Using Hyperbaric Oxygen to Improve the Radiosensitivity of Human U251 Glioma Cells

Summary

This protocol shows that hyperbaric oxygen can enhance the proliferation inhibition and apoptosis of U251 glioma cells treated with X-ray irradiation by blocking the cells in the G2/M phase. This improves the radiosensitivity of human glioma cell lines.

Abstract

The aim of this study was to explore the use of hyperbaric oxygen to enhance the radiosensitivity of human glioma cells. Sub-cultured U251 human glioma cells were randomly divided into four groups: an untreated control group, cells treated with hyperbaric oxygen (HBO) only, cells treated with X-ray irradiation (X-ray) only, and cells treated with both HBO and X-ray. Cell morphology, cell proliferation activity, cell cycle distribution, and apoptosis were observed in these groups to evaluate the role of HBO in improving the radiosensitivity of glioma cells. With the increase in X-ray doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy), the survival fraction (SF) of glioma cells gradually decreased.

Significantly lower SF was observed for the cells treated with the HBO and X-ray together than in the X-ray group for each dose (all P < 0.05). The proliferation inhibition was significantly higher in the HBO combined with X-ray group than in the X-ray group for each dose (all P < 0.05) for the U251 cell line. The percentage of G2/M phase cells was significantly higher in the HBO combined with X-ray (2 Gy) group (26.70% ± 2.46%) and the HBO group (22.36% ± 0.91%) than in the control group (11.56% ± 2.01%) and X-ray (2 Gy) group (10.35% ± 2.69%) (all P < 0.05). U251 cell apoptosis was significantly higher in the HBO combined with X-ray (2 Gy) group than in the HBO group, the X-ray (2 Gy) group, and the control group (all P < 0.05). We conclude that HBO can enhance the proliferation inhibition and apoptosis of glioma U251 cells by blocking glioma cells in the G2/M phase and improve the radiosensitivity of U251 glioma cells.

Introduction

Glioma is a primary intracranial tumor that originates from central nervous system glial cells¹. The current treatment strategy for glioma is surgery combined with radiotherapy and chemotherapy. Postoperative radiotherapy for glioma can provide survival benefits (grade I evidence), and early postoperative radiotherapy can effectively prolong patient survival (grade II evidence)². For higher-grade gliomas (grade III or IV), especially highly malignant and invasive glioblastoma (grade III evidence)³, postoperative radiotherapy should be performed as early as possible (<6 weeks). However, despite....

Protocol

All study methods were approved by the Institutional Review Board and Ethics Committee of the Second Hospital Affiliated with Lanzhou University and were performed in accordance with relevant guidelines and regulations.

1. Treatment of glioma cells

NOTE: The U251 glioma cell line was used in this experiment.

1. U251 cell culture
   1. Seed U251 cells in multiple dishes with DMEM containing 10% fetal bovine serum (FBS) and culture them at 37 °C with 5% CO₂.
   2. Upon reaching 50%-60% confluency, dissociate the cells using trypsin-EDTA solution (0.25%, without p....

Results

Culture of U251 glioma cells
U251 glioma cells had a fusiform shape 24 h to 48 h after culture in DMEM and were adherent. These cells were used for further study (Figure 1).

Glioma cell morphology and count
The cell counts for the U251 glioma cells in the HBO combined with X-ray (2 Gy) group were significantly lower than that for the X-ray (2 Gy) group after 24 h, 48 h, and 72 h of cell culture (all P < 0........

Discussion

The glioma cell line U251 is one of the most classical human glioma cell lines and is widely used as a glioma model in many studies.

Effects of HBO on U251 glioma cell proliferation
HBO typically refers to breathing pure oxygen (100% oxygen concentration) in a sealed chamber with a pressure 1.5-3-fold higher than normal atmospheric pressure, which can increase oxygen content in microvascular plasma¹⁴. For patients with glioma, combining radiothera.......

Materials

Name	Company	Catalog Number	Comments
Binding Buffer	Dickinson and Company	RH10 9RR
CCK-8 test kit	DOJINDO	NJ	Cell counting assay
CELL FIT			cell cycle analysis (DNA content)
CELLQUEST			apoptotic cell analysis
DMEM and Annexin V-FITC	Gibco BRL
flow cytometer	Dickinson
Glioma U251 and U87 cell line	Shanghai Institute of Cell Biology
hyperbaric oxygen chamber	Hongyuan Institute
medical linear accelerator	Elekta Limited Company
microplate reader
MOD FITLT formac v1.01			cell analysis--cell cycle phase
trypsin	Hyclone Laboratories Inc