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Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells

Daniela Schilling^*,,1, Mathias Gehrmann^*,,1, Claudia Steinem, Antonio De Maio, Alan G. Pockley^||, Michael Abend^¶, Michael Molls^*, and Gabriele Multhoff^*,,2

^* Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany;

Helmholtz Center Munich, German Research Center for Environmental Health–Institute for Pathology, Munich, Germany;

Institute for Organic and Biomolecular Chemistry, Georg-August University, Göttingen, Germany;

Department of Surgery, University of California, San Diego, La Jolla, California, USA;

^|| School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK;

^¶ Federal Armed Forces Medical Academy, Institute of Radiobiology, Munich, Germany

² Correspondence: Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Ismaningerstrasse 22, 81675 Munich, Germany. E-mail: gabriele.multhoff{at}lrz.tu-muenchen.de

Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/27/3<5/5<4/6<3/7<2/8)_. In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC₅₀ of Hsp70=85 µg/ml) and hypoxic (EC₅₀ of Hsp70=55 µg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 µg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.—Schilling, D., Gehrmann, M., Steinem, C., De Maio, A., Pockley, A. G., Abend, M., Molls, M., Multhoff, G. Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells.

Key Words: nonapoptotic tumor cell death • stress proteins • ionizing radiation • cancer therapy

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