Enhancement of Nile blue derivative-induced photocytotoxicity by nigericin and low cytoplasmic pH.

The mechanism of photocytotoxicity mediated by a lysosomotropic photosensitizer, Nile blue derivative (NBS-61), in relation to lysosome destruction was examined by lowering the intracellular pH with low extracellular pH and an ionophore, nigericin. The treatment performed after photoirradiation had minimal effect on the cytotoxicity. However, when the treatment was initiated before photoirradiation, it caused a three orders of magnitude enhancement on cytotoxicity with a two orders of magnitude enhancement by nigericin alone. This effect on cytotoxicity resembles closely that observed on photosensitization mediated by chloroaluminum phthalocyanine. The enhancement in this case has been attributed to the synergistic interaction between photodamage and perturbation of ion transports across mitochondrial or plasma membranes by nigericin. Because these are not the main sites of localization for Nile blue photosensitizers nor their initial targets of photocytotoxic action, data from the present study suggest the possibility of an intracellular dye translocation induced by nigericin, which redistributes the Nile blue photosensitizer from lysosomes to other sites, as a possible cause of the enhancement of cytotoxicity.

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells.

Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.

Photosensitization, uptake, and retention of phenoxazine Nile blue derivatives in human bladder carcinoma cells.

The overall goal of our research is to develop effective new photosensitizers for tumor-selective photodynamic therapy. Phenoxazine dyes, including several Nile blue analogues, are known to localize selectively in animal tumors. Structural modifications yielded several series of analogues with substantially higher 1O2 yields and different photochemical and physicochemical properties. This study examined the photosensitization potency, cellular uptake, and retention of these derivatives in human bladder carcinoma cells (MGH-U1) in culture. Nile blue derivatives containing halogens and/or sulfur substitutes were selected to exhibit different 1O2 yields, pKa values, and hydrophobicities. The effectiveness of these derivatives in mediating photokilling of tumor cells in vitro corresponded well with the 1O2 yields of these compounds, indicating that structural modifications which resulted in increased 1O2 yields enhanced potency in mediating photocytotoxicity in vitro. Using derivatives (sat-NBS and sat-NBS-61) with the highest 1O2 quantum yield (0.35 and 0.821), over 90% cell kill was achieved at a sensitizer concentration of 5 x 10(-8) M, about 3 orders of magnitude more effective than hematoporphyrin derivative, the only sensitizer currently available clinically. This result suggests that some of the oxazine derivatives could potentially be effective photosensitizers. The correspondence between 1O2 yield and photosensitizing potency, together with results showing enhanced photocytotoxicity in the presence of D2O and reduced photocytotoxicity under hypoxic conditions, strongly suggests that the generation of 1O2 is a major mechanism mediating the photocytotoxic effect. The uptake of Nile blue derivatives by cells in culture exhibited a pattern of rapid initial uptake followed by a gradual increase in cellular dye contents. The uptake does not correlate directly with the individual pKa values or hydrophobicities of the derivatives, indicating that the structural modifications that increased 1O2 yields did not significantly alter the uptake and retention of Nile blue derivatives. The highly concentrative uptake by and slow efflux from dye-loaded cells were consistent with an active mechanism for the cellular accumulation of these dyes. On the other hand, the retention of the compounds was directly proportional to dye concentration in the medium over a 1000-fold range of concentrations, and the uptake could proceed at temperatures below 2 degrees C; these observations excluded endocytosis or a carrier-mediated mechanism for the uptake. The uptake was also unaffected by the presence of serum in the medium. Based on these results, we hypothesize that Nile blue derivatives transport across the cell membrane possibly as deprotonated forms and, upon entering the cell, either partition into lipophilic areas of the cell membranes and/or become sequestered in certain intracellular organelles.

Subcellular localization of hematoporphyrin derivative in bladder tumor cells in culture.

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)

Photodynamic effect in an experimental bladder tumor treated with intratumor injection of hematoporphyrin derivative.

Photodynamic therapy (PDT) is an experimental treatment modality for malignant tumors. It is based on the principle that a photosensitizer, such as hematoporphyrin derivative (HPD), is retained in higher concentrations in tumors than in surrounding nonmalignant tissues and that photoactivation of the sensitizer can be used to evoke tumor destruction. However, retention of the systemic injection of HPD is not limited to malignant tissues. This lack of specific tumor localization thus reduces the therapeutic ratio of the treatment and causes skin photosensitivity and possible systemic toxicity. Injection of HPD directly into the tumor, on the other hand, has been shown to yield higher levels of the drug in the tumor and lower levels in normal tissues, in comparison with systemic administration. In this study, we examined the photodynamic effect on s.c. implanted mouse bladder tumors subjected to intratumor (i.t.) and i.p. HPD injections. Tumor cell killing, measured by cell survival, was observed in both the it. and i.p. groups and was dependent on fluence and HPD dosage. However, no significant enhancement of cell killing was observed in the i.t. injected tumors, despite the higher porphyrin levels in these tumors. Histological examination of the effect of PDT on the blood vessels indicated that while cell death accompanied severe hemorrhage in the i.p. injected tumors, in the i.t. tumors there was much less hemorrhage and intact blood vessels remained. This observation suggests that with i.t. administration, direct photodynamic action may play a significant role in the tumor cell killing, in contrast to systemic administration, in which destruction of the blood vessels is believed to be the main cause of tumor destruction.

Tumor-localizing and photosensitizing properties of hematoporphyrin derivative in hamster buccal pouch carcinoma.

The tumor-localizing and photochemotherapeutic properties of hematoporphyrin derivative (HPD) were examined in 7, 12 dimethylbenzanthracene (DMBA)-induced oral cancers in the Syrian hamster. Oral tumors in hamsters injected with HPD (50 micrograms per gram of body weight) exhibited bright salmon pink fluorescence when exposed to long-wave ultraviolet light 24 hours after intraperitoneal HPD injection. Adjacent tumor-free mucosa did not fluoresce. Similarly, tumors not treated with HPD, normal mucosa treated with HPD, and normal mucosa not treated with HPD did not fluoresce. Tumors in animals that received HPD and photochemotherapy (PCT) were examined for gross and microscopic pathologic changes following the phototreatment. Tumors displayed edema, hemorrhage, and cellular necrosis that progressed with the time of sampling after photochemotherapy. Complete tumor necrosis was evident in the majority of oral tumors 24 hours after HPD PCT.

Cellular effects of hematoporphyrin derivative photodynamic therapy on normal and neoplastic rat bladder cells.

HPD is known to localize in neoplastic cells and when exposed to the appropriate wavelength of light causes cytotoxicity. The authors have established a rat urothelial cell model for use in comparing and contrasting the effects of HPD photodynamic therapy (PDT) in normal (RBL-01) and transitional cell carcinoma (AY27) bladder cell lines. Uptake, toxicity, and morphologic damage following exposure to HPD PDT were evaluated. Trypan blue exclusion was used for determination of the toxicity of several HPD concentrations (1, 10, 25, and 50 micrograms/ml) with increasing duration of incubation with HPD (0, 1, 2, 4, 12, 24, and 48 hours). Both cell lines displayed increased toxicity with higher concentrations of HPD; however, the AY27 cells were more susceptible to the toxic effects of HPD PDT than the RBL-01 cells at the higher HPD doses studied (25 and 50 micrograms/ml). Viability decreased with increased duration of HPD incubation in RBL-01 cells up until 4 hours, after which it showed a steady increase. Viability decreased in the AY27 cells with increased duration of HPD incubation. An increase in serum concentration in the medium resulted in an increase in viability for both cell lines. Both cell lines demonstrated fast initial uptake of HPD followed by slower uptake over the time studied. By 24 and 48 hours the AY27 cells contained twice the amount of methanol-extractable porphyrins as the RBL-01 cells. The initial morphologic change following HPD PDT was damage to mitochondria. Mitochondrial damage occurred immediately after PDT in the AY27 cells and 30 minutes after PDT in the RBL-01 cells. Both cell lines exhibited a similar progression of cell injury; however, morphologic damage was observed earlier after PDT and appeared more extensive in the AY27 cells.

Morphologic studies of bladder tumors treated with hematoporphyrin derivative photochemotherapy.

The morphologic changes that occurred in transplanted rat bladder tumors after treatment with hematoporphyrin derivative (HPD) and/or phototherapy were investigated. Transitional cell bladder tumors were initiated subcutaneously in male F344 rats by injection of AY27 cells. When tumors reached 1 cm in diameter, the rats received either HPD (10 mg/kg body weight) photochemotherapy, HPD only, phototherapy only, or no treatment. Tumors were sampled immediately (0 time), 1/2, 1, 2, 4, and 24 hours after phototreatment for light and electron microscopy. Tumors receiving HPD-photochemotherapy displayed progressive injury to both tumor cells and endothelial cells. Early changes (0-2 hours) included focal tumor and endothelial cell vacuolation and swelling as well as sloughing of tumor cells into papillary spaces. Tumor cells and endothelial cells displayed vacuolization and damage to cell mitochondria immediately after phototreatment. Intercellular spaces also increased in size. Lethally injured cells were apparent in papillary spaces. At 4 hours after phototherapy, tumor cells and endothelial cells exhibited extensive cell damage, including mitochondrial destruction, endoplasmic reticulum swelling, polyribosome disaggregation, and plasma membrane blebbing. By 24 hours after phototherapy, the majority of cells within the tumor were necrotic. Untreated tumors and those treated with phototherapy-only did not exhibit these changes. Tumors that received HPD only exhibited focal areas of cell swelling and focal mitochondrial vacuolization in both tumor and endothelial cells. These changes, unlike the HPD-light-treated group did not progress and were reversible.