With Chloroaluminum Phthalocyanine) by Treatment of V79 Cells with the Lonophore Nigericin1

(CANCER RESEARCH 50. 1620-1625. March 1. I990| Enhancement of Photodynamic Cell Killing (with Chloroaluminum Phthalocyanine) by Treatment of V79 Cells with the lonophore Nigericin1

Marie E. Varnes,2 Marian E. Clay, Keith Freeman, Antonio R. Antunez, and Nancy L. Oleinick Divisions of Biochemical Oncology and Radiation Oncology, Department of Radiology, Case Western Resen'e University School of .Medicine and i'niversity Hospitals of Cleveland, Cleveland, Ohio 44106

ABSTRACT Addition of 1.0 Â¿Â¿Mnigericinduring the time of heating reduces The K' 11' ionophore nigericin dramatically increases killing of V79 survival to 0.3% at pHc 6.6, whereas there is little effect when cells are heated at pHc 7.4. Because both heat and PDT are cells by photodynamic therapy (PDT), when cells pretreated with 1 MM Chloroaluminum phthalocyanine are incubated with nigericin before ex believed to kill cells in part by damaging membranes (1,2, 6), posure to red light. Nigericin affects primarily the shoulder of the PDT and because both are more effective when pH(. is mildly acidic dose-response curve, reducing the surviving fraction from 0.90 to 0.02 (2, 7, 8), it was of interest to determine whether or not nigericin after a fluence of 7 k.l/m ' and from 0.80 to 0.0003 after a fluence of 12 would enhance the effects of PDT. This study deals with the k.l in. Optimal enhancement of PDT occurs when cells are incubated effects of combined treatment of V79 Chinese hamster lung with 2 MMnigericin, at pi I, 6.7, for 30 to 60 min before irradiation. cells with nigericin, AlPcCI, and red light. As with hyperther However, significant enhancement of PDT also occurs when nigericin is mia, a pHc-dependent potentiation of PDT was observed. Bio added immediately before irradiation. Treatments with Chloroaluminum chemical studies have been undertaken in an effort to under phthalocyanine and nigericin, nigericin alone, or nigericin and red light stand the mechanism by which nigericin treatment interacts are not toxic to cells. Cells treated with the combined agents display a synergistically with PDT. rounded morphology 2 h after light exposure and lyse within 12 h. However, rounding of cells is not accompanied by severe depletion of ATP or by permeabilization of the plasma membrane to trypan blue. MATERIALS AND METHODS These results, together with known metabolic effects of nigericin, suggest that nigericin potentiates PDT by perturbing ion transport across either Cell Culture and Clonogenic Survival Assays. Chinese hamster V79- mitochondria! or plasma membranes. 379 lung fibroblasts were grown as monolayers in McCoy's 5A medium supplemented with 10% calf serum and buffered with 2.2 g/liter bicar INTRODUCTION bonate and 20 HIMHepes, pH, 7.4. Cells in mid-log phase of growth (2 x 10*/25-cm2 flask) were used for experiments, unless otherwise PDT1 is a relatively new modality being developed for treat indicated. For survival assays cells were trypsinized and plated into ment of neoplasms which can be irradiated with visible light fresh medium. After 1 week the flasks were stained with crystal violet, (1-3). Photosensitizers under investigation at present include and colonies of 50 cells or more were scored as survivors. HPD and its active fraction, Photofrin II, and the phthalocy- Drug Treatment and Light Exposure. Nigericin was obtained from anines. Some of the latter appear promising for eventual clinical Sigma Chemical Co. (St. Louis, MO) and prepared as a 1 HIMstock use because they are readily synthesized in pure form and absorb solution in dimethyl sulfoxide. AlPcCI (purchased from Eastman Ko strongly in the 600-700 nm region of the spectrum (2, 3). dak Co., Rochester, NY, and recrystallized) was prepared as a 1 HIM stock solution in dimethylformamide. Cells were incubated with 1 JIM While the potential for selective killing of tumors with PDT AlPcCI in the dark for 18 h in order to allow optimal uptake of alone is substantial, efforts to improve PDT treatment by taking photosensitizer (9). At various times before irradiation, the medium advantage of unique metabolic properties of tumor tissue or by was replaced with HBSS, buffered with 5 mivisodium bicarbonate and exploiting interactive toxic mechanisms are under way. For either 20 m\i Hepes (pHc 7.5-6.7) or 20 mivi piperazine-/V.7V'-bis(2- example, Thomas and Girotti (4) have recently shown that ethanesulfonic acid) (pi 1, 6.4), Â±nigericin at selected concentrations. PDT, with HPD as photosensitizer, more effectively kills tu Cells were irradiated in HBSS in the presence or absence of nigericin mors in rats made hyperglycÃ©mieby glucose injection. Hyper- and then either refed with fresh complete medium (for photography glycemia lowers tumor pHc, resulting in increased uptake of and biochemical experiments) or trypsinized and plated into fresh HPD. The synergistic interaction of PDT and hyperthermia medium (for survival assays). Cells were kept in the dark after irradia has long been recognized and may be due to heat-induced tion, until the time of biochemical assay or staining for clonogenic survival. inhibition of repair of sublethal PDT damage (1). We have recently reported that the K+/H+ ionophore nigeri Red light irradiation was performed as described previously (9) using a 500-W tungsten-halogen lamp equipped with a 600 nm high-pass cin enhances both heat killing and radiation killing of Chinese filter (Vincent Lighting, Cleveland, OH). The fluence rate was approx hamster ovary cells in vitro and does so in a pHc-dependent imately 0.074 kJ/nr/s at the level of the monolayer and varied by no manner (5). In the case of hyperthermia, incubation of cells at more than 0.003 kJ/m2/s across the area of the monolayer. 42.1"C for 30 min at pHc 6.6 has no effect on cell survival. Intracellular ATP. ATP in cells was measured using a premixed bioluminescence assay kit (Sigma). Cells were trypsinized in Ca2*- and Received 8/7/89; revised 11/13/89; accepted 11/15/89. Mg2*-free HBSS containing 5.5 mM glucose. Two aliquots of suspended The costs of publication of this article were defrayed in part by the payment cells (approximately 1x10' cells each) were immediately added to a of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tube containing 2 ml of boiling 20 mM Tris buffer, pHc 7.75. Tubes 'This research was supported by Research Grant ROI Ã‡A40516 and by were capped loosely and boiled for 10 min. Samples were then cooled Cancer Center Support Grant P30 CA 43703 from the National Cancer Institute, to 4Â°C,and extracts were centrifuged to remove cell debris. A third Department of Health and Human Services, and by a generous gift from the aliquot of trypsinized cells was used for cell counting. Samples were Marguerite M. Wilson Foundation. *To whom requests for reprints should be addressed, at Division of Biochem assayed in vials containing 10-200 //I of cell extract, 3.0 ml Tris buffer, ical Oncology. Department of Radiology, University Hospitals of Cleveland, 2058 and 50 n\ of a 1:5 dilution of the ATP assay mix. Chemiluminescence Abington Road. W'earn B-21, Cleveland. OH 44106. was measured using a Packard 3522 Tri-Carb scintillation counter, out 5The abbreviations used are: PDT. photodynamic therapy; HPD, hematoporphyrin derivative; AlPcCI. Chloroaluminum phthalocyanine; HBSS, Hanks' bal of coincidence, with the adjustable discriminator set at 50-70 divisions, anced salt solution; Hepes. Af-2-hydroxyethylpiperazine-A'-2-ethanesulfonic acid; 100% gain (10). The reaction was initiated by adding an aliquot of pi I., extracellular pH; pHi. intracellular pH. either standard ATP or cell extract to vials containing buffer and 1620

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1990 American Association for Cancer Research. CYTOTOXIC INTERACTION OF PHOTOTHERAPY AND NIGERICIN enzyme. After a 30-s delay, continuous counts were recorded for 12 s. during the light exposure and were subcultured into fresh Trypan Blue Exclusion. At various times after exposure to red light, medium immediately afterward. Under the conditions of this cells were trypsinized, resuspended in fresh medium, mixed with 0.4% assay, control cells were incubated for 1 h in HBSS at pHt. 6.7 trypan blue, and allowed to stand for 2-3 min. This mixture was without nigericin, before light treatment. These cells are more transferred to a hemocytometer and the percentage of stained and resistant to PDT than we had previously reported (9); the unstained cells was determined. AlPcCl Retention by Cells. Effects of incubation of AlPcCl-treated fluence that reduces the surviving fraction to 0.37 is approxi cells in buffer, with or without nigericin. were determined as described mately 23 kJ/nr, compared to approximately 12 kJ/nr for the previously (11), except that cells were collected by trypsinization rather same AlPcCl concentration but without preincubation in buffer. than by scraping. Briefly, approximately 5 x IO6 cells/assay were Several subsequent experiments in which PDT response with collected on a glass fiber filter, washed with buffer, and allowed to dry. and without incubation of cells in HBSS was compared (e.g., AlPcCl was extracted with 100% ethanol, and the absorption at 674 Figs. 2 and 4) confirm the protective effect of buffer pretreat nm (Â«= 2.93 x 10') was measured. The efficiency of extraction was ment. similar for ethanol, butanol, and dimethylformamide and appeared to The striking feature of the data of Fig. 1 is the markedly be greater than 90%, since no detectable 674 nm-absorbing material increased cytotoxic response to AlPcCl and red light by cells remained in cells after extraction. which have been treated for 1 h, at pHc 6.7, with 2 //M nigericin. Absorption Spectra of AlPcCl in Hypotonically Swelled Cells. Cells treated with AlPcCl and then incubated in HBSS, in the presence or In contrast, none of the following treatments were cytotoxic: absence of nigericin, were trypsinized and resuspended, at a density of nigericin alone, for 1 h, up to 5 ^M; 1 Â¿ÃiviAlPcClfor 18 h, about 1 x 106/ml, in phosphate-buffered saline (pHc 7.4). The spectra followed by 1 h in HBSS with or without nigericin; 2 pM were scanned from 450 to 750 nm in a DW-2 spectrophotometer (SLM nigericin in HBSS at pHt. 6.7, followed by exposure to 12 kJ/ Instruments Inc./American Instrument Co., Urbana, IL). rrr red light. In addition, neither the 0.2% dimethyl sulfoxide used as a vehicle for nigericin nor the 0.1 % dimethylformamide RESULTS used as a vehicle for AlPcCl affected the response to PDT (data not shown). Effect of Nigericin on the Response of V79 Cells to PDT. Fig. Effects of Extracellular pH. We previously found that niger 1 shows that incubation of AlPcCl-treated V79 cells with ni icin increases hyperthermic killing and radiation response of gericin, at pHc 6.7 for l h prior to red light irradiation, markedly cells more effectively as pHc is lowered (5). Fig. 2 shows the sensitizes the cells to photodynamic killing. The effect of niger response of cells exposed to 12 kJ/m2 red light following 1 h icin is primarily on the shoulder of the dose-response curve. incubation in HBSS, with and without nigericin, at pHt.s varying The average surviving fraction (from ten separate experiments) from 7.50 to 6.40. For all pHt. values, variation over the course for cells pretreated with l UM AlPcCl and irradiated in the of the treatments was no greater than 0.02 pH unit. Although presence of 2 /Â¿Mnigericin is 0.00025 Â±0.00008 (SEM), com the toxicity of nigericin has been reported to increase with pared to 0.77 Â±0.10 for cells irradiated at 12 kJ/nr in the decreasing pHt. (13), nigericin itself was not toxic under any of presence of AlPcCl alone. The dose-response curve for cells the incubation conditions used in these experiments. Since the treated with both nigericin and PDT exhibits a small shoulder, response of cells to the combined actions of nigericin and PDT a steep exponential region, and a much reduced slope after a is nearly optimal at pH0 6.7 and since mildly acidic pHc values light fluence of about 20 kJ/nr. Reasons for the latter change have been reported for several tumors (14, 15), subsequent in slope are discussed below. experiments were performed at this pHc. Because nigericin binds to serum (12), experiments were performed by preincubating cells in bicarbonate- and Hepes- 10Â°r buffered HBSS ("Materials and Methods"). Cells were in HBSS AlPcCl, HBSS

. n AlPcCl 10"

AlPcCl, HBSS Â»Nigericin I IO'3

AlPcCl, HBSSÂ»Nigericin

IO'5 75 73 7.1 6.9 67 65 63 5 10 15 20 25 30 pH of Buffer Light Fluence (kj/m2) Fig. 2. pH dependence for enhancement of PDT by nigericin. Cells were Fig. 1. Effect of nigericin on response of V79 cells to PDT. Cells were exposed exposed to 1 H.MAlPcCl for 18 h; then the medium was replaced with HBSS with to 1 n\i AlPcCl for 18 h; then the medium was replaced with HBSS, pHt 6.7, (â€¢)or without (O) 2 /IM nigericin, at various pHe values between 7.5 and 6.4. with (â€¢)or without (O) 2 /IM nigericin. After 1 h. cells were exposed to various After 1 h cells were exposed to 12 kJ/nr red light. Some AlPcCl-treated cells fluences of red light. Cells were plated for survival immediately after light (D) were irradiated immediately after replacement of medium with HBSS. pHc treatment. A. A. average values for 10 separate experiments |Â±SEM (Aars)] for 7.5. Data are averages of quadruplicate measurements from a representative treatment with AlPcCl and 12 kJ/nr red light with (A) or without (A) nigericin. experiment. 1621

Effects of Nigericin Concentration and Preincubation Time. In 10Â° the experiment of Fig. 3, AlPcCl-treated cells were incubated AlPcCl, HBSS in HBSS containing various concentrations of nigericin up to

5.0 /Â¿M,for l h before exposure to 12 kJ/irr red light. The IO'1 results show that a concentration of 2.0 UM, which has been used for all other experiments in this report, gives nearly optimal sensitization to PDT. Concentrations above 5.0 Â¿IM were not tested because of reports of nonspecific membrane S io'2 permeabilization at higher drug concentrations (16). Previously I we found that nigericin is readily absorbed into the plastic of culture flasks (5). Since the nigericin concentration cannot be ,1 IO'3 AlPcCl, HBSS * Nigericin monitored readily, we cannot preclude the possibility that the concentration curve of Fig. 3 in part reflects scavenging of drug by the support matrix. The effects of nigericin on pHÂ¡,cation distribution, and IO"' energy metabolism of cells are time dependent (5, 12, 13). It was of interest, therefore, to determine the time dependence for sensitization of V79 cells to PDT by nigericin. Fig. 4 shows that survival is reduced by somewhat more than 1 order of IO"' IO 20 30 40 50 60 magnitude even when nigericin is added to cultures immediately Incubation Time (min) before irradiation. However, optimal sensitization to PDT oc curs after about 30 min preincubation of AlPcCl-treated cells Fig. 4. Time course for enhancement of PDT by nigericin. Conditions were as described for Fig. 1, except that the time interval between addition of 2 MM with nigericin. Although sensitization to PDT does not increase nigericin and irradiation with 12 k.l m ' red light was varied. substantially with a further 30 min incubation, 1-h pretreat- ments were used in most experiments for technical convenience. AlPcCl and light but no nigericin (Fig. 5). Cytoplasmic vacuoles Nigericin-induced Changes in Cell Morphology and Membrane or deformations appeared in cultures exposed to nigericin for Permeability. Nigericin acts by penetrating plasma and intra- 1 h, but these are not associated with cell killing in the absence cellular membranes, where it facilitates exchange of H+, and of PDT, since cells recovered upon removal of the nigericin- possibly Na+, for K+ (12, 17). Dramatic changes in membrane containing HBSS and addition of fresh medium. Vacuoles permeability may result in visible changes in cell morphology remained in cells that were exposed to red light in the presence or in failure of living cells to exclude trypan blue. To test these of nigericin and AlPcCl, and by 2 h after PDT most of these possibilities, cells were treated with AlPcCl and nigericin in cells appeared shrunken and rounded. By 6 h after PDT these HBSS and exposed to light as described for Figs. 1-4 but were cells were largely detached from the plastic substrate. Lysis was subsequently refed with fresh medium and kept in the dark, observed within 12 h of PDT. rather than being trypsinized (Fig. 5; Table 1). Cells treated with PDT alone or with nigericin and PDT were Cell monolayers were inspected in a phase-contrast micro assayed for ability to exclude trypan blue (Table 1). Less than scope at various times after addition of HBSS (with or without 1% of untreated cells or cells treated with AlPcCl and red light nigericin), and at times up to 2 h after exposure to light. No incorporated dye. Treatment with nigericin and PDT slightly morphological changes were observed for cells treated with increased the fraction of cells taking up dye, to a maximum of 3% of the total cell population. However, effects on dye exclu ioc sion were not pronounced until 8 h after exposure of cells to AlPcCl, HBSS the combined treatment. This is long after cell shrinkage and detachment had occurred. IO'1 Changes in Intracellular Levels of ATP. Since there is evi dence that PDT may mediate cell death by inhibiting mito chondria! ATP production (18, 19), and since nigericin is known to affect mitochondrial function by eliminating the pH AlPcCl, HBSS* Nigericin component of the proton-motive force (13, 16, 20), we hypoth esized that the synergistic interaction between PDT and niger icin might be due to severe inhibition of cellular ATP produc 5 IO'J tion. ATP levels were measured for cells treated with AlPcCl, incubated for 1 h in HBSS, with or without nigericin, exposed to light, and then refed with fresh medium. These were com pared to ATP levels of untreated cells (680 Â±70 pmol/105 10- cells) (Fig. 6). Exposure of AlPcCl-treated cells to 12 kJ/nr red light resulted in a slight decrease in ATP. These cells recovered their ATP upon being refed with fresh medium, which IO'Â» is consistent with the low toxicity of PDT treatment at 12 kJ/ 001 010 1.00 10.0 m2. Cells exposed to AlPcCl and then to nigericin in HBSS Nigericin ( .ttM) Fig. 3. Effect of varying the nigericin concentration. Cells were treated with 1 lose about 30 and 65% of their ATP after 30 and 60 min in (

Fig. 5. Morphological changes in V79 cells treated with AlPcCl, nigcricin. and 12 kj/m2 red light. A. cells treated with PDT but no nigcricin, as described for Fig. I. Cells were refed with fresh growth medium immediately after irradiation and kept in the dark at 37Â°C.Cells were photographed 2 h later and were indistinguishable in appearance from untreated cells. In B and C, cells were treated with 1 uM AlPcCl, 2 /Â¿Mnigericinfor I h, and red light and then rcfcd and incubated as described above. Cytoplasmic vacuoles were observed 30 min after irradiation (B). and by 2 h postirradiation the majority of cells were shrunken and displayed a rounded morphology (C). Phase-contrast microscopy, x 400.

Table 1 Effects of various combinations of AlPcCl, nigericin, and red light on uptake oftrypan blue in V79 cells Treatment conditions were as described for Fig. 5. % of uptake oftrypan blue at following times conditionControlTreatment min0.03.160 min0.71.9120min0.02.6480 min0.4 (no treatment) AlPcCl. 12kJ/m2 0.6 0.6 HBSS, pH. 6.7, 1 h 0.00.61.630 (1.6 AlPcCl. HBSS at pH, 6.7 0.6 AlPcCl, HBSS with nigericin, pH, 6.7. 12 kJ/m2Omin0.3 23.6* a Only about 50% of the cells were recoverable for assay, based on total cell counts in control flasks.

(5 mg/ml); therefore addition of fresh medium to the cells namic treatment with AlPcCl and red light. The effect of should have reduced or stopped nigericin-induced metabolic nigericin is most pronounced at light fluences below 20 kJ/m2 perturbations. (Fig. 1). Nigericin and PDT interact synergistically, since treat Effect of Incubation in HBSS, with or without Nigericin, on ments with nigericin alone, nigericin and red light, or nigericin Cellular Content of AlPcCl. The protective effect of incubation and AlPcCl are not toxic to cells. A change in slope of the in HBSS before red light irradiation was described for Figs. 2 nigericin-PDT dose-response curve is observed such that the and 4. Since medium containing AlPcCl was removed before surviving fraction is similar for 20 and 30 kJ/m2 light exposure. addition of HBSS, we tested the possibility that this protection This might be due to the difficulty of exposing cells at the is due to reduction in the cellular content of AlPcCl. Table 2 periphery of the culture flasks to the full dose of light, or it shows that such a mechanism is unlikely since incubation in might be due to the presence of a small, resistant subpopulation buffer does not alter the total amount of AlPcCl extractable of cells. In either case, it should be noted that the reduced effect from cells. is observed for a relatively insignificant subset of cells (1 in With respect to nigericin-induced sensitization to PDT, a IO5). possible mechanism is ionophore-mediated redistribution of Because nigericin may be inactivated by serum (12), these AlPcCl to highly sensitive sites within the cells. We have not studies were performed by incubating AlPcCl-treated cells in yet tested this by subcellular fractionation; however, we found HBSS, with or without nigericin, just before light exposure. As no effect of nigericin on the cellular content of AlPcCl (Table described in "Results," cells incubated in HBSS were less 2) and no effect of nigericin on the spectrum of AlPcCl in intact photosensitive than counterparts which had not been incubated cells measured after trypsinization and swelling in hypotonie in buffer. Ben-Hur and Rosenthal (21) have shown that the saline (data not shown). photosensitivity of AlPcCl-treated cells is linearly dependent on dye concentration. Therefore we tested the effect of incuba DISCUSSION tion in HBSS on total content of AlPcCl (Table 2). No differ This study shows that V79 cells exposed to the K+/H+ ences in total dye content were observed; however, this does ionophore nigericin are dramatically sensitized to photody- not preclude the possibility that HBSS induces a redistribution 1623

tial for increasing the selectivity of PDT for tumor over normal tissue. Possible cellular targets for PDT include the plasma mem brane, mitochondria! membrane, microsomes, and the nucleus (1,2,9, 19). In the case of both HPD and AlPcCl phototherapy, there is substantial evidence that mitochondrial damage is a primary cause of phototoxicity (18, 19, 25). Fig. 5 demonstrates that nigericin-PDT combinations induce a metabolic rather than a reproductive cell death, since cells are rounded up within 2 h of light exposure and lysed within 12 h, even when the buffer containing nigericin is replaced with fresh medium im mediately following PDT. Nigericin is known to affect mito chondrial function by permeabilizing mitochondrial mem branes to K+ and H+ (12, 13, 20). We observed that nigericin inhibits respiration in Chinese hamster ovary cells (5). Since PDT can inhibit mitochondrial enzymes (19, 24, 26), we rea soned that the combined effects of nigericin and PDT might cause a drastic reduction in intracellular ATP levels, resulting in death by energy deprivation. Fig. 6 shows that nigericin 30 60 90 120 treatment alone lowers ATP levels and that PDT produces a Mm Before or After Start of Light Exposure transient decrease in ATP in both control and nigericin-treated Fig. 6. Effect of PDT. with (â€¢)or without (O) nigericin. on cellular ATP cells. However, cells treated with nigericin and PDT recover levels. Cells were incubated for 18 h with AlPcCl and then for up to 60 min in HBSS with or without nigericin. , time of irradiation with 12 kJ/m2 red their ATP levels after being refed with fresh medium, although light. Cells were left on the monolayer and refed with fresh medium immediately not to the extent of cells treated with PDT alone. ATP levels after irradiation, as described for Fig. 5. Data represent an average of triplicate measurements Â±SEM (bars). were measured for up to 2 h after light exposure, at which time morphological changes were pronounced. These data indicate

Table 2 Effect of incubation of AlPcCl-treated cells in HBSS, with ana without that the morphological changes occurring in cells destined to nigericin, on cellular content ofAIPcCI die are not due to energy deprivation. However, the data do not nmol preclude the possibility that mitochondria are the prime targets AIPcCl/107 of the combined therapy. ATP levels may be maintained by Treatment condition cells glycolysis for a considerable time after mitochondrial ATP 0.01Â° Control (untreated) production has been compromised. AlPcClfor 18h 0.91 Â±0.16 AlPcCl for 18 h, followed by l h in 0.99 Â±0.12 In addition to lowering pHÂ¡of cells and destroying the mi HBSS at pH, 6.7 tochondrial electrochemical potential, nigericin induces a rapid AlPcCl for 18 h, followed by l h in 0.95 Â±0.17 equilibration of intracellular and extracellular K+ and Na+ (5, HBSS containing 2 /;\i nigericin. pH. 6.7 13). Nigericin potentiates PDT significantly even when added ' A low level of background absorption was not subtracted from the values to cells just prior to light exposure, and the effects of this drug presented. Data represent an average value from three or more flasks, from one are optimal after 30 min preincubation (Fig. 4), a time when experiment, Â±SEM. the effects on ATP levels are minimal (Fig. 6). This suggests that the effects of nigericin may be at the plasma membrane as of AlPcCl to less sensitive sites within cells or permits associ well as at the mitochondrial membrane. However, nigericin ation of AlPcCl monomers into less photoactive aggregates does not induce permeability to trypan blue in a significant (22). fraction of cells (Table 1). Fig. 2 shows that potentiation of PDT by nigericin is re Another possibility for potentiation of AlPcCl phototoxicity markably pHt. dependent. We previously reported that nigericin by nigericin is that the drug causes a change in distribution of enhances heat killing of cells preferentially under acidic condi AlPcCl within cells, or within cell membranes, so that more tions and that it inhibits repair of potentially lethal radiation susceptible targets are exposed. Nigericin does not affect overall damage when pHc is acidic (5). Both of these effects were AlPcCl content (Table 2), nor does it change the absorption attributed in part to the ability of nigericin to equilibrate pHÂ¡ spectrum of AlPcCl in intact cells which have been swelled by with pHe when the latter is mildly acidic (13, 23). In our incubation in hypotonie buffer ("Results"). However, the effects experiments PDT with AlPcCl and 12 kJ/m2 red light was only slightly toxic at pHc 7.5 and was not increased by lowering pHc of nigericin on distribution of AlPcCl within various subcellular (Fig. 4). However, Ben-Hur and Rosenthal (24) observed an organelles have not yet been determined. effect of pHc on the response of V79 cells to AlPcCl and light In vivo, destruction of tumors by PDT is related to vascular under more toxic treatment conditions. These authors reported damage as well as to cellular damage (1,2,19). The experiments a dose modification factor of approximately 1.3 for cells incu reported here do not address vascular mechanisms of PDT bated in phosphate-buffered saline at pHe 6.5 for 30 min prior action. However, they provide strong evidence for a component to light treatment, as compared to incubation at pHs 7.5. Thus of cellular damage which can be enhanced by membrane-active the pHÂ«dependence for enhancement of PDT by nigericin may drugs. We are currently testing the ability of other ionophores be due to effects of this drug on pHÂ¡.The pHe of both animal and mitochondrial inhibitors to interact synergistically with and human tumors is, on the average, 0.5 unit lower than the PDT. Our objectives are to elucidate the mechanism for syn- pHc of surrounding normal tissue (14, 15). Thus drugs which, ergistic interaction and to determine the limits to which PDT- like nigericin, act in a pHe-dependent manner may have poten induced cellular damage can be enhanced by metabolic controls. 1624

Oleinick, N. L. Cytotoxic and mutagenic effects of the photodynamic action ACKNOWLEDGMENTS of chloroaluminum phthalocyanine and visible light in LSI 78Y cells. Pho tochem. Photobiol., 49: 43-47. 1989. The authors wish to thank the following persons: Dr. James K. V. 12. Grinstein, S., Cohen, S., and Rothstein, A. Cytoplasmic pH regulation in Willson, Division of Hematology/Oncology, Department of Medicine, thymic lymphocytes by amiloride-sensitive Na*/H* antiport. J. Gen. Physiol., University Hospitals of Cleveland, for advice on photomicroscopy; 83: 341-369, 1984. 13. Rotin, D., Wan, P., Grinstein, S., and Tannock, 1.Cytotoxicity of compounds George Sebold of the Packard Instrument Co., for assistance with that interfere with the regulation of intracellular pH: a potential new class of chemiluminescence assays using the Packard 3522 Tri-Carb scintilla anticancer drugs. Cancer Res., 47: 1497-1504, 1987. tion counter; Drs. Helen Evans and Oddvar Nygaard, Division of 14. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1990 American Association for Cancer Research. Enhancement of Photodynamic Cell Killing (with Chloroaluminum Phthalocyanine) by Treatment of V79 Cells with the Ionophore Nigericin

Marie E. Varnes, Marian E. Clay, Keith Freeman, et al.

Cancer Res 1990;50:1620-1625.

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