Dwarakanath B S, Adhikari J S, Jain V
Department of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Delhi, India.
Int J Radiat Oncol Biol Phys. 1999 Mar 15;43(5):1125-33. doi: 10.1016/s0360-3016(98)00465-9.
Two deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolysis, has been shown to differentially inhibit the repair of radiation damage in cancer cells by reducing the flow of metabolic energy. Since hematoporphyrin derivatives (Hpd) inhibit certain enzymes of the respiratory metabolism, resulting in an increase in the glucose usage and glycolysis, Hpd could possibly enhance the energy-linked radiosensitizing effects of 2-DG in cancer cells. The purpose of the present work was to verify this suggestion.
Two human tumor cell lines (cerebral glioma, BMG-1 and squamous cell carcinoma, 4197) and a murine tumor cell line (Ehrlich ascites tumor [EAT], F-15) in vitro were investigated. A commercially available preparation of Hpd, Photosan-3 (PS-3) was used in the present studies. Cells incubated with 0-10 microg/ml PS-3 for 0-24 h before irradiation were exposed to 2.5 Gy of Co-60 gamma rays and maintained under liquid holding conditions for 1-4 h to facilitate repair. 2-DG (0-5 mM) added at the time of irradiation was present during the liquid holding. Radiation-induced cytogenetic damage (micronuclei formation) and cell death (macrocolony assay) were analyzed as parameters of radiation response. Effects of these radiosensitizers on glucose usage and glycolysis were also studied by measuring the glucose consumption and lactate production using enzymatic assays.
The glucose consumption and lactate production of BMG-1 cells (0.83 and 1.43 pmole/cell/h) were twofold higher than in the 4197 cells (0.38 and 0.63 pmole/cell/h). Presence of PS-3 (10 microg/ml) enhanced the rate of glycolysis (glucose consumption and lactate production) in these cells by 35% to 65%, which was reduced by 20% to 40% in the presence of 5 mM 2-DG. In exponentially growing BMG-1 and EAT cells, presence of 2-DG (5 mM; equimolar with glucose) for 4 hours after irradiation increased the radiation-induced micronuclei formation and cell death by nearly 40%, whereas no significant effects could be observed in 4197 cells. In EAT cells, radiation was also observed to induce apoptotic death, which was significantly increased in the presence of the combination (PS-3 + 2-DG). The combination (PS-3 + 2-DG) enhanced the radiation damage in all three cell systems by 60-100%. Furthermore, the radiosensitizing effects of the combination (PS-3 + 2-DG) were higher at pH 6.7 as compared to pH 7. 4. In the plateau phase, presence of 2-DG alone did not significantly influence the radiation response of either BMG-1 or of 4197 cells, whereas in combination with PS-3, 2-DG enhanced the radiation damage in both these cell lines by 40% to 50%. Furthermore, in BMG-1 cells, the effects of 2-DG were observed to be reversible to a very great extent, while that of the combination were mostly irreversible.
The hematoporphyrin derivative PS-3 enhances the radiosensitizing effects of 2-DG in cancer cells, possibly by further reducing the energy supply leading to an irreversible inhibition of DNA repair, and increased cytogenetic damage and cell death. Since both these compounds have been used in clinical practice, further studies to investigate their use in improving radiotherapy of tumors are warranted.
2-脱氧-D-葡萄糖(2-DG)是一种葡萄糖转运和糖酵解抑制剂,已被证明可通过减少代谢能量流动来差异性地抑制癌细胞中辐射损伤的修复。由于血卟啉衍生物(Hpd)可抑制呼吸代谢的某些酶,导致葡萄糖利用和糖酵解增加,Hpd可能会增强2-DG在癌细胞中的能量相关放射增敏作用。本研究的目的是验证这一推测。
对两种人肿瘤细胞系(脑胶质瘤,BMG-1和鳞状细胞癌,4197)以及一种小鼠肿瘤细胞系(艾氏腹水瘤[EAT],F-15)进行体外研究。本研究使用了市售的Hpd制剂Photosan-3(PS-3)。在照射前将细胞与0-10μg/ml PS-3孵育0-24小时,然后暴露于2.5 Gy的钴-60γ射线,并在液体保持条件下维持1-4小时以促进修复。在照射时添加的2-DG(0-5 mM)在液体保持期间存在。分析辐射诱导的细胞遗传损伤(微核形成)和细胞死亡(大集落测定)作为辐射反应的参数。还通过酶促测定法测量葡萄糖消耗和乳酸产生,研究了这些放射增敏剂对葡萄糖利用和糖酵解的影响。
BMG-1细胞的葡萄糖消耗和乳酸产生(0.83和1.43 pmole/细胞/小时)比4197细胞(0.38和0.63 pmole/细胞/小时)高两倍。PS-3(10μg/ml)的存在使这些细胞中的糖酵解速率(葡萄糖消耗和乳酸产生)提高了35%至65%,而在5 mM 2-DG存在下降低了20%至40%。在指数生长的BMG-1和EAT细胞中,照射后4小时存在2-DG(5 mM;与葡萄糖等摩尔)使辐射诱导的微核形成和细胞死亡增加了近40%,而在4197细胞中未观察到显著影响。在EAT细胞中,还观察到辐射诱导凋亡死亡,在联合使用(PS-3 + 2-DG)时显著增加。联合使用(PS-3 + 2-DG)在所有三种细胞系统中使辐射损伤增加了60-100%。此外,与pH 7.4相比,联合使用(PS-3 + 2-DG)在pH 6.7时的放射增敏作用更高。在平台期,单独存在2-DG对BMG-1或4197细胞的辐射反应没有显著影响,而与PS-3联合使用时,2-DG使这两种细胞系中的辐射损伤增加了40%至50%。此外,在BMG-1细胞中,观察到2-DG的作用在很大程度上是可逆的,而联合使用的作用大多是不可逆的。
血卟啉衍生物PS-3增强了2-DG在癌细胞中的放射增敏作用,可能是通过进一步减少能量供应,导致对DNA修复的不可逆抑制,并增加细胞遗传损伤和细胞死亡。由于这两种化合物都已用于临床实践,因此有必要进一步研究它们在改善肿瘤放射治疗中的应用。