Santini Maria Teresa, Romano Rocco, Rainaldi Gabriella, Indovina Paola, Ferrante Antonella, Motta Andrea, Indovina Pietro Luigi
Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161, Rome, Italy.
Radiat Res. 2006 Nov;166(5):734-45. doi: 10.1667/RR0635.1.
The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.
利用高分辨率质子核磁共振（(1)H-NMR）光谱技术，研究了MG-63骨肉瘤三维肿瘤球体在辐射诱导凋亡过程中随时间变化的代谢变化。具体而言，将暴露于5 Gy电离辐射后24、48和72小时收集的MG-63球体的(1)H-NMR光谱与其各自对照组的光谱进行比较。使用无缺氧中心的小球体（直径约50-80微米）。通过用Hoechst 33258染料对球体DNA进行染色以及在所检查的三个时间点测定半胱天冬酶3酶活性来验证凋亡。结果表明，随着暴露于电离辐射后凋亡百分比随时间增加，MG-63球体中发生的代谢变化遵循非常精确的时间动态。特别是，观察到许多作者认为是凋亡标志物的CH(2)和CH(3)可移动脂质均随时间显著增加。此外，还观察到含胆碱代谢物、还原型谷胱甘肽（GSH）、谷氨酰胺/谷氨酸、牛磺酸、丙氨酸、肌酸/磷酸肌酸和乳酸的时间变化。这些数据表明，除了CH(2)和CH(3)脂质外，其他代谢物对于更深入理解辐射诱导凋亡的时间动态也可能极其有用。这种理解在球体中尤为重要，球体是一种极其复杂的细胞模型，比单层培养更接近体内肿瘤。最终，希望此类研究能够有助于更准确地评估放射治疗方案的结果。
