Abstract C240: Protein photoadducts of acridine photosensitizers

Abstract

Abstract Photodynamic therapy is used to treat many cancers and some non-cancerous conditions. It exploits photosensitizers, compounds that are only toxic to cells when light exposure causes them to generate reactive oxygen species that damage many cellular molecules and organelles. A common observation is the shrinkage of distant unirradiated tumors as the result of photodynamic treatment of a single tumor. This systemic response against tumors is thought to be immunological and to involve inflammation and cytokines generated by the treated tumor in response to photodynamic damage. The possible role of photodynamic damage to proteins in such a global immune response has been underexplored. We hypothesized that photodynamically damaged tumor cell proteins might act as immunological adjuvants, stimulating an immune response to tumor specific proteins so that the photodynamically treated tumor acts as a tumor vaccine. Photoaddition of the photosensitizing molecule to cellular proteins could be one molecular mechanism for this since such adducts would be expected to produce highly immunogenic haptens on tumor cell proteins. In a test of this hypothesis, we have investigated photoaddition of acridines to cellular proteins. Cultured mammalian cells were treated with radiolabeled proflavine or acriflavine and light. SDS gel electrophoresis and gel fluorography revealed that a wide range of cellular proteins were radiolabeled. Proteinase treatment of samples before SDS PAGE resulted in label migrating with the dye front. Proteins from cells treated with the acridines but not exposed to light were not radiolabeled. Cesium chloride ultracentrifugation of cell lysates also confirmed that the only macromolecules labeled were proteins. The singlet oxygen quencher histidine and the singlet oxygen stabilizer deuterium oxide had little effect on photolabeling of cellular proteins, suggesting that the mechanism does not rely on singlet oxygen. Experiments with purified proteins and fluorography of SDS polyacrylamide gels demonstrated photoadducts of proflavine and acriflavine to these proteins. Finally, proflavine was shown to form photoadducts to angiotensin by proteomic analysis. The detailed photochemistry and the identity of the amino acid residues involved in photoadduct formation are currently being studied. We conclude that photodynamic treatment of cells can result in photoaddition of the photosensitizing drug to cellular proteins and can generate potentially immunogenic haptens. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C240.