[CANCER RESEARCH 43, 716-720, February 1983] 0008-5472/83/0043-0000502.00 Selective Toxicity of Rhodamine 123 in Carcinoma Cells in Vitro Theodore J. Lampidis, 1 Samuel D. Bernal, 2 lan C. Summerhayes, and Lan Bo Chen a Sidney Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT ing appears to be unique, since chemotherapeutic agents currently in clinical use have not been shown to be particularly The study of mitochondria in situ has recently been facilitated selective between tumorigenic and normal cells in vitro. through the use of rhodamine 123, a mitochondrial-specific fluorescent dye. It has been found to be nontoxic when applied MATERIALS AND METHODS for short periods to a variety of cell types and has thus become an invaluable tool for examining mitochondrial morphology and Cell Cultures. All cell types and cell lines were grown in Dulbecco's function in the intact living cell. In this report, however, we modified Eagle's medium supplemented with 10% calf serum (M. A. demonstrate that with continuous exposure, rhodamine 123 Bioproducts, Walkersville, Md.) at 37 ~ with 5% CO2. The following cell selectively kills carcinoma as compared to normal epithelial lines were obtained from the American Type Culture Collection: cells grown in vitro. At doses of rhodamine 123 which were CCL 105, CCL 15, CRL 1550, CRL 1420, CCL51, CCL77, CCL185, toxic to carcinoma cells, the conversion of mitochondrial-spe- CCL 34, PtK-1, CV-1, and BSC-I. The human bladder carcinoma line cific to cytoplasmic-nonspecific localization of the drug was (E J) was provided by Dr. L. M. Frank (Imperial Cancer Research Fund). observed prior to cell death. At 10 /~g/ml, >50% cell death EJ and mouse bladder epithelial cells were isolated and grown as described previously (10). MCF-7 was provided by Dr. M. Rich (Mich- occurred within 7 days in all nine of the carcinoma cell types igan Cancer Foundation). Human breast epithelial cells were gifts from and lines of different origin studied, while six of six normal Dr. N. S. Yang (Michigan Cancer Foundation) and Dr. M. Stampfer epithelial cell types and lines remained unaffected. Cotreating (Peralta Cancer Research Institute). carcinoma cells with 2-deoxyglucose and rhodamine 1 23 en- Cytotoxicity and Clonogenic Survival Assays. For the cytotoxicity hanced the inhibition of growth by rhodamine 123 alone in assays, cells were seeded at 5 x 10 ~ cells/60-mm plate and incubated clonogenic survival assays. The observation of the selective at 37 ~ in 5% CO2. Rhodamine 123 (Eastman Kodak, Rochester, N. Y.) toxicity of rhodamine 1 23 appears to be unique in view of the was applied the following day at the indicated doses, and cell numbers absence of selective toxicity reported in vitro for the various were monitored at various periods during continuous drug exposure by antitumor agents currently in clinical use. Preliminary results trypsinizing the appropriate cultures and counting the number of cells with rhodamine 1 23 in animal tumor systems indicate antitumor by hemocytometer. The exclusion of trypan blue (0.2%) was used as an indicator of the number of live cells. For the clonogenic assays, 300 activity for carcinomas. cells were seeded in 60-mm dishes and incubated at 37 ~ in 5% CO2. On the following day, drug was applied at the indicated doses, and INTRODUCTION CFU ~ were determined 2 weeks later by staining with methylene blue (0.2%). Rhodamine 1 23, a cationic fluorescent dye, has been used Fluorescence Localization. Cells were grown on 12-mm round glass previously to specifically localize mitochondria in living cells coverslips (Rochester Scientific, Rochester, N. Y.), treated with rho- (9). It has been a convenient and useful tool for mitochondrial damine 123 for various time periods, and rinsed and mounted on glass studies in situ, since their detectability is enhanced and the dye slides with silicone rubber chambers in rhodamine-free medium as is relatively nontoxic in most of the cell types studied (1-3, 5- described previously (9). Stained cells were examined by epifluores- 9, 1 1, 1 2). Recently, however, rhodamine 1 23 has been found cent illumination at 485 nm on a Zeiss Photomicroscope III equipped with a Zeiss Planapochromat objective lens (x40). to inhibit oxidative phosphorylation in isolated mitochondria at high doses" and has been reported to have a cytostatic effect in L1 21 0 cells (4). Since we had observed that carcinoma cells RESULTS accumulate and retain more rhodamine 123 than do normal Chart 1 illustrates the difference in the effect of rhodamine epithelial cells, fibroblasts, myoblasts, chondrocytes, and lym- 123 on the growth of transformed and normal epithelial cell phocytes (1 1), we were prompted to investigate the cytotoxic lines. Chart 1, A and C, illustrate the marked difference be- effects of this agent on carcinoma cells. In this report, we tween treated and untreated cultures in 2 tumor-derived epi- demonstrate that rhodamine 1 23 selectively inhibits the growth thelial cell lines, human pancreatic carcinoma line CRL 1420 and kills carcinoma-derived epithelial cells in vitro, while the and human breast carcinoma line MCF-7. At 2 days of treat- growth of normal epithelial cells remains unaffected. This find- ment, a marked cytotoxic effect is seen in cell line CRL 1420, 1 Supported by a Young Investigators Award (CA24771). To whom requests while MCF-7 cells are inhibited in their growth. With further for reprints should be addressed, at Sidney Farber Cancer Institute, Harvard incubation, 3 days for CRL 1420 and 7 days for MCF-7, the Medical School, 44 Binney Street, Boston, Mass. 02115. 2 Supported by a Postdoctoral Fellowship (CA06943) from the National Cancer cell number is reduced to less than 103 per plate in each of the Institute. rhodamine-treated cultures. In marked contrast to these re- 3 Recipient of an American Cancer Society Faculty Research Award. Sup- ported by grants from the National Cancer Institute, CA 22427, and the American sults, CV-1 and PtK-1, derived from normal African green Cancer Society, CD 92B. monkey kidney (epithelial) and normal marsupial kidney (epi- "T. J. Lampidis, C. Salet, G. Moreno, and L. B. Chen. Effects of the mito- thelial), respectively, show no cytotoxicity when exposed to the chondrial probe rhodamine 123 and related analogues on the function and viability of pulsating myocardial cells in culture, submitted for publication. Received June 21, 1982; accepted November 5, 1982. The abbreviation used is: CFU, colony-forming units. 716 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. Rhodamine 123 Kills Carcinoma Cells in vitro same dose of rhodamine 123 (Chart 1, B and D). With even hanced by cotreating with 2-deoxyglucose. longer exposure (2 weeks at 10 #g/ml) or at higher doses (50 To determine whether the selective toxicity of rhodamJne /zg/ml for 7 days), cell growth of CV-1 and PtK-1 remains 123 is a general phenomenon shared by other tumorigenic relatively unaffected. epithelial cells, a number of carcinoma lines were studied. In the course of these experiments, it was noted that a Table 2 is a composite of 15 different cell lines tested for significant number of cells had detached from the culture dish sensitivity to rhodamine 123 when exposed continuously to surface after drug treatment (3 days for CRL 1420 and 7 days various doses. It can be seen that all 9 tumorigenic carcinoma- for MCF-7). We followed the fate of these cells in the presence derived epithelial cell lines used in this study were susceptible of drug with increasing time and found that they converted to the cytotoxic effects of rhodamine 1 23 at 10 or 50/zg/ml. gradually from trypan blue negative to trypan blue positive. In The nontumorigenic epithelial cell lines derived from normal addition, we have tested the ability of these detached cells, tissues were unaffected by treatment at these doses and con- while still trypan-blue negative, to proliferate in drug-free me- tinued to grow normally as untreated cells. Thus, a dose of dium and found that they are no longer able to reattach to the --<10/~g/ml, at which 50% of the treated cells were killed, was culture dish surface nor divide in suspension. found for each of the carcinoma cell lines tested, whereas the The selective toxicity of rhodamine 123 for carcinoma cells dose at which 50% of the treated normal epithelial cell lines was also observed when clonogenic survival assays were per- were killed was >50/zg/ml. To confirm the epithelial origin of formed. Chart 2 illustrates the difference in CFU between the cell lines tested, immunofluorescence staining with keratin rhodamine 1 23-treated normal epithelial (CV-1) and carcinoma antibody was used. All cell lines desiqnated in this report as (1420) cell lines. At 5 /~g/ml, the carcinoma cells form no epithelial or carcinoma were keratin positive. colonies while the normal cells remain relatively unaffected In conjunction with these experiments, each cell line was (CFU are 83% of control). Thus, although there is a slight tested for its sensitivity to the potent, broad-spectrum antitumor reduction in normal colony formation, the same pattern of agent Adriamycin. Although some of the carcinoma cell lines carcinoma sensitivity versus normal cell resistance to rhoda- did appear to be more sensitive to Adriamycin than did the mine 1 23 treatment as seen in the growth experiments prevails. untransformed lines, the complete insensitivity of normal epi- Since rhodamine 123 inhibits oxidative phosphorylation in thelial cells to rhodamine 1 23 at high doses (50 #g/ml) and for isolated mitochondria, 4 we were interested in testing whether prolonged exposure times (7 days) suggests a stronger selec- an inhibitor of glycolysis, 2-deoxyglucose, could affect the selective toxicity of rhodamine 123 for carcinoma cells.