Bromine indirubin FLIM/PLIM sensors to measure oxygen in normoxic and hypoxic PDT conditions.

BACKGROUND: The induction of phototoxicity during photodynamic therapy (PDT) is dependent on oxygen availability. For this reason, the development of sensors to measure oxygen and oxygen consumption is extremely important. APPROACH: In this project we have used Fluorescence Lifetime imaging (FLIM) and Phosphorescence Lifetime Imaging/ delayed Fluorescence Lifetime Imaging (PLIM/dFLIM) to investigate the ability of bromine indirubin derivatives as oxygen sensors. RESULTS: The oxygen sensitivity of bromine indirubins was detected through PLIM/dFLIM. Moreover, we have observed, by measuring nicotinamide adenine dinucleotide (NADH) FLIM, that bromine indirubin has a significant impact on cellular metabolism by shifting the SCC-4 Cells metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. CONCLUSIONS: In conclusion, this study successfully achieves its goals and provides important insights into the use of indirubin as a potential oxygen consumption sensor with the capability to identify and differentiate between normoxic and hypoxic regions within the cells.

Functional characterization of Fospeg, and its impact on cell cycle upon PDT of Huh7 hepatocellular carcinoma cell model.

BACKGROUND: Although several treatment options are available for hepatocellular carcinoma (HCC), their application is mostly restricted to early diagnosed cases or includes liver transplantation, which is rarely available due to donor scarcity. The attractiveness of PDT as a cancer treatment does not only come from its minimal invasiveness, but also from the high selectivity due to tumor localization that can be applied. Precise focusing of light on tumor lesions will result in tumor-specific PDT activation. Novel photosensitizers can be applied in such low concentrations that cells not subjected to irradiation remain healthy. The lethal effect and mechanism of death induction of the photosensitizer Fospeg has never been studied on hepatocellular carcinoma. The aim of the present study is to functionally analyze the impact of PDT on Huh-7 HCC cell line, as well as to analyze its impact on cell cycle protein expression. METHODS: Cellular viability, and proliferation assays were conducted via MTT and BrdU assay, respectively. Transfected cell models of Huh7 with different constructs harboring cell cycle genes and downstream reporter luciferase gene were generated. RESULTS: Our results show a statistically significant decrease in both viability and proliferation of Huh-7 cells following PDT, while maintaining Fospeg and laser concentrations far below toxic levels. Proliferative cell cycle genes show a tendency of inhibition, while p53 levels show a significant increase following PDT. CONCLUSION: Fospeg-mediated PDT is a promising strategy for treatment of hepatocellular carcinoma and needs to be further explored in vivo.

Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein.