Motexafin Gadolinium Causes Apoptosis in Lymphoma Cells by Increasing Intracellular Free Zinc and Disrupting Redox Balance.

Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that is being studied in clinical trials in various types of cancer including hematologic malignancies. MGd has been shown to generate reactive oxygen species and induce apoptosis in myeloma and lymphoma cells, and to cause an increase in intracellular levels of free zinc. We now report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in human B-cell lymphoma and other hematologic cell lines. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. In cultures treated with MGd and zinc, 21% of Ramos cells were apoptotic within 8 hours of treatment as demonstrated by Annexin V staining. The apoptotic fraction increased to 30% within 12 hours and 68% by 24 hours. Analogous results were obtained using JC-1, with 38% of Ramos cells exhibiting non-aggregated (green) JC-1 fluorescence characteristic of mitochondrial dysfunction within 8 hours of combined treatment with MGd and zinc. This fraction increased to 52% by 12 hours and 74% by 24 hours. MGd and zinc similarly increased cell death in other B-cell lines (Raji, DB, DHL-4 and HF-1) tested. We also found that increased levels of oxidative stress and intracellular free zinc preceded and correlated with cell cycle arrest and apoptosis. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1) and hypoxia inducible transcription factor 1 (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 and also additional transcripts regulated by NF-E2-related transcription factor 2 (NRF-2). HIF-1 activation can have negative consequences for cell growth and survival by induction of targets linked to apoptosis, whereas NRF-2 activation is characteristic of cells responding to disruption of redox balance. These data demonstrate that MGd increases intracellular free zinc and oxidative stress levels in lymphoma cells that activate adaptive survival responses but eventually lead to cell death.