Single high dose irradiation induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cells through the Ras/Raf/MEK/ERK pathways

Abstract

Purpose: 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.

Materials and methods: Firstly, HepG2 cells were divided into three groups: control group, 3.5 Gy*8f group (L group), and 15 Gy*1f 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.

Results: 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.

Conclusions: 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.

Keywords:

• Hepatocellular carcinoma
• G2/M phase arrest
• apoptosis
• Ras/Raf/MEK/ERK pathway

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The project was supported by the Nanjing Science and Technology Development Foundation [Grant numbers. 201503028, 201303014, No. YKK18148] and the Nanjing Medical Science and Technology Development Foundation, Nanjing Health Commission [No. ZDX16008].

Notes on contributors

XiaoQiang Tian

XiaoQiang Tian, MD, is a doctor of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

Jie Geng

Jie Geng is a doctor in-charge of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

Qin Zheng

Qin Zheng, MD, is an associate chief physician of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

LiXue Wang

LiXue Wang, MD, is an associate chief physician of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

PeiLin Huang

PeiLin Huang, MD, is a professor of oncology at the School of Medicine of Southeast University, Nanjing, China.

JinLong Tong

JinLong Tong, MD, is an associate chief physician of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.

ShengQin Zheng

ShengQin Zheng is a doctor in-charge of Department of Oncology, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.