Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines

We report the effect of motexafin gadolinium (MGd) and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, cell cycle, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptotic response. Treatment with exogenous zinc increased oxidative stress and, conversely, oxidative stress resulted in increased intracellular free zinc levels. To better understand the molecular basis of these results, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of MTF-1 and HIF-1 regulated genes. Cultures co-treated with MGd and zinc acetate displayed further increases in the levels of MTF-1 and HIF-1 regulated transcripts as well as additional transcripts regulated by NRF-2. Overall, these studies suggest that co-treatment of Ramos cells with MGd and zinc acetate increase intracellular zinc levels with a concomitant rise in oxidative stress levels that activate adaptive survival responses but eventually lead to cell death. Keywords: Dose response Ramos cells were treated with MGd alone or along with different concentrations of zinc acetate. Each treatment was done in triplicates. Expression was compared to that of cells treated with mannitol.

本研究报道了莫特沙芬钆（motexafin gadolinium, MGd）与外源性锌对指数生长期人类B细胞淋巴瘤及其他血液系统细胞系的细胞内游离锌水平、氧化应激、细胞增殖、细胞周期与细胞死亡的影响。研究发现，氧化应激与细胞内游离锌水平升高先于细胞周期阻滞与凋亡应答出现，且二者与后者存在显著相关性。外源性锌处理可提升氧化应激水平；反之，氧化应激亦可导致细胞内游离锌水平上升。为进一步阐明上述实验结果的分子机制，本研究对经MGd和/或乙酸锌（zinc acetate）处理的拉莫斯（Ramos）细胞培养物开展了基因表达谱分析（gene expression profiling）。单独使用MGd或乙酸锌处理的细胞培养物，其转录应答以金属调节因子1（MTF-1）与缺氧诱导因子1（HIF-1）调控的基因诱导为特征。联合使用MGd与乙酸锌处理的培养物，不仅MTF-1和HIF-1调控的转录本水平进一步升高，还出现了核因子红细胞2相关因子2（NRF-2）调控的额外转录本。总体而言，本研究表明，联合使用MGd与乙酸锌处理拉莫斯细胞，可升高细胞内锌水平，同时伴随氧化应激水平上升，后者可激活适应性存活应答，但最终诱导细胞死亡。关键词：剂量反应（Dose response）。本研究将拉莫斯细胞分为单独MGd处理组，以及联合不同浓度乙酸锌处理组；每组处理均设置三次生物学重复，以甘露醇（mannitol）处理组的细胞基因表达水平作为对照。