No abstract

To gain a better understanding of the mechanism of action of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer. (Cancer Res 2005; 65(9): 3837-45)

Supplementary Methods from Motexafin Gadolinium Disrupts Zinc Metabolism in Human Cancer Cell Lines

<div>Abstract<p>To gain a better understanding of the mechanism of action of the metal cation–containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (<i>ZnT1</i>) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer.</p></div>

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 (Biaglow, Anal. Biochem.281:77, 2000). 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.