ALBERT

Description: Sapphyrins are pentapyrrolic metal-free expanded porphyrins that localize to tumors. We have previously demonstrated that sapphyrins induce apoptosis in a variety of hematologic tumor cell lines including lymphomas (Ramos, DHL-4, HF-1), leukemias (Jurkat, HL-60), and myelomas (8226/S, 1-310, C2E3, 1-414). Through chemical modification of the parent compound, PCI-2000, a number of derivatives were generated and tested for induction of apoptosis in Ramos cells. PCI-2000 and one of the more potent apoptosis-inducing derivatives, PCI-2050, were injected into CD-1 nude mice bearing Ramos xenografts. Animals were sacrificed 48 hrs after injection and analyzed for drug uptake in the tumor, liver and spleen using flow cytometry. For PCI-2000, the relative uptake was spleen〉tumor〉liver. For PCI-2050 the relative uptake was tumor〉spleen〉liver, suggesting that PCI-2050 preferentially localizes in tumors compared to PCI-2000. Tumor cells isolated from PCI-2050 treated animals grew less well in culture and had more apoptotic cells than those derived from PCI-2000 or control animals. Uptake of PCI-2050 into xenograft tumor cells and tumor cell killing was dose dependent. PCI-2050 (10 umol/kg x 2 days in a row) was administered to Ramos xenograft bearing animals that were then monitored for tumor growth. In both minimal tumor (animals treated before tumor was palpable) and established tumor (palpable tumor) models, PCI-2050 reduced tumor growth by 60–75%. Alternative dosing strategies revealed that split dosing (allowing 1 or more days between doses) was more efficacious in tumor control than dosing 2 days in a row. At the doses used in this study, there was no myelosuppression or lymphosuppression, hepatic or renal abnormalities as assessed by complete blood count and comprehensive serum chemistry analysis, respectively. Our work demonstrates that PCI-2050 induces apoptosis in tissue culture and inhibits tumor growth in an animal tumor model while exhibiting minimal toxicity. PCI-2050 and other sapphyrin derivatives will be further evaluated as potential anti-cancer agents.

Description: 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.