Delayed hyperresistance of endothelial cells to photodynamic inactivation after contact with hemin.

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a potential source of prooxidant iron in heavily vascularized tumors. We have evaluated hemin's effects on photodynamic inactivation of bovine artery endothelial cells, using a partially purified oligomeric fraction of hematoporphyrin derivative (HPD-A) as the sensitizing agent. Confluent cells in 5% serum/RPMI medium showed a progressive loss of thiazolyl blue (MTT)-detectable viability when irradiated with broadband visible light in the presence of HPD-A. Cells pretreated with desferrioxamine (DFO) were substantially less sensitive to photokilling, implying that non-heme iron plays a role in cytotoxic activity. Hemin (10-20 microM) had remarkably different effects on photokilling, depending on the time interval between adding it to cells and exposing them to photodynamic action. For example, cells were more sensitive when photostressed immediately after 1 h hemin treatment and washing but much more resistant when photostressed 23 h later. Similar responses were observed when cells were challenged with glucose oxidase. Immunoblot analysis following hemin treatment revealed a progressive induction of the heavy (H) subunit of ferritin that paralleled the development of hyperresistance. After incubation with saturating levels of the synthetic iron donor [55Fe]ferric-8-hydroxyquinoline, hemin-stimulated cells contained about four times more immunoprecipitable ferritin 55Fe than controls. This is consistent with the notion that sequestration of toxic iron as a result of induction of H-chain-enriched ferritin is a key factor in hyperresistance. Inflammatory injury in tumor vasculatures could expose endothelial and neoplastic cells to chronic hemoglobin-derived iron. Consequent upregulation of ferritin could impact negatively on the efficacy of photodynamic therapy and other oxidant-based cancer therapies.

Role of hydrogen peroxide in the cytotoxic effects of UVA/B radiation on mammalian cells.

Effects of selenium (Se) deficiency on the sensitivity of murine leukemia L1210 cells to broad band UVA/B radiation (310-400 nm) have been investigated. Cells rendered glutathione peroxidase (GPX) deficient by shortterm (2-3 week) growth in 1%, serum/RPMI medium without added Se [L.Se(-) cells] were found to be much less resistant to clonally assessed UVA/B lethality than Se-supplemented controls [L.Se(+) cells]. By contrast, long-term ( > 20 week) Se-deprived [L'.Se(-)] cells whose catalase (CAT) activity was elevated > 100-fold were far more resistant to UVA/B than L.Se(+) cells. Similar trends were observed for cells irradiated in 1% serum/RPMI or Hank's medium. Whereas the CAT inhibitor 3-amino-1,2,4-triazole had no effect on L.Se(+) photosensitivity, it produced a large increase in L'.Se(-) photosensitivity. These findings are consistent with H2O2 intermediacy in photokilling and suggest that L1210 cells depend mainly on GPX for protection against this species but switch to overexpressed CAT after chronic Se deprivation. In agreement with this, steady-state H2O2 levels measured by H2O2 electrode during UVA/B exposure were higher in L.Se(-) than L.Se(+) suspensions but much lower (barely detectable) in L'.Se(-) suspensions. Cytotoxic effects of UVA/B and variations thereof resulting from Se manipulation could be mimicked by treating cells with glucose oxidase in the presence of D-glucose, providing further support for H2O2 involvement. Whether UVA/B-generated H2O2 is directly cytotoxic or gives rise to a more damaging species such as hydroxyl radical (HO) is presently unknown.