Oxidants and Cell Death

摘要：我们正在研究过氧化氢等炎性氧化剂如何杀死肿瘤细胞，以及它们如何影响免疫系统对肿瘤细胞的识别和清除。大多数化疗药物通过诱导细胞凋亡来杀死肿瘤细胞。实体瘤常常被炎性吞噬细胞渗透，在肿瘤组织内产生氧化应激。此前，我们发现在过氧化氢存在的情况下，人类Burkitt淋巴瘤(BL)细胞无法对癌症化疗药物做出反应而发生凋亡，而是以坏死的形式死亡。过氧化氢和化疗药物相互作用的最重要后果之一是，在共培养的巨噬细胞膜溶解之前，细胞不会被吞噬。这可能会导致不受欢迎的炎症反应，从而使肿瘤细胞枯竭进一步复杂化。过去一年开展的研究旨在确定过氧化氢抑制巨噬细胞摄取垂死肿瘤细胞的分子机制。在诱导细胞凋亡后不久，死亡的细胞通过将磷脂酰丝氨酸(PS)从质膜的内叶转移到外叶而被吞噬。我们发现，即使BL细胞在质膜表面表达PS，双氧水也能抑制其吞噬功能。过氧化氢处理并不抑制蛋白质(膜联蛋白)与PS的结合，也不改变PS在凋亡细胞膜上的空间分布(通过共聚焦显微镜测定)。这些结果表明，PS对巨噬细胞识别和摄取凋亡细胞是必要的，但还不够。此外，他们还表明，过氧化氢通过改变凋亡细胞表面一种单独的、迄今尚未确定的吞噬标记来发挥作用。在另一系列研究中，我们试图确定过氧化氢杀死细胞的机制是否在炎症细胞产生的所有氧化剂中都是典型的。此前，我们发现过氧化氢诱导人B淋巴瘤细胞的主要死亡方式是非凋亡性的。在最近的研究中，我们研究了另外两种已知由激活的吞噬细胞产生的氧化剂杀死细胞的机制。用细胞核形态、半胱氨酸天冬氨酸氨基转移酶活性和PS翻转来评价细胞的凋亡和坏死。我们发现，黄嘌呤氧化酶产生的超氧化物完全通过形成过氧化氢来杀死细胞，使细胞主要通过固缩/坏死而死亡。相反，HOCl通过细胞凋亡来杀死细胞。H_2O_2和HOCl之间的差异似乎在于它们对ATP水平的影响；H_2O_2使细胞内的ATP降到对照水平的25%以下，而在HOCl处理后，ATP保持在对照的50%以上。当通过抑制聚(ADP)核糖聚合酶活性来维持ATP水平时，过氧化氢也会通过凋亡来杀死细胞。人们正在寻找这些氧化剂的分子靶标。

Summary: We are investigating how inflammatory oxidants such as hydrogen peroxide (H2O2) kill tumor cells and how they may affect tumor cell recognition and elimination by the immune system. Most chemotherapeutic agents kill tumor cells by inducing apoptosis. Solid tumors are often infiltrated by inflammatory phagocytes which can generate oxidative stress within the tumor tissue. Previously, we found that in the presence of H2O2, human Burkitt's lymphoma (BL) cells are unable to undergo apoptosis in response to cancer chemotherapy drugs and die instead by a form of necrosis. One of the most important consequences of the interaction between H2O2 and the chemotherapy drugs is that the cells do not become phagocytosed by co-cultured macrophages until after their membranes have lysed. This can lead to an undesirable inflammatory reaction which can further complicate tumor cell depletion. Studies carried out during the past year were aimed at identifying the molecular mechanism whereby H2O2 inhibits uptake of dying tumor cells by macrophages. Shortly after the induction of apoptosis, dying cells are targeted for phagocytosis via the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. We found that H2O2 inhibits phagocytosis of BL cells even when they express PS on the exofacial surface of the plasma membrane. H2O2 treatment does not inhibit protein (annexin) binding to PS, nor does it modify the spatial distribution of PS on the apoptotic cell membrane (as determined by confocal microscopy). These results indicate that PS is necessary, but is not sufficient for recognition and uptake of apoptotic cells by macrophages. Further, they suggest that H2O2 acts by modifying a separate, as yet unidentified phagocytic marker on the surface of apoptotic cells. In a separate series of studies, we sought to determine whether the mechanism of cell killing by H2O2 is typical of all oxidants generated by inflammatory cells. Previously, we found that the primary mode of death induced in human B lymphoma cells by H2O2 is non-apoptotic. In more recent studies, we examined the mechanism of cell killing by the two other oxidants known to be generated by activated phagocytes. Apoptosis and necrosis were assessed by nuclear morphology, caspase activity, and PS flipping. We found that superoxide generated by xanthine oxidase kills cells entirely through formation of H2O2 such that the cells die primarily by pyknosis/necrosis. In contrast, HOCl kills the cells by apoptosis. The difference between H2O2 and HOCl appears to lie in their effects on ATP levels; H2O2 causes intracellular ATP to drop below 25% of control levels whereas ATP is maintained above 50% of control after HOCl treatment. When ATP levels are maintained by inhibiting poly(ADP)ribose polymerase activity, H2O2 also kills the cells by apoptosis. The molecular targets for these oxidants are being sought.