Prolonged lipid oxidation after photodynamic treatment. Study with oxidation-sensitive probe C11-BODIPY581/591.

Photodynamic treatment (PDT) is an emerging procedure for the therapy of cancer, based on photosensitizers, compounds that generate highly reactive oxygen species on illumination with visible light. Photodynamic peroxidation of cellular lipids is a consequence of PDT associated with cytolethality. We used chloromethyl dichlorodihydrofluorescein diacetate and a novel fluorescent ratiometric oxidation-sensitive probe, C11-BODIPY581/591 (C11-BO), which reports on lipid peroxidation, for visualizing oxidative stress in cells subjected to PDT with a phthalocyanine photosensitizer Pc4. With C11-BO loaded into the cells before or immediately after PDT, we observed a prolonged oxidation, which continued up to 30 min after illumination. In contrast, H2O2 caused oxidation of C11-BO only when the cells were in direct contact with H2O2. PDT-induced oxidative stress was most pronounced in vesicular perinuclear organelles, most likely photodamaged lysosomes. We hypothesize that the lysosomal localization of the prolonged oxidative stress is a consequence of the presence of redox-active iron in lysosomes. In conclusion, we have found that oxidative stress induced in cells by PDT differs from one induced by H2O2 in respect of induction of prolonged oxidation of lipids.

Determination of the stability of the noncovalent phospholipid transfer protein-lipid complex by electrospray time-of-flight mass spectrometry.

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein alpha (PI-TPalpha) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPalpha, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPalpha. This may explain why PI-TPalpha preferentially binds PI from a membrane interface.

Clofibrate-induced relocation of phosphatidylcholine transfer protein to mitochondria in endothelial cells.

The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 microM) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.