Raju Uma, Nakata Eiko, Yang Peiying, Newman Robert A, Ang Kian K, Milas Luka
Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Int J Radiat Oncol Biol Phys. 2002 Nov 1;54(3):886-94. doi: 10.1016/s0360-3016(02)03023-7.
Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system.
Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined.
The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 microM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 microM, 3 days) removed the "shoulder" region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 microM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry.
SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug's concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage.
据报道，选择性环氧化酶-2抑制剂可增强肿瘤在体内对辐射的反应，但放射增敏作用的细胞机制尚不清楚。在本研究中，我们使用小鼠肉瘤细胞培养系统研究了几种可能的机制。
从小鼠肉瘤中分离得到的细胞，命名为NFSA，在体外培养，并在有或无选择性环氧化酶-2（COX-2）抑制剂SC-236（4-[5-(4-氯苯基)-3-(三氟甲基)-1H-吡唑-1-基]苯磺酰胺）预处理的情况下，接受不同（单次或分次）剂量的辐射。检测细胞的克隆形成存活率，以确定SC-236的放射增敏作用。此外，进行MTT法和TUNEL法检测，以确定SC-236和辐射对细胞存活及细胞周期分布的影响。使用编码选定细胞周期调节蛋白（如细胞周期蛋白和细胞周期蛋白依赖性激酶）的探针，对总RNA提取物进行核糖核酸酶保护分析。为监测COX-2活性程度及其在放射增敏中的作用，还测定了前列腺素E2的细胞含量，前列腺素E2是COX-2对花生四烯酸活性的主要代谢产物。
细胞克隆形成存活率分析表明，SC-236显著增强了肿瘤细胞的放射敏感性：在0.1细胞存活水平时，50μM的SC-236使其提高了1.51倍。用SC-236（50μM，3天）处理消除了辐射存活曲线上的“肩”区，表明该药物抑制了亚致死性辐射损伤的修复。分次剂量实验证实了这种抑制作用，即相隔4小时给予两剂（各3 Gy）辐射。仅接受辐射的细胞将损伤修复了1.44倍，而接受SC-236加辐射处理的细胞仅将损伤修复了1.1倍。虽然SC-236诱导了这些NFSA细胞凋亡，但辐射未诱导凋亡。当细胞同时暴露于SC-236和辐射时，未观察到凋亡进一步增加，这表明SC-236未使肿瘤细胞对辐射诱导的凋亡更敏感。核糖核酸酶保护分析表明，SC-236（50μM，3天）抑制了细胞周期蛋白A和B以及细胞周期蛋白依赖性激酶-1的表达。通过流式细胞术测定，SC-236对这些细胞周期调节元件的抑制与细胞在放射敏感的G2-M期（67%）停滞有关。
SC-236显著增强了肿瘤细胞的放射敏感性；敏感程度取决于药物浓度。可能的机制包括细胞在细胞周期的放射敏感G2-M期积累以及抑制亚致死性辐射损伤的修复。
