Establishment of new molecular-targeted therapy for leukemia mediated through HIF-1 regulation by ROS

白血病的治疗方法以化疗为基础，但所用药物的副作用和各种并发症有时是致命的。近年来，分子靶向治疗在临床上受到重视，因此，我们一直在寻找新的药物，以建立对白血病患者更无创的治疗。我们报道了活性氧（ROS）是绿色茶多酚（-）-表没食子儿茶素-没食子酸酯（EGCG）诱导细胞凋亡的关键介质。表没食子儿茶素没食子酸酯能迅速诱导髓过氧化物酶阳性白血病细胞系和急性髓细胞白血病新鲜标本的凋亡，但对髓过氧化物酶阴性白血病细胞系和急性髓细胞白血病新鲜标本的凋亡无明显影响。MPO阳性白血病细胞与MPO特异性抑制剂4-氨基苯甲酸酰肼（ABAH）和血红素生物合成抑制剂琥珀酰丙酮（ScAc）的预孵育导致加入EGCG后ROS产生的抑制和细胞凋亡的诱导。为了研究MPO在EGCG诱导的细胞凋亡中的作用，我们将全长MPO cDNA转染到人肝癌细胞系中， ...更多信息 并将空载体导入MPO阴性的K562细胞。与对照细胞相比，MPO转染细胞增强MPO活性和ROS产生，并使EGCG抗性K562细胞对ROS产生剂诱导的凋亡敏感。此外，一个酶失活的MPO突变体表达K562（K562/H502 A）细胞不能响应EGCG，这表明MPO是重要的决定敏感性，EGCG诱导的氧化应激。此外，值得注意的是，在用EGCG刺激后，负载APF和HPF的髓性白血病细胞的荧光强度均显著增加，表明EGCG在MPO阳性白血病细胞中产生高毒性ROS。这些结果表明，H_2O_2/MPO/卤化物系统产生的高毒性活性氧（如羟自由基）可诱导细胞凋亡，这些活性氧可能是氧化应激诱导MPO阳性白血病细胞凋亡的直接介质。Affiplatin基因芯片的基因表达谱显示HIF-1α及其下游基因（RTP 801、EGR-1、BNIP 3和VEGF）表达上调，提示HIF-1α通路在ROS介导的细胞凋亡中起重要作用。少

The therapeutic approach to leukemia is based on chemotherapy, but the side effects of the drugs used and various complications are sometimes fatal. Recently, molecular-targeted therapy is pay attention in the clinical setting ; therefore, we have sought out new agents to establish the more non-invasive therapy for the patients with leukemiaWe have reported that reactive oxygen species (ROS) are key mediators of apoptosis induced by a green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG). EGCG rapidly induced apoptosis in MPO-positive, but not MPO-negative leukemia cell lines as well as fresh samples from AML. Pre-incubation of MPO-positive leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and the heme biosynthesis inhibitor, succinylacetone (ScAc), resulted in inhibition of the ROS production and induction of apoptosis following addition of EGCG. To investigate the role of MPO in EGCG-induced apoptosis, we transfected the full length MPO cDNA … More and empty vector into MPO-negative K562 cells. In contrast to control cells, MPO transfected cells enhanced MPO activity and ROS production, and sensitized EGCG-resistant K562 cells to apoptosis induced by ROS-producing agents. In addition, an enzymatically inactive MPO mutant expressing K562 (K562/H502A) cells could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. Further, it is noteworthy that the fluorescence intensity of both APF-and HPF-loaded myeloid leukemic cells significantly increased upon stimulation with EGCG suggesting that EGCG generated highly toxic ROS in MPO-positive leukemic cells. Taken together, these observations indicate that highly toxic ROS such as hydroxyl radical generated via the H_2O_2/MPO/halide system induce apoptosis, and that such ROS may be the direct mediators of oxidative stress-induced apoptosis in MPO-positive leukemic cells. Gene expression profiling with Affimetrix gene chips demonstrated HIF-1α and its down-stream genes (RTP801,EGR-1,BNIP3, and VEGF), that were up-regulated, suggesting that HIF-1α pathways will be important for ROS-mediated apoptosis. Less