磷酸鸟苷环化水解酶1去磷酸化突变体对食管癌放射敏感性的影响及机制研究 |
| Exploring the impacts and mechanisms of GCH1 dephosphorylated mutants on the radiosensitivity of esophageal cancers |
| 投稿时间：2024-03-04 |
| DOI：10.3760/cma.j.cn112271-20240304-00084 |
| 中文关键词: 食管癌 电离辐射 三磷酸鸟苷环化水解酶1 四氢生物蝶呤 |
| 英文关键词:Esophageal cancer Ionizing radiation GTP cyclohydrolase 1 (GCH1) Tetrahydrobiopterin (BH4) |
| 基金项目:江苏省卫生健康委面上项目(H2023027);无锡市“太湖人才计划”优秀医学专家团队(2021-9);江阴市“暨阳名医”消化内镜领航专家团队(2023-37);无锡市卫生健康委青年人才项目(Q202206)及中青年后备拔尖人才资助计划(HB2023105);四川省科技计划项目(2022NSFSC0797,2023NSFSC0648) |
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| 中文摘要: |
| 目的 探讨体内从头合成四氢生物蝶呤(BH4)过程中的限速酶三磷酸鸟苷环化水解酶1(GCH1),关键磷酸化位点突变体(GCH1-S81A)对食管癌放射敏感性的影响及机制。方法 构建不同磷酸化水平的GCH1稳定过表达食管鳞癌(KYSE-150)细胞系,按照GCH1磷酸化水平及照射剂量,将实验分为空白对照组、空载病毒组、空载+照射组、野生GCH1组、GCH1磷酸化组、GCH1去磷酸化组、GCH1去磷酸化+照射组、回补验证组、回补验证+照射组。通过Western blot验证感染效率,CCK-8法检测各组肿瘤细胞存活率,克隆形成实验检测肿瘤细胞放射敏感性变化,流式细胞术检测各组肿瘤细胞凋亡率、活性氧(ROS)及脂质过氧化物含量变化,Western blot检测铁死亡相关蛋白表达变化。结果 GCH1去磷酸化组感染48 h后细胞内GCH1-S81磷酸化蛋白表达水平明显降低(t=9.35、16.57,P<0.05)。与空载+照射组相比,GCH1去磷酸化+照射组细胞在10 Gy X射线照射24 h后存活率明显降低(t=26.97,P<0.05)。在10 Gy X射线照射8 h后KYSE-150细胞中ROS含量,以及照射48 h后细胞凋亡率和脂质过氧化物水平,均上升明显(t=17.89~131.10,P<0.05),且在加入GCH1-S81A突变体后进一步加剧(t=157.06~97.81,P<0.05),这一现象能被补充野生型GCH1后抑制(t=66.38~23.08,P<0.05)。与空载+照射组相比,去磷酸化+照射组在各个X射线剂量(2、4、6和8 Gy)照射下克隆形成能力均有所降低(t=7.31~8.16,P<0.05)。GCH1-S81A突变能够降低铁死亡相关蛋白GPX4的表达(t=4.55,P<0.05),且在10 Gy的X射线照射后表达量进一步降低(t=12.98,P<0.05)。结论 GCH1突变为GCH1-S81A去磷酸化后,可抑制食管癌细胞KYSE-150的生长并提高其放射敏感性,GCH1-S81A突变体有望为提高食管癌放疗疗效提供新的方式。 |
| 英文摘要: |
| Objective To investigate the impacts and mechanisms of the GCH1-S81A mutants of the rate-limiting enzyme GTP cyclohydrolase 1 (GCH1) at key phosphorylation sites on the radiosensitivity of esophageal cancers during the de novo synthesis of tetrahydrobiopterin (BH4). Methods The KYSE-150 cell lines of esophageal squamous cell carcinoma with stable GCH1 overexpression at different phosphorylation levels were constructed in this study. Based on GCH1's phosphorylation levels and radiation doses, the experimental groups were divided into the blank control group, the empty virus group, the empty virus + irradiation group, the wild-type GCH1 group, the GCH1 phosphorylation group, the GCH1 dephosphorylation group, the GCH1 dephosphorylation + irradiation group, the validation group, and the validation + irradiation group. The Western blot and the CCK-8 assay were employed to detect the infection efficiency and the survival rates of tumor cells in various groups, respectively. The flow cytometry was applied to detect the changes in the apoptosis rate, reactive oxygen species (ROS) level, and lipid peroxide level of tumor cells in various groups. The colony formation assay was used to detect the changes in the radiosensitivity of tumor cells. Besides, the Western blot was performed to detect the changes in the expression of ferroptosis-related proteins. Results The GCH1 dephosphorylation group showed a significantly decreased expression level of phosphorylated GCH1-S81 protein in the cells at 48 h after infection (t=9.35, 16.57,P<0.05). Compared to the empty virus + irradiation group, the GCH1 dephosphorylation + irradiation group exhibited a significantly decreased cell survival rate 24 h after 10 Gy X-ray irradiation (t=26.97,P<0.05). The ROS levels in KYSE-150 cells at 8 h after 10 Gy X-ray irradiation, and the apoptosis rates and lipid peroxide levels at 48 h after irradiation, all showed a significant increase (t=17.89-131.1,P<0.05), which was further aggravated following the addition of GCH1-S81A mutants (t=157.06-97.81,P<0.05). This phenomenon could be inhibited by complementing wild-type GCH1 (t=66.38-23.08,P<0.05). Compared to the empty virus + irradiation group, the GCH1 dephosphorylation + irradiation group manifested decreased colony formation capacity under various X-ray doses (2, 4, 6 and 8 Gy, t=7.31-8.16,P<0.05). The GCH1-S81A mutation reduced the expression level of the ferroptosis-related protein GPX4 (t=4.55,P<0.05), which was further decreased after 10 Gy X-ray irradiation (t=12.98,P<0.05). Conclusions The GCH1-S81A dephosphorylated mutants can inhibit the growth of esophageal carcinoma cells KYSE-150 and enhance their radiosensitivity, which may hold promise as a novel approach to improve the efficacy of radiotherapy for esophageal cancers. |
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