| Backgrounds & Objectives:Nasopharyngeal carcinoma (NPC) is one of the most common human cancers, and is highly prevalent in Southern China. Radiotherapy is the primary treatment of NPC. Intensity-modulated radiation therapy (IMRT) has become common as a new radiotherapy technique for treatment of malignancies. Compared with conventional 3D conformal radiotherapy (3D-CRT), Intensity-modulated radiation therapy allows greater dose conformity to the tumor target and normal tissues sparing; meanwhile, IMRT optimizes dose distribution of radiotherapy, which thereby could increase the dose of the target tissues and decrease the dose of the surrounding normal tissues at risk. All these technical innovations have led on the one hand to delivering a much higher dose to the target volumes, thus possibly increasing local tumor control, while on the other hand minimizing the undue dose delivered to the surrounding normal tissues, thus possibly decreasing treatment morbidity. IMRT has obvious dosimetric superiority. Over the past few years, IMRT appears to be more and more commonly used for routine treatment, especially in head and neck (HN) malignancies, prostate cancer, breast cancer and lung cancer. However, only a few randomized trials have demonstrated its superiority over conventional treatments. This technique often requires 15～30 min or a longer time in one treatment session for precise positioning of patient. Several studies suggested that the radiobiological effectiveness of IMRT that require considerably long beam interruption might be less than that of continuous irradiation with the same dose owing to the recovery from sublethal damage in tumor cells. It is known that sublethal damage repair (SLDR), cell cycle and apoptosis influence the radiosensitivity of tumor cells. To our knowledge, however, no systematic research has been carried out regarding the influence of IMRT on radiosensitivity,apoptosis and cell cycle and radiosensitivity-related genes expression in cultured tumor cells. Methods:Radiosensitivity was analyzed by Courtenay assay. Cell growth inhibition was measured by MTT. Apoptosis and cell cycle were analyzed by flow cytometry. RT-PCR was used to detect mRNA expressions of damage repair -related genes: Ku80,ATM; Cell cycle regulation genes: Cyclin D1,Cyclin B1.Apoptotic regulation genes: Bcl2,Bax; Western blotting was used to analyze protein expression.Results:1.Courtenay assay1)α/βof CNE-2 was 13.177Gy.when we delivered 4 different radiation doses including 2Gy,4Gy,6Gy,8Gy, Surviving Fractions (SF) of convention radiation (CR) group were 0.2253±0.0177,0.013±0.002,0.0026±0.0006 and 0.0002±0.0001. Surviving Fractions (SF) of Simulation IMRT (SIMRT) group were 0.3353±0.0167, 0.0607±0.0093, 0.0069±0.0009 and 0.0006±0.0003, respectively. Compared with SIMRT group, SF of convention radiation group was lower in every dose point.2) Fitting cell survival curve by Linear quadratic equation and Multi-target one-shot model, the values ofα,β,N,D0,Dq of CR group were 0.674,8,0.051,2,1.890,0,0.885,0 and 0.563 0. The values ofα,β,N,D0,Dq of SIMRT group were 0.537,7,0.026,8,2.610,0,0.986,0 and 0.946,0, respectively; andαCR>αSIMRT,βCR>βSIMRT, NCR0.05). Western blotting showed the similar results on the protein level expressions.6.RT-PCR and Western blotting were employed to detect the expression of Cyclin B1,Cyclin D1. RT-PCR results showed: when we delivered 4 different radiation doses including 2Gy,4Gy,6Gy,8Gy1) The Cyclin B1 mRNA level expressions of CR group were 0.745±0.068,0.624±0.100,0.518±0.054,0.428±0.038.The Cyclin B1 mRNA level expressions of SIMRT group were 0.881±0.075,0.711±0.054,0.617±0.061,0.528±0.046; respectively. Compared with SIMRT group, the Cyclin B1 mRNA level expressions of CR group were lower in every dose point (P<0.05). Western blotting showed the similar results on the protein level expressions.2) The Cyclin D1 mRNA level expressions of CR group were 0.857±0.090,0.810±0.327,0.798±0.303,0.802±0.038.The Cyclin D1 mRNA level expressions of SIMRT group were 0.822±0.063,0.701±0.213,0.812±0.093,0.801±0.064, respectively. Compared with SIMRT group, the Cyclin D1 mRNA level expressions of CR group had no significant difference in every dose point (P>0.05). Western blotting showed the similar results on the protein level expressions.7.RT-PCR and Western blotting were employed to detect the expression of Bcl2,Bax. RT-PCR results showed: when we delivered 4 different radiation doses including 2Gy,4Gy,6Gy,8Gy1) The Bcl2 mRNA level expressions of CR group were 0.298±0.029,0.308±0.024,0.290±0.033,0.273±0.025. The Bcl2 mRNA level expressions of SIMRT group were 0.313±0.030,0.297±0.028,0.294±0.021,0.293±0.025; respectively. Compared with SIMRT group, the Bcl2 mRNA level expressions of CR group had no significant difference in every dose point (P>0.05). Western blotting showed the similar results on the protein level expressions. 2) The Bax mRNA level expressions of CR group were 0.618±0.061,0.706±0.032,0.817±0.064,0.915±0.076.The Bax mRNA level expressions of SIMRT group were 0.513±0.051,0.606±0.087,0.662±0.132,0.721±0.086, respectively. Compared with SIMRT group, the Bax mRNA level expressions of CR group is higher in every dose point (P<0.05). Western blotting showed the similar results on the protein level expressions.Conclusions:1.IMRT that require considerably long beam interruption may be one of the mechanisms responsible for altered radiosensitivity.2.Radiation-induced G2 arrest correlated with radiation dose and IMRT that required considerably long beam interruption weaken the effect of G2 arrest. Radiation-induced early and late apoptosis ratios were increasing when radiation dose raised and IMRT that required considerably long beam interruption weaken the effect of apoptosis and increased the rate of viable cells.3.Using RT-PCR and Western blotting to detect the expression of ATM,Ku80,Cyclin D1,Cyclin B1,Bcl2,Bax,and calculate the Bcl2/Bax ratio maybe a new approach to evaluate the altered IMRT radiobiological effect. And this unique radiobiological effect of IMRT is worthy of further research. |
PDF Full Text Request