Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells1

[CANCER RESEARCH 51, 2710-2719, May 15, 1991] Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells1

Chi-Wei Lin,2 Janine R. Shulok, Sandra D. Kirley, Louis Cincotta, and James W. Foley

Urology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 [C-W. L., J. R. S., S. D. KJ, and Rowland Institute for Science, Cambridge, Massachusetts 02142 Â¡L,C., J. W. F.]

ABSTRACT tosensitizers with high tumor selectivity will enable effective treatment of multiple, infiltratili!*, and invisible tumors, thus Nile blue derivatives have been shown to be potentially effective expanding the utility of PDT as a useful tool in cancer therapy photosensitizers for photodynamic therapy of malignant tumors. Results with intent to cure. Active research is under way to search for of a previous study suggested that the high accumulation of these dyes more tumor-selective sensitizers (4-6) and to improve the sen- in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, sitizer delivery system for better tumor targeting (7-10). results of studies are presented from an investigation of the subcellular Several early studies with animal tumor models have shown localization and mechanism of accumulation of these dyes in cells in that benzophenoxazines, including several Nile blue analogues, vitro. A video-enhanced fluorescence microscopy was used, and a punctate constitute a special class of dyes that are selectively localized in pattern of fluorescence was seen, most of which was localized in the tumors (11-15). Results of recent work have demonstrated that perinuclear region with extracellular dye concentrations between 1 to 100 Nile blue A can be converted to derivatives with substantially UM.These particles resembled characteristic particles identified by stand increased photoactivity (16-18). Furthermore, structural mod ard lysosomal dyes. At higher dye concentrations (1 pM or above), ifications of the parent dye can result in analogues having fluorescence in the perinuclear region was too intense to resolve into substantially altered pKa values and hydrophobicities, proper discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher ties which may be significant in dye localization in tumors. In dye concentrations (10-100 MM).Nile blue derivatives were seen with a a previous study (19, 20), we showed that Nile blue derivatives having high "O^ yields are effective in mediating photocytotox- light microscope as blue particles, the size and location of which resem icity in vitro. Derivatives with "O2 quantum yields of 35-80% bled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) can mediate a 90% in vitro photocytotoxicity with extracellular histochemical staining of dye-loaded cells with the lysosomal marker dye concentrations as low as 5 x 10~8M. This is about 3 orders enzyme acid phosphatase, which showed similar localization of the lower than with hematoporphyrin derivative. The finding thus enzyme-staining and dye-containing particles, (b) phototreatment of dye- suggests that these compounds are potentially effective photo loaded cells which obliterated the majority of the acid phosphatase- sensitizers for PDT. The cellular uptake of Nile blue derivatives stained particles, and (c) treatments with agents affecting the membrane is rapid, highly concentrative, and directly proportional to the pH gradient reduced the uptake and enhanced the efflux of dyes, while extracellular dye concentration. The uptake can proceed at agents that alter cellular membrane potentials had no effect on dye temperatures below 2Â°C,thus excluding endocytosis or a car accumulation. The uptake of the dyes was partially inhibited by inhibitors rier-mediated mechanism for the uptake. The overall results of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results show suggest that high cellular accumulation of these dyes may result ing that the cellular uptake of these dyes is highly concentrative and from dye aggregation, partition in membrane lipids, and/or proportional to the extracellular dye concentration over a wide range, are sequestration in certain intracellular organelles (20). consistent with the hypothesis that the dyes are mainly localized in the In the present study, the intracellular localization of Nile lysosomes via an ion-trapping mechanism. Results of the present study blue derivatives and the mechanism of their accumulation in also suggest that the lysosomes may be an intracellular target for pho human bladder carcinoma cells were examined. Findings from todynamic killing of tumor cells mediated by Nile blue photosensitizers this study suggest that the lysosome is the main site of localiza and that lysosomotropic photosensitization may be a strategy for effective tion and ion trapping is likely the process by which Nile blue and selective destruction of tumor cells. dyes are accumulated in cells.

INTRODUCTION MATERIALS AND METHODS PDT' is an investigational treatment procedure for malignant Nile Blue Derivatives. Previous reports (16-18) indicated that struc tumors (1-3). The effectiveness of the treatment relies, to a tural modifications of Nile blue A yielded derivatives with enhanced great extent, on the tumor selectivity of the photosensitizer photoactivity as well as different photochemical properties. The six Nile blue derivatives used in this study and their designations are shown which, upon photoactivation, imparts a photodynamic action in Table 1. The photochemical properties of these derivatives have been for cytotoxicity and tumor destruction. The availability of pho- described previously (16-20). All six derivatives were examined in subcellular localization studies using fluorescence and light microscopy. Reccived 12/19/90; accepted 3/6/91. The costs of publication of this article were defrayed in part by the payment In studies involving uptake and sequestering mechanisms, derivatives of page charges. This article must therefore be hereby marked advertisement in NBA and NBA-6I were used to represent derivatives with different pK. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. values and hydrophobicities. In studies involving photodynamic treat 1This work was supported by grants from the National Cancer Institute (CA ment of the cell, derivatives with moderate photoactivity, NBA-6I and 32259), the Beinecke Foundation, the Thomas Anthony Pappas Charitable Foun NBS-6I, were used. dation, and the Rowland Institute for Science. 2To whom requests for reprints should be addressed, at Urology Research Tumor Cells. The cell line used for this study, MGH-U1, is a Laboratory, Massachusetts General Hospital, Boston, MA 02114. subculture of I 24. a well established human bladder carcinoma cell 3The abbreviations used are: PDT, photodynamic therapy; PBS. phosphate- line (21). The cells were grown routinely in McCoy's 5A medium buffered saline at pH 7.4: DPBS. Dulbecco's phosphate-buffered saline at pH 7.4; 'Oi. singlet oxygen; SIT, silicon-intensified-target; TPP, tetraphenyl-phos- supplemented with 5% fetal calf serum. phonium: FCCP.p-trifluoromethoxyphenyl hydrazone; 2,4-DNP, 2,4-dinitrophe- Microscopic Observations of Nile Blue Derivatives in Cells. Both nol. The designations of the Nile blue derivatives are listed in Table 1. fluorescence and light microscopies were used to examine the subcel- 2710

Table 1 Structures and designation of Nile blue derivatives used in this study examining dye localizations in cells (22), subconfluent cells grown on with their absorption maxima, pK,, and partition coefficients" coverslips were incubated for 30 min at 37Â°Cwith 10 ml of dye Designation Structure , (nm) pK* P/ solutions; dye concentrations ranging from 10 MMto 0.1 nM were used. At the end of the incubation, the cells were removed from the dye NBA (C2H5)2N 623 10.0 173 solution, washed with DPBS, mounted on Lab-Tek chambers, and observed under the fluorescence microscope with the aid of a Hama- matsu C2400 SIT camera connected to a high resolution color monitor and a Sony UP-5000 video printer. At dye concentrations >10 MM,Nile blue dyes in ceils can be directly NBA-6I (C2H5)2N 642 6.6 5625 seen under the light microscope. This was performed by incubating subconfluent cells, grown on glass slides, in 100 mm culture dishes with 10 ml of various dye solutions at 20 MM,at 37Â°Cfor 10-30 min to permit dye uptake. Cells were rinsed to remove residual dye solution, mounted with dye-free medium, and observed immediately under the NBS 645 10.0 356 microscope. In experiments designed to examine the intracellular trans- location of the dyes under conditions of short uptake times and rela tively high dye concentration, cells grown on 22-mm coverslips were incubated with 100 /il of 10-100 MMdye solutions at 37Â°Cfor 5 min. Cells were rinsed twice to remove residual dye, placed in dye-free NBS-6I 660 6.5 5027 medium, and returned to the incubator for specified times before microscopic observation. Acid Phosphatase Staining. To verify that the blue stained particles seen under the light microscope were lysosomes, cells were stained with Sat-NBS 628 11.0 109 a standard lysosomal marker enzyme, acid phosphatase, to identify the organelle. In this experiment, cells were permitted to take up NBA-61 (20 MMat 37Â°Cfor30 min), and locations of the dye-containing particles were recorded by photomicrograph. The cells were immediately fixed in cold 3% glutaraldehyde in 60% acetone for 3 min at -20Â°C,washed Sat-NBS-6I (C2HS)2N 637 9.5 1752 in deionized water, and air dried. Staining of acid phosphatase was carried out with naphthol AS-BI phosphate (0.4 mg/ml) and Fast Garnet GBC (0.3 mg/ml) in acetate buffer at pH 5.2 for 60 min at 37Â°C.When the reaction was completed, the cells were washed in * Values of Aâ€ž,â€ž,t,pKâ€žand Pc are taken from Refs. 16-20. deionized water, air dried, and counterstained with mÃ©thylÃ¨neblue.The * Solvent, methanol:acetic acid, 250:1. ' Partition coefficients between 2-octanol and phosphate-buffered saline at pH stained slides were examined under the microscope to compare the sites of dye localization with the pattern of the acid phosphatase-stained 7.4. lysosome particles. Photodynamic Treatment of Cells. Cells to be treated were grown on 16 standard microscopic glass slides by placing one slide in a 100 mm cell 16 culture dish and adding 2 x IO6cells in 10 ml McCoy's 5A medium to NBA-61 NBA the dish. After the cells were allowed to attach overnight, the medium 12 â€¢12 was removed and the cells were washed once with DPBS. Ten ml of DPBS containing photoactive Nile blue derivative, either 0.5 MMNBA- 61 or 0.2 MMNBS-6I, which was the extracellular dye concentration â€” 8 determined from a previous study (20) to effect a 90% cell kill upon photoirradiation, was added to the dish, and the cells were allowed to take up the dye for 30 min at 37Â°C.Extracellular dye was removed and the cells were rinsed once with PBS. The cells adhering to the slide were placed in a new dish and covered with 8.5 ml of PBS, and light treatment was performed immediately. The light source was a Polaroid projector equipped with 590- to 700-nm band pass filters. The power density of the light source was 8-10 mW/cm2, the light dose for the 500 600 700 500 600 700 treatment was 4.8 J/cm2, and the total treatment time was 8-10 min. Wavelength (nm) After photoirradiation, the cells were placed in medium for 10 min Fig. 1. Excitation ( ) and emission ( ) fluorescence spectra of NBA and NBA-61 in MGH-U1 cells. Monolayer cells were allowed to take up dyes by before being fixed and stained for acid phosphatase to identify the incubating at 37"C with 2 ml of dye solution at 2.5 x 10"' M in serum-free lysosomal particles. McCoy's 5A medium for 30 min. Cells were then removed from culture plates, Dye Uptake and Quantitation. The uptake of the Nile blue derivatives resuspended in DPBS, and placed in a cuvette equipped with a stirring device to by MGH-LJ1 cells was determined by plating 2 x IO6cells/60-mm dish maintain cells in suspension. Excitation spectra were obtained with 725 nm and allowing them to attach overnight. Dye solutions, 2 ml at 2.5 MM detection and emission spectra with 600 nm excitation. Autofluorescence was in phenol-red free and serum-free McCoy's 5A medium, were added to corrected from the spectra by using cells without dye. the cells at 37Â°Cand cellular dye concentrations were determined at different time intervals as previously described (20). Briefly, cells were lular localization of Nile blue derivatives. A Zeiss epifluorescence removed from the cultured plates with 0.1% EDTA, dissolved in microscope equipped with a xenon light source was used to observe the concentrated HC1, and extracted with acidified chlorofornrmethanol fluorescence of the Nile blue dyes in cells. Based on the excitation and (1:1). Concentrations of the dye in the extracts were measured by emission spectra of Nile blue dyes in cells (Fig. 1), a filter system fluorescence spectroscopy. consisting of a band pass excitation filter centered at 633 Â±10 nm Effect of Membrane Potential on Dye Uptake. Cellular membrane (mean Â±SD), a dichroic beam splitter at 650 nm, and a barrier filter potential has been shown to affect the uptake and retention of cationic at 675 nm, was used to permit specific observation of the fluorescence dyes (23-25). We therefore examined the effects of this parameter on emitted by these dyes. As described previously for the procedure of the uptake of the Nile blue analogues by subjecting the cells to three 2711

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS conditions known to reduce cell membrane potential: treatment with have been shown to localize, became dominant, and fluores valinomycin or ouabain or exposure to high K* medium. In the exper cence in other cytomembrane structures, including mitochon iments in which the effects of valinomycin and ouabain were examined, monolayers of cells at 2 x IO6cells/60-mm dish were pretreated with dria, endoplasmic reticulum, and plasma membrane, became visible (Fig. 2B). At even higher dye concentrations (>1 MM), either valinomycin (1 fig/mi) or ouabain (1 HIM)for 30 min prior to the addition of NBA or NBA-6I (2.5 Ã•Ã•Minphenol-red free and serum-free fluorescence in the perinuclear area was too intense to resolve McCoy's 5A medium), and the uptake was performed at 37Â°Cin the into discrete cellular structures, while fluorescence in mito chondria! and cytomembrane structures was clearly visible (Fig. presence of the agents. The cells were then removed from the plates 2("). Examinations of these cells under the light microscope with 0.1% EDTA, and cellular dye was extracted and quantitated as described above. TPP, a lipophilic cation, which has been used in a revealed the appearance of blue particles in the perinuclear area variety of cellular and subcellular systems to measure membrane poten where intense fluorescence was seen. tials (23), was used to verify that our experimental conditions produced Other Nile blue derivatives showed similar punctate patterns the expected changes in membrane potentials. Experiments in which of fluorescence at dye concentrations between 1 and 10 nivi; at the effect of high K medium on dye uptake was examined were carried out in DPBS with KC1substituted for NaCI. The concentrations of K+ higher dye concentrations, cytomembrane structures, including were 139 mM for the high and 3 mM for the low K+ DPBS. Cells were the plasma membrane and mitochondria, began to fluoresce pretreated with high or low K* DPBS for 30 min before addition of the (Fig. 2, D-F). dyes (NBA or NBA-61 at 2.5 UM). With light microscopy, all six Nile blue derivatives appeared Effects of Membrane pH Gradient Modulators on Dye Uptake and mostly as blue particles localized at the perinuclear area (Fig. Efflux. Uptakes of many cationic compounds, particularly those that 3). The location and the size of these particles resembled the are weak bases, are known to be affected by agents which reduce the punctate fluorescence particles seen under the fluorescence pH gradient or increase the intralysosomal pH (26, 27). Effects of these microscope. With NBA, NBA-61, and NBS-6I, the paniculate agents on uptakes of NBA and NBA-61 were thus examined. Uptake localization was highly exclusive and the sequestering occurred experiments were performed as described above for valinomycin with almost immediately after the 5-min dye uptake period. With the exception that nigericin (5 jig/ml), monensin (6 and 25 jiM), or NBS, sat-NBS, and sat-NBS-6I, however, diffuse cytoplasmic FCCP (1 and 10 JIM)was used. and mitochondrial stains were also seen, and the sequestering The effect of nigericin on efflux of Nile blue dyes was also examined. Monolayer cells plated at 2 x 106/60-mm dish were incubated with 2 of the dye into particulate localizations usually required 15-40 ml of serum-free McCoy's 5A medium containing 2.5 fiM of NBA or min after the dyes entered the cells. Plasma membrane staining NBA-61 for 30 min at 37Â°Cto allow uptake of the dye. The medium can also be seen in cells treated with sat-NBS and sat-NBS-6I; nucleolar staining can be observed in cells with NBS and sat- was removed, and the cell culture plates were washed twice in DPBS. Dye-free medium with or without 5 Â¿ig/mlof nigericin was added to NBS. the plates and cellular dye concentrations were determined as above at Histochemical Identification of the Dye-containing Particles. various times intervals for up to 80 min. Further evidence for the lysosomal nature of Nile blue analogue- Effects of Oxidative Phosphorylation Inhibitors on Uptake. To deter containing particles was obtained from histochemical staining mine whether the uptake of Nile blue derivatives is an energy-dependent of the dye-loading cells with a standard lysosomal marker process, effects of agents that inhibit oxidative phosphorylation on the enzyme, acid phosphatase. The stained slides were examined to uptake of NBA and NBA-61 were examined in the absence of glucose to reduce glycolysis as an alternative source of energy. Cells, 2 x IO6/ determine whether the localization of blue particles matched that of the acid phosphatase-staining particles. As shown in 60-mm dish, were washed with DPBS twice, incubated with DPBS without glucose for 30 min to deplete endogenous glucose, and then Fig. 4, in most cells, particles containing both stains were pretreated with each of the inhibitors (2,4-DNP at 0.1 mM and sodium localized in the same areas. In some instances, individual azide at 10 mM) for 30 min in DPBS before the addition of dye (2.5 matching particles were identified with both stainings. This jiM). Uptake studies were performed at 37Â°Cin the presence of the result supports the overall conclusion that the dye-containing inhibitor. particles observed under the light microscope were indeed lysosomes. RESULTS Several technical difficulties prevented us from obtaining a perfect match in localization between the dye-containing blue Subcellular Localizations of Nile Blue Derivatives. The intra- particles and the acid phosphatase-staining particles. First, cellular localization of Nile blue dyes was observed with both since cells were viable before fixation, some movement of the fluorescence and light microscopy. A video-enhanced fluores lysosomes undoubtedly occurred during the time that lapsed cence microscope equipped with a filter system for specific Nile (2-5 min) between the photographic recordings of the blue blue dye excitation and emission was used, and punctate fluo particles and fixation. Second, the fixation step tended to cause rescence was seen in the cell with extracellular dye concentra shrinkage which may have altered the localization of subcellular tions as low as 1 nM. Fig. 2, A-C, shows MGH-U1 cells stained structures. Third, not all lysosomes took up adequate dye to be with 10 nM to 1 (Â¿Mof NBA, taken with the SIT camera, seen under the light microscope. Thus, some lysosomes identi demonstrating the dye distribution in these cells. At dye con fied by the acid phosphatase staining may not have been visu centrations of 1-10 nm, the fluorescence was seen as punctate alized as blue particles. Finally, the pattern of localization particles localized at the perinuclear area of the cell. These recorded by photomicrograph is dependent on the depth of particles resembled, both in the location and size, the particles focus under the microscope. Thus, different patterns of local identified by standard lysosomal fluorescence dyes such as ization can exist for the same cell when a different layer of the Lucifer Yellow CH (28, 29) and acridine orange (30-32), as cell is in focus. well as particles identified by acid phosphatase staining shown Photodynamic Evidence for Lysosomal Dye Localization. As in Figs. 4 and 5. Aside from these particles, essentially no another test of the lysosomal localization of Nile blue dyes, fluorescence was observed in other parts of the cell in this MGH-U1 cells were treated with NBA-61 or NBS-6I at concen concentration range. As the dye concentration increased to 0.1 trations previously determined to elicit a 90% cell kill when Â¿/M,fluorescence in the perinuclear region, where lysosomes photoirradiated. The consequential elimination of the dye-eon- 2712

Fig. 2. Localizations of Nile blue derivative NBA in MGH-U1 cells seen under a video-enhanced fluorescence microscope equipped with a filter system for specific excitation and emission of the dyes. Cells were incubated with various concentrations of dyes. At the end of the incubation, dye solution was removed and cells were washed with DPBS and observed immediately. Photographs were taken with a Hamamatsu C2400 SIT camera connected to a color monitor and a Sony UP-5000 video printer. In A, with 10 nM of NBA, most of the fluorescence appeared as particles concentrated in the perinuclear region and an area around the nucleus. In B, with 100 nM of NBA, the dye appeared mostly as punctate fluorescence, concentrated in the perinuclear region (arrows). Cytomembrane and mitochondria-like structures also fluoresced. In C with 1 JJMof NBA. fluorescence at the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in mitochondria (arrows) and cytomembrane structures was clearly visible. In A with NBA-61 at 10 nM, punctate fluorescence, characteristic of lysosomes, appeared as the main fluorescent structures. In E, with NBA-61 at I

taining lysosomes after photoirradiation of the cells may pro particles remained visible in the perinuclear area and virtually vide another indication for the lysosomal localization of these all the peripheral lysosomes were no longer present. In about dyes acting as photosensitizers. Lysosomal particles, identified 10% of the cells, complete elimination of lysosomes was by the acid phosphatase staining, in untreated cells were seen observed. distributed throughout the cells but, in most cases, were con Effects of Membrane Potential and pH Gradient on Dye centrated in the perinuclear area (Fig. 5A). Photoirradiation of Accumulation. Previous studies have shown that cationic dyes, dye-free cells (Fig. 5B) or dye treatment alone without photoir such as rhodamine 123 and AyV'-bis(2-ethyl-l,3-dioxolane)- radiation (photograph not shown) did not result in a noticeable kryptocyanine, which localize in the mitochondria are accu reduction in number or alteration in distribution of lysosomal mulated through an action of the mitochondrial membrane particles. However, most of the acid phosphatase particles were potential. Alterations of the membrane potential changes the obliterated with photoirradiation of dye-containing cells (Fig. uptake of these mitochondrial localizing dyes (23-25). Other 5, C and D). In these cells, only a small number of lysosomal cationic dyes such as acridine orange, which localize in lyso- 2713

Fig. 3. Localizations of Nile blue derivatives observed under the light microscope. Cells grown on glass slides were incubated with 20 //\i dye solutions at 37"C for 10-30 min. Cells were rinsed, mounted with dye-free medium, and observed immediately. Blue particles (arrowheads) signify sites of dye localization. Arrows, mitochondria-like structures without noticeable stain. A, NBA, 30-min dye incubation: fi, NBA-6I, 30-min dye incubation; C, NBS, 20-min dye incubation; D, NBS- 61, 30-min dye incubation: E, sat-NBS. 30-min dye incubation: F, sat-NBS-6I. I0-min dye incubation.

somes, are accumulated through the action of the pH gradient permeability changes, studies with higher ionophore concentra across the lysosomal membrane. Agents perturbing the pH tions have not been performed. gradient will affect the uptake of these compounds (26, 27). In Effects of cellular uptake of NBA and NBA-6I by valinomy the present study, effects of agents and conditions which alter cin, ouabain, and high K+ medium were then examined. All either transmembrane potential or pH gradient on the uptake these are known to reduce the transmembrane potential but by of Nile blue analogues were studied in order to provide insight different mechanisms (23-25). Valinomycin is a membrane into the mechanism of cellular dye uptake. ionophore which facilitates the efflux of K* ion. Ouabain is a To verify that agents such as valinomycin, ouabain, and specific inhibitor for the membrane Na-K ATPase which pro nigericin caused the expected changes in membrane potentials vides the energy for maintaining the membrane potential. High under our experimental conditions and cell system, a prelimi k medium eliminates the transmembrane K" gradient and nary experiment was conducted to test the effects of these agents hence reduces the potential. As shown in Fig. 6, none of these on MGH-U1 uptake of TPP, a well-studied lipophilic cation treatments caused any significant reduction in dye uptake, whose uptake is highly dependent on membrane potential (23). suggesting that the uptake of Nile blue dyes is not membrane Under the conditions used, valinomycin (1 Mg/ml) and ouabain potential dependent. This result also implies that the mitochon (1 ITIM)caused, respectively, a 20 and 33% reduction of TPP dria is not likely to be the main site of localization of these uptake, while nigericin (1 ^g/ml), which reduces the pH gra dyes. dient and induces a compensatory increase in membrane poten Effects of nigericin, FCCP, and monensin on the uptakes of tial, caused a >2-fold increase in TPP uptake. Since high NBA and NBA-6I by MHG-U1 cells were then examined. All concentrations of ionophores tend to produce nonspecific of these agents are known to reduce the lysosomal transmem- 2714

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS of NBA and NBA-6I from dye-loaded cells (Fig. 7; P < 0.0001 for both NBA and NBA-6I at the end of 80 min). This result suggests that the transmembrane pH gradient has a significant effect on the retention of Nile blue dyes and further substanti ates the hypothesis for the lysosomal localization of these dyes. pH-dependent Dye Accumulation at Different Concentrations. Since microscopic results described above indicate that the intracellular distribution of the dyes may vary with different dye concentrations and that nigericin appeared to be partially effective in inhibiting the accumulation of dyes in the lysosomes, nigericin was used to examine whether lysosomal dye accumulation varies as a function of dye concentration. Effects of 5 ng/m\ of nigericin on 30-min uptakes of NBA and NBA- 61 were examined at different extracellular dye concentrations ranging from 0.156 to 40 pM. Results (Fig. 8) show that, for both NBA and NBA-6I, inhibitions of uptake by nigericin were approximately 40-50% at dye concentrations <1 ^M, 60-80% inhibitions between 1 and 10 ÃŸM,and 60-70% inhibitions between 10 and 40 //M. Although there appears to be higher distributions of the dye in nigericin-sensitive sites at higher dye concentrations, the overall result indicates that the pH-dependent accumulation constitutes a major portion of the total cell ular uptake over the entire dye concentration range examined. Effects of Oxidative Phosphorylation Inhibitors on Dye Up take. To determine whether the uptake of Nile blue derivatives is energy dependent, we examined the effects of two oxidative phosphorylation inhibitors, 2,4-DNP and sodium azide, on the uptakes of NBA and NBA-6I. Experiments were performed in the absence of glucose to reduce glycolysis as an alternative energy source. Both inhibitors caused significant reductions (P < 0.0001 for all values) in the overall accumulation of dyes (Fig. 9). At the end of the 20-min incubation, sodium azide and 2,4-DNP reduced NBA uptake by 32.9 and 49.5%, respectively, and NBA-6I uptake by 28.2 and 50.0%, respectively. The cell morphology at the end of the uptake period appeared to be normal when observed by light microscopy, indicating that the decreased uptakes with inhibitors of oxidative phosphorylation B were due to an energy-dependent process and not to cell death. Although 2,4-DNP can also cause a reduction in mitochondria! Fig. 4. Acid phosphatase staining of NBA-61-loaded cells as histochemical membrane potential, the observed decrease in uptake can imply verification of the h sosomal nature of dye-containing particles. Cells were per mitted to take up the dye (20 MMat 37Â°Cfor 30 min) and the locations of the a mitochondria! localization of these dyes. Yet, the inhibition dye-containing particles were recorded by photomicrograph as shown in A. Cells did not occur in the presence of other energy source such as were immediately fixed and stained for acid phosphatase. The stained slides (B) glucose (data not shown), suggesting that the effect is related were examined under the microscope to compare the localizations of the dye- loaded particles and the acid phosphatase-containing lysosome particles. Imm \. to energy dependency rather than to membrane potential. several matched particles. DISCUSSION brane pH gradient via different mechanisms (26, 27). Nigericin In a previous study (20), we suggested that the highly concen- is a membrane ionophore which facilitates the exchange of K+ trative accumulation of Nile blue derivatives by tumor cells in and H+. FCCP is an ionophore which facilitates the efflux of culture may be the result of dye self-aggregation, partition of H+. Monensin acts by binding H+, thus increasing the intraly- the highly lipophilic dyes into cellular lipids, and/or sequester sosomal pH. All of these agents caused significant reductions ing of the dyes into certain subcellular organdÃes. The first two of the uptakes of the Nile blue dyes examined (Table 2). At the mechanisms are energy-independent processes, while the last is end of a 30-min incubation, nigericin (5 ng/m\) reduced NBA likely energy dependent. Since the results of the present study uptake by 72%, FCCP (10 ^M) by 75%, and monensin (25 MM) indicate that dye accumulation is energy dependent, it is un by 54%. NBA-6I uptake was reduced 65% by nigericin, 67% by likely that molecular aggregation or lipophilicity is the primary FCCP, and 40% by monensin (P < 0.0001 for all values, in mechanism for the cellular accumulation of these dyes, although comparison to uptake without any of the agents). These levels lipophilicity is undoubtedly an important factor contributing to of reductions were considerably greater than those reported for the uptake of the dye. The overall results of the present study these agents with /V-dodecyl-(Ci2)-imidazole, a lysosomal local indicate that the lysosome is the main site of intracellular izing compound, in which reductions following 30-min uptakes localization of Nile blue derivatives. At high dye concentrations were 26 and 23% by nigericin (1.25 Mg/ml) and monensin (25 (1 fiM or higher), the cytomembranes, including plasma and ), respectively (28). mitochondrial membranes, are additional sites of dye Nigericin also caused a rapid and significant increase of efflux localization. 2715

,â€¢.-* l' %

Fig. 5. Photodynamic evidence for lysosomal dye localization. Dye-treated MGH-U1 cells were photoirradiated (590-700 nm, 4.8 I/cm2), fixed 10 min later, and stained for acid phosphatase. The elimination of acid phosphatase-staining particles (arrows) by this process is considered an indication for the lysosomal localization of these dyes. In /. in untreated cells, the particles arc distributed throughout the cells but, in most cases, concentrated in the perinuclear area. B, cells treated with photoirradiation alone without dye. C and D, cells treated with NBA-6I (0.5 JIM)or NBS-6I (0.2 /IM), respectively, and at 10 min postphotoirradiation showing elimination of the majority of the particles.

The evidence for lysosomal localization for all of the dyes of than that required for mitochondria or other cytomembranes the present study is based on results which show (a) the simi and (b) nigericin significantly inhibits uptakes of NBA and larity in locations, distributions, and morphologies of the Nile NBA-6I at various dye concentrations ranging from 0.156 to blue-containing particles observed using fluorescence and light 40 MM(Fig. 8). microscopy to those identified by known lysosomotropic dyes, Nigericin and FCCP affect both the lysosomal pH gradient including acridine orange and Lucifer Yellow CH (28-32), (b) as well as the mitochondrial membrane potential. Nigericin the similarity in localization between dye-containing particles reduces lysosomal pH gradient and induces a compensatory and acid phosphatase-stained lysosomal particles, (c) the results increase in mitochondrial membrane potential. Thus, it can of photoirradiation of dye-loaded cells which obliterated most inhibit the uptake of lysosomal but enhance the uptake of of the lysosomes identified by acid phosphatase staining, (d) mitochondrial localizing compounds. Since the overall effect of the actions of nigericin which inhibited rather than enhanced nigericin on dye localization to the two sites are opposite, the the uptake and which promoted the efflux of the dyes, and (e) inhibition on Nile blue uptake observed above can be clearly the alterations in dye uptake by other agents which reduce attributed to the reduction of lysosomal pH gradient. On the lysosomal pH gradients but not by those which change plasma other hand, FCCP is a protonophore which increases the mem and/or mitochondria! membrane potentials. Other evidence brane permeability to protons and allows them to reach electro supporting this conclusion come from the results of our pre chemical equilibration across the membrane. The overall effect vious study (20) which showed (a) highly concentrative cellular of FCCP is reductions of both lysosomal pH gradient and accumulations of Nile blue derivatives and (b) a linear increase mitochondrial membrane potential. Therefore, the inhibition of uptake proportional to the dye concentration in the medium on the uptake of Nile blue dyes can be due to the reduction of over a wide concentration range. Further supporting this sup pH gradient and/or membrane potential. Since the former position that the majority of dyes are localized in the lysosomes interpretation agrees with other findings in this study, while are: (a) under the same experimental conditions with video- the latter does not, we attribute the FCCP action on Nile blue enhanced fluorescence microscopy, the minimum dye concen uptake to its effect on the lysosomal pH gradient. tration required for observing NBA fluorescence in lysosomes Although many cationic dyes are known to be localized in was about two orders of magnitude less (IO'9 versus 10~7 M) the mitochondria (4, 25), there are a great number of other 2716

NBA NBA-61

-Â» NBAonly -*- VaJjnomycin Â» Ouabajn o.

wo Nigericin w/ Nigericin 10 20 30 40 0 10 20 30 40 i â€¢w/ Nigericin Uptake Time (min)

.1 1 10 100 1 10 100 Extracellular Dye Concentration (uM) Fig. 8. Effect of nigericin on uptakes of NBA (left) and NBA-61 (right) at different dye concentrations. Cells were pretreated with 5 Mg/ml of nigericin for 30 min prior to the addition of the dye (NBA or NBA-61, from 0.156 to 40 MM). The uptake was performed for 30 min at 37Â°Cin the presence of nigericin. For comparison, uptakes of dyes at different concentrations but without nigericin were also performed. Point, mean of 3 determinations; bar, SD when exceeding symbol size.

10 20 30 40 O 10 20 30 40 Uptake Time (min) 1 Fig. 6. Effects of valinomycin (top) and ouabain (top) and high K* medium (bottom) on uptakes of NBA and NBA-61. For the valinomycin and ouabain studies, cells were pretreated with valinomycin (1 Mg/ml) or ouabain (1 mM) for 30 min prior to the addition of the dye. Uptake of NBA or NBA-61 (2.5 MM)was performed for various time intervals at 37Â°Cin the presence of valinomycin or ouabain. Uptake experiments in high K+ medium were carried out in DPBS with KC1 substituted for NaCl. The K+ concentrations were 139 mM for the high and 3 mM for the low K+ DPBS. Cells were pretreated with high or low K+ DPBS for 30 min before addition of the dyes (NBA or NBA-61 at 2.5 UM)to the medium. Point, mean of 3 determinations; bar, SD.

Table 2 Percentage inhibitions by nigericin, FCCP, and monensin on uptakes of NBA and NBA-61 by MGH-U1 cells" 30 40 0 10 20 30 40

uptake71.6Â± uptake64.5 Uptake Time (min) 2.0* Fig. 9. Effects of oxidative phosphorylation inhibitors, sodium azide and 2,4- Nigericin (5 Mg/ml) Â±0.8 DNP, on uptake of NBA and NBA-61. Cells, 2 X 10'/60-mm dish, were washed FCCP(1 MM) 36.4 Â±0.6 30.8 Â±2.0 with DPBS twice, incubated with DPBS without glucose for 30 min to deplete FCCP (10 MM) 75.4 Â±2.1 66.9 Â±0.7 the endogenous glucose, and pretreated with 2,4-DNP (0.1 mM) or sodium azide Monensin (6 MM) 42.3 Â±2.6 33.3 Â±0.5 (10 mM) for 30 min in DPBS before the addition of dye (2 ml, 2.5 MM).Uptakes Monensin (25 MM)NBA 54.3 Â±2.3NBA-61 40.1 Â±0.4 were performed at 37'C in the presence of the inhibitor. Point, mean of 3 " Uptake time: 30 min. determinations; bar, SD when exceeding symbol size. * Mean of 3 determinations Â±SD.

cationic compounds that are localized in the lysosomes (33- 37). Multiple factors may dictate the subcellular localization of these compounds. Subtle changes in the chemical structure of dyes can have profound influence on their localization. For example, alkylation of acridine orange, a well-known lysosom- o otropic cationic fluorescent dye, can yield derivatives that are localized specifically in the mitochondria. And the derivatives can have different distributions between these two organelles, depending on the degree of alkylation and apparently due to the increase in hydrophobic interaction between the alkylated derivatives and the mitochondrial membrane (32). The localization of Nile blue derivatives in lysosomes may be related to their capability of existing as uncharged weak bases when deprotonated from the cationic species. This is common 0 20 40 60 800 20 40 to many weakly cationic "lysosomotropic agents" which become Efflux Time (min) Fig. 7. Effects of nigericin on efflux of NBA and NBA-61. Cells (2 X 10') were highly concentrated within the lysosome because they can dif incubated with 2 ml of serum-free medium containing 2.5 MMdye for 30 min at fuse across cell membranes in their uncharged, nonprotonated 37Â°C.The medium was removed, the cell culture plates were washed twice, and form but become trapped in their protonated, nondiffusible dye-free medium with (bottom) and without (top) 5 Mg/ml of nigericin was form in the membrane-bound low pH compartment of the replaced. Cellular dye concentrations were determined at various time intervals. Point, mean of 3 determinations; bar, SD. lysosome (34). This ion-trapping mechanism is dependent on 2717

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENS1TIZERS the pH gradient of the subcellular compartment and the ability intracellular sites for effective PDT of tumors (4, 24, 25, 38, of the dye to undergo protonation-deprotonation conversion. 42). For example, the mitochondria has been considered an From the findings of the present study and based on the ion- important target for its possible high cationic sensitizer accu trapping mechanism, we offer the following hypothetical mulations in tumors, because of its higher membrane potentials scheme for the uptake, distribution, and accumulation of Nile in tumor cells (23, 25, 43) and its importance in supplying blue derivatives in MGH-U1 cells. Dye molecules enter the cell energy for critical cell functions (38). Recently, the photosen by diffusing across the plasma membrane in neutral nonproton- sitizers mono-L-aspartyl chlorin ce and sulfonated teraphenyl- ated forms. After they enter the cell, some of the dyes may porphyins have been found to be localized in the lysosomes, remain associated with the membrane and are distributed although the uptake of these photosensitizers may occur throughout various cytomembrane systems. This part of the through endocytosis (39-40). These findings, taken with the dye distribution process probably relies on the lipophilicity of results of the current study, suggest the possibility of targeting the dye. Hence, it is not energy dependent and the steady-state lysosomes as another strategy for PDT. There are a number of concentration depends on the extracellular dye concentration. advantages to this approach. One is the ability of the organelle On the other hand, dye molecules which partition into the to accumulate very high concentrations of cationic photosensi cytoplasm are quickly sequestered into and accumulated in the tizers. As shown in our previous study (20), with an initial lysosomes. This process is mediated by the action of an ion- extracellular dye concentration of 2.5 Â¿tMtheratio of intracel trapping mechanism common to many weakly basic compounds lular to extracellular Nile blue derivative concentrations after a which concentrate in subcellular sites of lower pH. This process 30-min incubation was on the order of 1000-6000. If most of relies on the pH gradient across the lysosomal membrane and the dye is localized in the lysosomes, which constitute only a is maintained by an energy-dependent proton pump. Intrinsic small fraction of the cell volume, the intralysosomal dye con to this process is the ability of dyes to undergo protonation- centration can be many times higher. Our previous data (20) deprotonation conversions at different pH values. In the acidic also show that the capacity of the cell to take up these dyes can environment of the lysosomes, the dyes are protonated and be extended to 100 pM without reaching saturation, further cannot readily diffuse across the membrane. The difference in indicating the high capacity of lysosome to accumulate these transmembrane permeability coefficients between the proton sensitizers. This permits the use of a lower sensitizer dose to ated and deprotonated forms is the main reason for the high produce low systemic toxicity and effective cell killings, as exemplified by the low (5 x 10~8 M) extracellular sensitizer accumulation of dyes in the lysosomes (34). Although the lysosome appears to be the major site of local concentration needed to cause a 90% cell kill upon photoirra ization for all of the Nile blue dyes examined, derivatives with diation for some of the Nile blue derivatives. Another advantage minor differences in structure also have subtle variations in for targeting lysosomes is the potential of low dark toxicity. their distribution to other cell structures relative to the lyso Dye in lysosomes, if not activated by light, is less likely to cause some. Sulfur substitution and D-ring saturation reduce, while damage to cells. Cells containing fairly high concentrations of iodination increases, both the degree of specificity and speed of acridine orange, which is highly photoactive, can survive for sequestering into the lysosome. Of note, iodination also causes days in the dark (30, 31). Still another advantage is the destruc reductions in pKa values and increased hydrophobicities of the tive nature of this organelle. Release of the hydrolytic enzymes resulting derivatives. Both of these properties may influence resulting from photodamage to lysosomes has been proven to the ion-trapping process. However, the significance of pKa may be effective in causing cell degeneration and death (44). be limited only to the ability of the dye to be protonated. As Although results of the present study indicate that the lyso long as the pKa of the derivative is higher than the pH of the some is the main localization site and implies that this may be lysosome, a sufficient amount of the dye will be in the proton the main target of photodynamic action for Nile blue deriva ated form and trapped in the lysosome. The increase in hydro- tives, they have not ruled out the presence of these dyes in other phobicity, however, may increase the ratio of permeability subcellular structures, such as plasma membrane and mito coefficients between the deprotonated and the protonated forms chondria, as well as the significance this presence in the process moving across the lysosomal membrane and thus increase up of photocytotoxicity mediated by these sensitizers. In a recent take and accumulation of the dye in the lysosome. report, we suggested that a reduction of mitochondrial function Since the subcellular localization of many photosensitizers is due to either the presence of a small amount of sensitizers or a currently a topic of active investigation (22, 38-41), it is im secondary effect from lysosomal damage may potentiate the portant to note that the present study demonstrated the de lysosomotropic photosensitization action of Nile blue deriva pendence of apparent dye localization on both dye concentra tives (45). tion and method of detection. For example, under the light microscope, Nile blue dye in cells can only be detected at high extracellular concentrations (>10~5 M), which indicated mostly ACKNOWLEDGMENTS lysosomal localization. However, under fluorescence micros The authors wish to thank Carl F. Schanbacher and Cindy Bachelder copy, the fluorescence in the perinuclear region and area around for technical assistance and Dr. Irene Kochevar for critical reading of the nucleus was too intense to discern lysosomes at dye concen the manuscript. trations between 10~6 and 10~7 M, while fluorescence in mito chondria and cytomembranes was clearly visible. Only when REFERENCES lower dye concentrations were used did the punctate lysosomal 1. Dougherty, T. J., Weishaupt, K. R., and Boyle, D. G. Photodynamic therapy stain become evident, which was confirmed by histochemical, and cancer. In: V. J. DeVita, S. Hellman, and S. A. Rosenberg (eds.), photodynamic, and biochemical studies. The use of multiple Principles and Practices of Oncology, Ed. 2, pp. 1836-1844. Philadelphia: methods at a variety of dye concentrations to establish subcel J. B. Lippincott, 1985. 2. Dougherty, T. J. Photosensitizers of malignant tumors. Semin. Surg. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells

Chi-Wei Lin, Janine R. Shulok, Sandra D. Kirley, et al.

Cancer Res 1991;51:2710-2719.

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