Department of Oncology, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
Department of Pathology, Medicine School of Southeast University, Nanjing, Jiangsu, China.
Int J Radiat Biol. 2020 Jun;96(6):740-747. doi: 10.1080/09553002.2020.1694188. Epub 2020 Feb 10.
Stereotactic body radiation therapy (SBRT) is emerging as a new noninvasive treatment in patients with primary liver carcinoma or liver-confined metastatic cancer. However, the radiobiological targets remain a subject of debate. Here, we investigated the potential biological effects of the radiation on the human hepatocellular carcinoma HepG2 cells. Firstly, HepG2 cells were divided into three groups: control group, 3.5 Gy8f group (L group), and 15 Gy1f group (H group). After treatment, cell proliferation was examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and plate colony formation assays. Cell cycle and apoptosis were assessed using propidium iodide and Hoechst 33258 staining, respectively. Furthermore, the mechanisms underlying irradiation-induced cell cycle arrest and cell apoptosis were investigated by Western blot assay. Irradiation could effectively inhibit the proliferation and colony formation of HepG2 cells, and the single high dose irradiation showed stronger inhibitory effects. Irradiation-induced cell cycle arrest at G2/M phase in HepG2 cell, during which the expression levels of cyclin B1, CDK1, and p-CDK1 proteins were downregulated, whereas expression of p21 was upregulated in the irradiated HepG2 cells. After irradiation, typical morphological changes of apoptosis in HepG2 cells were observed; the number of cell apoptosis and the expression of apoptosis associated proteins were significantly increased in HepG2 cells by high dose irradiation compared with low dose irradiation. Additionally, compared with low dose irradiation, high dose irradiation significantly downregulated the phosphorylated proteins in the Ras/Raf/MEK/ERK signaling pathway. Our results suggest that irradiation applied in SBRT, particularly single high dose irradiation, mediates its anti-tumor effects by inducing cell cycle arrest and apoptosis via modulation of the Ras/Raf/MEK/ERK signaling pathway.
立体定向体部放射治疗（SBRT）作为一种新的非侵入性治疗方法，正在原发性肝癌或肝转移性肿瘤患者中得到应用。然而，放射生物学靶点仍然存在争议。在这里，我们研究了辐射对人肝癌 HepG2 细胞的潜在生物学效应。首先，将 HepG2 细胞分为三组：对照组、3.5Gy8f 组（L 组）和 15Gy1f 组（H 组）。治疗后，通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐和平板集落形成实验检测细胞增殖。通过碘化丙啶和 Hoechst 33258 染色分别评估细胞周期和细胞凋亡。此外，通过 Western blot 实验研究了照射诱导细胞周期阻滞和细胞凋亡的机制。照射能有效抑制 HepG2 细胞的增殖和集落形成，且单次高剂量照射表现出更强的抑制作用。照射诱导 HepG2 细胞周期阻滞在 G2/M 期，在此期间，cyclin B1、CDK1 和 p-CDK1 蛋白的表达水平下调，而 p21 在照射的 HepG2 细胞中表达上调。照射后，观察到 HepG2 细胞凋亡的典型形态学变化；与低剂量照射相比，高剂量照射显著增加了 HepG2 细胞的细胞凋亡数量和凋亡相关蛋白的表达。此外，与低剂量照射相比，高剂量照射显著下调了 Ras/Raf/MEK/ERK 信号通路中磷酸化蛋白的表达。我们的研究结果表明，SBRT 中应用的照射，特别是单次高剂量照射，通过调节 Ras/Raf/MEK/ERK 信号通路诱导细胞周期阻滞和凋亡，发挥其抗肿瘤作用。
