Abstract
Summary Statement
A novel transcutaneous CO2 therapy significantly enhanced the antitumor effectiveness of X-ray irradiation in human MFH xenografts The results strongly suggest that transcutaneous CO2 therapy could be a novel therapeutic tool for overcoming radioresistance in human malignancies.
Introduction
Hypoxia contributes to tumor radioresistance. In the presence of oxygen, reactive oxygen species (ROS) play crucial roles in cellular apoptosis to irradiation. We previously showed that a novel transcutaneous application of CO2 can improve hypoxia and that it induces apoptosis and decreases the expression of HIF-1α in sarcoma. Therefore, we hypothesised that a transcutaneous application of CO2 may increase radiosensitivity in sarcoma by improvement of hypoxic condition and increasing ROS production in tumors. The purpose of this study is to examine the effect of transcutaneous application of CO2 on radiosensitivity in human malignant fibrous histiocytoma (MFH) cells.
Methods
Cells. We used a human MFH cell line, Nara-H in this study. X-ray irradiation. X-ray irradiation was performed at a dose rate of 0.64 to 0.66 Gy/min. Colony formation assay. In vitro cell viability after X-ray irradiation was assessed by colony formation assay. In vivo studies. Nara-H cells were subcutaneously implanted to 24 nude mice which were randomly divided into 4 groups; CO2 group, X-ray group, Combination group and Control group. CO2 therapy was performed as we previously reported (1, 2). In combination group, mice were treated twice a week by X-ray at 3.2 Gy shortly after CO2 therapy for 2 weeks. The changes in body weight and tumor volume were monitored for 14 days in all 4 groups. The implanted tumors were excised at the end of experiment. We also excised tumors on the first day of each treatment in all 4 groups, and examined apoptosis and ROS expression by FACS analysis. The animal experiments were approved by the Animal Committee in our institute. Immunoblot analysis. The protein expression of HIF-1α, ROS-related proteins (p38 and JNK/SAPK), and apoptosis-related proteins (caspase-3 and PARP) were assessed by immunoblot analysis. FACS analysis. DNA fragmentation and ROS production in tumors were assessed by FACS analysis. Statistical analysis. ANOVA with post hoc test to compare for continuous values. All tests were considered significant at p<0.05.
Results
Approximately 50% of Nara-H cells survived after a total of 3.2 Gy X-ray irradiation. Tumor volume in combination group was significantly reduced at the end of experiment (47% of that in X-ray group and 28% of that in control group). In Combination group, apoptosis with ROS production markedly increased when compared with those in Control, CO2 or X-ray group at 24 hours after treatment. Immunoblot analysis showed that, in combination group, the expression of phospho-p38, phospho-JNK/SPAK, and cleavage of both caspase-3 and PARP were increased compared with other groups, conversely, the expression of HIF-1α was decreased.
Discussion/Conclusion
In this study, we demonstrated that the combination therapy showed more significant effects on apoptosis and ROS production through improving hypoxia in MFH cells in vivo. Our findings strongly suggest that the combination therapy of CO2 and X-ray could be a novel therapeutic strategy for the treatment of human MFH, and that transcutaneous application of CO2 may be one of the best radiosensitisers.
