| Objective: Radiotherapy plays an important role in the treatment of malignancies, and it is involved in about 70% of total malignancies. The outcome of radiation therapy depends on the radiosensitivity of tumor cells and the tolerance of the tissue bed to radiation. The effect of radiation in some malignancies, including colorectal cancer , is not very satisfactory because of significant short-term and long-term toxicity. Radiosensitizing agents can enhance the killing effect of radiation to tumor cells,and is an important way to increace radiotherapy outcome without significant complications. Evidence from experiments in vitro and in vivo and clinical trials has showed selective cyclooxygenase-2 inhibitors act as potent radiosensitizers. The mechanisms of radiosensitivity enhancement of selective cyclooxygenase-2 inhibitors are complicated and not very clear. In this study I explored the radiosensitization potential of NS-398, a selective cyclooxygenase-2 inhibitor,on cyclooxygenase-2 positive colorectal adenocarcinoma cell line HT-29 and the involving mechanisms.This study help to develop efficient and safe radiosensitizing agents.Methods: MTT assay was used to measure the proliferation inhibition of selective COX-2 inhibitor NS-398 in different concentrations (25 μ mol/L, 50 μ mol/L, 100 μ mol/L, 500 μ mol/L) and durations ( 1d, 2d, 3d, 4d) on colorectal adenocarcinoma cell line HT-29. After HT-29 cells had been incubated with 25 μ mol/L NS-398 for 24h before X-ray irradiation(IR) at different doses(0,2,4,6,8Gy), clonogenic survival assay was carried out to determine the radiosensitizing effect of NS-398 on HT-29 with IR of 8 Gy X-ray, and clonogenic survival curve was constructed from at least three independent experiments. Flow cytometry was used to measure DNA content, and the percentages of different phases of cells and the apoptotic index were calculated. DNA fragmentation assay was performed to observe the cells apoptosis. RT-PCR and flow cytometric analysis were used to detect the expression of Bax and Bcl-2. Caspase 3,8,9 activation were measured by spectrophotometry.Results: HT-29 cells were incubated with NS-398 at doses of 25, 50, 100, and 500 μ mol/L for 1, 2, 3, and 4 days, respectively. MTT assay showed that the proliferation of cells was inhibited at a dose- and time-dependent manner. The COX-2 inhibitor NS-398 at the concentration of 25 μ mol/L enhancing the killing effect of IR on HT-29 cells. The sensitization enhancement ratio(SER) was 1.36, 1.27 according to Dq, D0. NS-398(25 μ mol/L) did not increase the percentage of G2/M phase cells, and induced a decrease of S phase cells. After HT-29 cells had treated by IR of 8 Gy X-ray, NS-398 (25 μ mol/L), and both of above, agrose electrophoresis of DNA showed a typicl ladder pattern, especially in the cells treated by combinaition of IR and NS-398. Compared with untreated cells, Bax mRNA abundance of HT-29 cells significantly increased when the cells were treated by 25 μ mol/L NS-398, IR of 8 Gy, and both of above, and it was most dramatic in cells with combined treatment. However, 25 μ mol/L NS-398 pretreatment for 24 hours had no impact on the Bcl-2 transcription of HT-29 cells. The Bcl-2 mRNA abundance of HT-29 cells pretreated by IR of 8 Gy decreased. The activity of caspase 3, 8, 9 of the cells with pretreatment of 25 μ mol/L NS-398 , IR of 8 Gy, or combined treatment increased, especially for caspase 3,9.Conclutions: These findings indicated that selective COX-2 inhibitor NS-398 inhibited the growth of colorectal adenocarcinoma HT-29 cells at a dose- and time-dependent manner. NS-398 enhanced the radiosensitivity of HT-29 cells, and the mechanisms for this action might include inhibition of sublethal injury repair, direct cellular proliferation inhibition, and increasing the sensitivity of HT-29 to X-ray irradiation-induced apoptosis.Cytochrome C might play an important role in the sensitization enhancement of NS-398 on HT-29 to irradiation-induced apoptosis. |
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