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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 ¿¿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), ±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 hyperglycémieby 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 ÇA40516 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 4°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, hematopor- phyrin 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 («= 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.
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