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 early intervention, glioma still has a high recurrence rate and poor prognosis after comprehensive treatment. These outcomes are mainly associated with the low radiosensitivity of glioma. Factors related to tumor radiosensitivity include the inherent radiosensitivity of tumor cells, hypoxic or non-hypoxic tumor cells, the proportion of hypoxic tumor cells, and the capacity of peritumoral tissue to repair radiation damage4.
Among these factors, hypoxic or non-hypoxic tumor cells and the proportion of hypoxic tumor cells have important effects on tumor radiosensitivity. Hyperbaric oxygen (HBO) can improve tissue oxygen storage by increasing tissue oxygen tension and blood oxygen diffusion. HBO may also produce a series of beneficial biochemical, cytological, and physiological effects5. For example, HBO has a marked reparative effect on radiotherapy-induced radiation damage. Although HBO combined with radiotherapy or chemotherapy is reported to improve the clinical efficacy of radiotherapy or chemotherapy for glioma6, there is considerable debate about how HBO alone affects malignant glioma growth. Ding et al.7 and Wang et al.8 both demonstrated that HBO promotes the growth of in situ glioma in mice via mechanisms that involve the inhibition of apoptosis and the promotion of tumor angiogenesis. Under physiological conditions, HBO is reported to promote tumor angiogenesis by inducing oxidative stress9.
However, one study indicated that short-term HBO exposure promotes tumor cell proliferation, whereas prolonged HBO exposure promotes apoptosis and inhibits proliferation10. Therefore, further studies are needed to explore whether HBO promotes or inhibits the growth of glioma and how HBO combined with radiotherapy or chemotherapy can induce therapeutic sensitization. In particular, mechanistic details about how HBO improves the radiosensitivity of glioma are needed. To explore how HBO improves the radiosensitivity of human U251 glioma cells in this study, we used HBO combined with X-ray irradiation on glioma cell proliferation and observed the effects on cell cycle distribution and apoptosis.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
None.
Materials
| 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 |
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