The study on the glutamate-induced glial cell death mechanism for modulation of apoptosis to necrosis by arachidonic acid-mediated lipid peroxidation

谷氨酸诱导的谷胱甘肽(GSH)耗竭导致C6大鼠胶质瘤细胞死亡，其机制是通过积累活性氧(ROS)或过氧化氢。在GSH耗竭条件下，花生四烯酸(AA)对12-脂氧合酶活性有显著的促进作用。AA促进谷氨酸诱导的细胞死亡，降低caspase-3活性，减少核小体DNA片段化。此外，AA降低细胞内NAD、ATP和膜电位，显示线粒体膜功能障碍。半胱氨酸酶抑制剂AC-DEVD不能抑制谷氨酸诱导的细胞溶解。这些结果表明，AA通过ROS或GSH耗竭过程中产生的脂质过氧化作用，通过诱导caspase-3非依赖性细胞凋亡而导致细胞死亡。其次，我们研究了AA对紫外线诱导的细胞死亡的影响。在致死剂量下，UV-C(254 Nm)辐射诱导细胞功能障碍，导致细胞凋亡或坏死。在T-24人膀胱癌细胞死亡过程中，观察到1-2MBP的DNA大片段，然后DNA片段进入大分子量100-800KBP的DNA片段，然后是梯状的核小体间DNA片段。不产生活性过氧化脂质或氧物种。相反，caspase-3表达增加，细胞内NAD和聚(ADP-核糖)聚合酶减少。UV-C辐射诱导T-24癌细胞DNA断裂，导致细胞凋亡，而不产生DCFH可检测到的活性氧，并伴随caspase-3的激活和核小体间DNA的断裂。

Glutamate induced glutathione (GSH) depletion leading to cell death in C6 rat glioma cells through accumulation of reactive oxygen species (ROS) or hydroperoxides. A significant increase of 12-lipoxygenase activities was observed in the presence of arachidonic acid (AA) under the GSH depletion. AA promoted the glutamate-induced cell death reducing caspase-3 activity and diminishing internucleosomal DNA fragmentation observed in apoptosis. Furthermore, AA diminished intracellular NAD, ATP and membrane potential revealing a dysfunction of mitochondrial membrane. Ac-DEVD, a caspase inhibitor, did not suppresse the glutamate-induced cytolysis. These results suggest that AA promotes cell death by inducing to necrosis from caspase-3 independent apoptosis through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during the GSH depletion in C6 cells.Next, we studied the effect of AA on UV-induced cell death. At lethal dose, UV-C (254 nm) radiation induces cell dysfunction leading to apoptosis or necrosis. During the cell death of T-24 human bladder carcinoma cells, 1-2 Mbp giant DNA fragmentation was observed and consequently the DNA fragmentation was proceeded into high molecular weight 100-800 kbp DNA fragmentation followed by ladder-like inter-nucleosomal DNA fragmentation. Reactive lipid peroxides or oxygen species were not produced. In contrast, increase of caspase-3 and reduction of intracellular NAD and poly (ADP-ribose) polymerase were observed. UV-C radiation induces giant DNA fragmentation leading to apoptosis associated without producing DCFH detectable reactive oxygen species and with activation of caspase-3 and internucleosomal DNA fragmentation in T-24 carcinoma cells.