In vitro and in vivo interactions of D-penicillamine with tumors.

D-penicillamine (PSH) is a copper chelator that generates hydrogen peroxide and inhibits neovascularization. As hydrogen peroxide is toxic to some tumor cells and to blood vessels, we reasoned that PSH plus copper would inhibit tumors in vivo and in vitro. To test this hypothesis, we first incubated murine J558L plasmacytoma cells with varying combinations of drug (PSH and/or copper sulfate) plus modulators (fetal calf serum, dithiothrietol, catalase, eosinophil peroxidase) and then used fluorescence microscopy to measure cell proliferation, necrosis, and apoptosis. We also incubated various types of human tumor cells with PSH plus copper for 24 hours and then measured the number of surviving cells 24 hours later. For the in vivo studies, we measured the effects of 7 daily i.p. injections of 10 mg of PSH on the growth rates of interleukin-5 genetransfected J558L tumors in 20 BALB/c mice. Our experiments demonstrated that PSH plus copper exerted a significant antiproliferative effect on tumor cells in vitro that was neutralized by protein or catalase and enhanced by adherent eosinophil peroxidase. Human acute myelogenous leukemia cells were especially sensitive to PSH plus copper. In vivo, however, PSH had no significant effect on the growth rates of J558L tumors that were infiltrated by eosinophils. We conclude that the interaction of PSH-copper with tumors is primarily antiproliferative, mediated by hydrogen peroxide, and inhibitable by protein. Therefore, for PSH to be an effective antineoplastic drug strategies will need to be developed to prevent its rapid neutralization by protein.