Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu2+ and Zn2+

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Abstract
Pyrrolidine dithiocarbamate (PDTC) has been found to induce or inhibit apoptosis in different cell types. Here we show that PDTC dose-dependently reduced the viability of rat smooth muscle cells (rSMC), human fibroblasts, and endothelial cells at low but not at high cell density. Endothelial cells were least sensitive, fibroblasts showed a medium sensitivity, and rSMC showed a high sensitivity to PDTC-mediated cell death. An early reduction in the mitochondrial membrane potential indicated a rapid onset of apoptosis in rSMC. Apoptosis was further confirmed by annexin V staining and DNA fragmentation analysis. Gel shift analysis demonstrated increased nuclear factor (NF)-κB activity in high-density rSMC compared with low-density cells. NF-κB has recently been shown to regulate the induction of anti-apoptotic proteins. Although PDTC is widely used as an inhibitor for NF-κB and a radical scavenger, our data show that PDTC rather enhanced NF-κB activity and, alone or in combination with menadione, induced oxygen radical generation. Notably, PDTC failed to reduce rSMC viability in medium without Cu2+ or Zn2+, and addition of Cu2+ or Zn2+resulted in a dose-dependent increase in PDTC-induced cell death. Addition of both Cu2+ and Zn2+ showed synergistic effects. Our results indicate that the induction of apoptosis by PDTC requires Cu2+ and Zn2+ and is dependent on cell type and density. Such differential effects may have implications for studies of PDTC as an anti-atherosclerotic or immunomodulatory drug.