Abstract 5656: Preclinical evaluation of cytotoxic effect of photosensitive fluorescent protein in human cancer cells

Photodynamic therapy (PDT) is an anticancer therapy to induce tumor-specific cytotoxicity by accumulation of photosensitizers (PSs) and following non-toxic light irradiation to tumor tissues. Non-toxic light irradiation induces the PS-mediated generation of reactive oxygen species (ROS) and subsequent cytotoxicity in tumor cells. For clinical application, some kinds of PSs, such as photofrin and laserphyrin, are intravenously injected before light irradiation in anticancer PDT. However, the lack of sufficient and continuous accumulation of PSs in tumor tissues makes difficult to eradicate tumor cells by PDT. Therefore, the development of novel PSs that accumulate to tumor tissues more efficiently and persistently is required to improve the antitumor effect of PDT. Recently, a bioengineered fluorescent protein KillerRed has been reported to generate ROS upon green light irradiation, suggesting a potential of KillerRed fluorescent protein as a novel PS. In this study, we investigated the antitumor effect of KillerRed fluorescent protein in human cancer cells in combination with PDT. Transient transfection with KillerRed expression vector induced the photosensitive cytotoxicity upon green light irradiation within a few hours in human cancer HeLa and OST cells. To further evaluate the cytotoxic effect of KillerRed fluorescent protein in human cancer cells, we obtained the stable transfectants expressing cytotoxic KillerRed or non-cytotoxic Katushka fluorescent protein from human lung cancer H1299 cells. Green light irradiation induced the cytotoxic effect within one hour in H1299-KillerRed cells, but not in H1299-Katushka or H1299 cells. H1299-KillerRed cells irradiated with green light showed significantly higher levels of ROS, which was confirmed by intracellular fluorescence level of 2′,7′-Dichlorodihydrofluorescein Diacetate, than H1299-Katushka or H1299 cells. Immunocytochemistry further revealed that green light-irradiated H1299-KillerRed cells showed the apoptotic cell death with the cleavage of caspase-3 24 hours after irradiation. These results suggest that KillerRed fluorescent protein is a potent novel PS inducing ROS generation and apoptotic cell death in tumor cells in anticancer PDT. The development of virus-mediated delivery system for tumor-specific induction of KillerRed fluorescent protein is under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5656. doi:1538-7445.AM2012-5656