DELAYED HYPERRESISTANCE OF ENDOTHELIAL-CELLS TO PHOTODYNAMIC INACTIVATION AFTER CONTACT WITH HEMIN

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemog lobin, is a potential source of prooxidant iron in heavily vascularize d tumors. We have evaluated hemin's effects on photodynamic inactivati on of bovine artery endothelial cells, using a partially purified olig omeric fraction of hematoporphyrin derivative (HPD-A) as the sensitizi ng agent. Confluent cells in 5% serum/RPMI medium showed a progressive loss of thiazolyl blue (MTT)-detectable viability when irradiated wit h broadband visible light in the presence of HPD-A, Cells pretreated w ith desferrioxamine (DFO) were substantially less sensitive to photoki lling, implying that nonheme iron plays a role in cytotoxic activity. Hemin (10-20 mu M) had remarkably different effects on photokilling, d epending on the time interval between adding it to cells and exposing them to photodynamic action. For example, cells were more sensitive wh en 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, Immunob lot analysis following hemin treatment revealed a progressive inductio n of the heavy (H) subunit of ferritin that paralleled the development of hyperresistance, After incubation with saturating levels of the sy nthetic iron donor [Fe-55]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 vascula tures could expose endothelial and neoplastic cells to chronic hemoglo bin-derived ir on. Consequent upregulation of ferritin could impact ne gatively on the efficacy of photodynamic therapy and other oxidant-bas ed cancer therapies.