Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that selectively localizes in tumors and promotes redox stress by oxidizing intracellular reducing species. MGd, at low concentrations, induces expression of metallothioneins (MT) and zinc transporter 1 (ZnT-1) transcripts in vitro. In the present study, we describe the effects of MGd on zinc ion homeostasis, thioredoxin reductase activity and cytotoxicity in lymphoma cell lines. Human lymphoma (Ramos, DHL-4, HF-1) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Zinc (0–100 μM) and 0–25 μM MGd were added for 4–6 hr. Medium was exchanged and thioredoxin reductase activity was assessed by measuring the rate of lipoate reduction (

Anal. Biochem.
). In other experiments, cells were treated with MGd and zinc, and analyzed by flow cytometry using annexin-V, propidium iodide, and the ion-specific fluorescent probe, FluoZin-3-AM™. RNA from treated cultures was harvested and metallothionein and ZnT-1 induction assessed by Northern blotting. Treatment with MGd and zinc led to synergistic increases in free intracellular zinc levels, inhibition of lipoate reduction, MT and ZnT-1 induction, and cytotoxicity. In DHL-4, exposure to 10 μM MGd and 50 μM zinc for 3 hr led to a 2.5-fold increase in FluoZn-3 fluorescence, relative to treatment with zinc alone. In Ramos, this treatment led to 1.4, 2.6, 5.2, and 30-fold increase in FluoZn-3 fluorescence after 2, 4, 6, or 24 hr. There was a 2-fold increase in annexin-V positive Ramos cells after 6 hr, and an 11-fold increase in propidium iodide permeable cells by 24 hr. The rate of lipoate reduction decreased to 69%, 47%, and 58% of control in Ramos, DHL-4, and HF-1 cell lines after 5 hr under these conditions. MT and ZnT-1 transcript levels were elevated within 4 hr after treatment with MGd, zinc, or the combination, and remained elevated for at least 24 hr. For example, in HF-1, MT transcript levels were increased 20, 40, and 173-fold by MGd, zinc, or the combined treatment after 8 hr. These observations support the characterization of MGd as a redox cycling agent that increases the intracellular availability of free zinc. This activity leads to inhibition of thioredoxin reductase and, ultimately, induction of cell death. The proposed mechanism of action supports the use of this agent alone or in combination with various chemotherapy agents that induce redox stress.

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