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.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
F
F
Lin
C J
CJ
Bertling
P G
PG
Geiger
A W
AW
Girotti
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004351:Drug Resistance; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005293:Ferritins / Q000378:metabolism; D017324:Hematoporphyrin Derivative / Q000494:pharmacology*; D006414:Hematoporphyrin Photoradiation; D006427:Hemin / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D018462:Photobiology; D017319:Photosensitizing Agents / Q000494:pharmacology*
D017319:Photosensitizing Agents; D017324:Hematoporphyrin Derivative; D006427:Hemin; D005293:Ferritins; D007501:Iron; D003676:Deferoxamine