Susceptibilities of Normal and Malignant Human Lung Cells in Culture to the Cytocidal Action of Antitumor Agents1
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[CANCER RESEARCH 38, 2529-2532, August 1978] 0008-5472/78/0038-0000$02.00 Susceptibilities of Normal and Malignant Human Lung Cells in Culture to the Cytocidal Action of Antitumor Agents1 Hohko Tsukeda, Satoshi Mizuno, and Kazuo Nitta Departments of Virology [H. T.¡and Antibiotics, [S. M.¡,National Institute of Health, Shinagawa-ku, Tokyo 14T, and Chemotherapy Division, National Cancer Center Research Institute [K. N.¡,Chuo-ku, Tokyo W4, Japan ABSTRACT normal and malignant human lung cell lines to 9 clinically efficacious antitumor agents. Cultured normal and malignant human lung cells were examined for their different sensitivities to the cytocidal action of nine antitumor and three non-antitumor agents. MATERIALS AND METHODS The malignant cells were killed preferentially at lower Cell Lines and Culture. Two cultured human cell lines concentrations and in a shorter time with each antitumor were used in these experiments. One is a diploid fibroblast agent, Adriamycin, neocarzinostatin, bleomycin, actino- derived from normal embryonic lung tissue and mycin D, mitomycin C, carboquone, 1-0-D-arabinofurano- designated HAIN-6 (15). The other is a cell line derived from sylcytosine, 5-fluorouracil, and 5-fluorodeoxyuridine. lung squamous carcinoma and designated PC-1 (14). PC-1 Among these, Adriamycin, 5-fluorouracil, and 5-fluoro cells produce anaplastic carcinomas when transplanted deoxyuridine exhibited a higher differential lethal action s.c. into nude mice (9). The HAIN-6 cells were grown as a on the malignant cells than did the other antitumor agents. monolayer in Eagle's basal medium (Grand Island Biologi The two cell lines exhibited little difference in cytolytic cal Co., Grand Island, N. Y.), as described by Hayflick and sensitivity to non-antitumor cytotoxic agents such as am- Moorhead (6). The PC-1 cells were grown as a mono- photericin B, G-strophanthin, and a-amanitin. layer in Eagle's minimum essential medium (2) (Nissui, Tokyo, Japan), with 10% fetal calf serum (Grand Island INTRODUCTION Biological Co.). The media contained kanamycin instead of penicillin and streptomycin. HAIN-6 cells were dispersed Cell culture techniques have been widely used to assay into a single-cell suspension with 0.25% trypsin (Difco the cytotoxic action of antitumor agents against several cell Laboratories, Detroit, Mich.; 1:250) in phosphate-buffered lines that were derived from malignant human and animal saline. PC-1 cells were scraped off into a single-cell suspen tissues. Those studies indicated that there exists a high sion. Each cell suspension was diluted, transferred to degree of correlation between cytotoxicity in vitro and growth medium, and cultured at 37°in a closed system. antitumor activity in vivo (10-12). When drugs are evaluated HAIN-6 cells were capable of 50 ±5 (S.D.) population for antitumor action in vitro, it may be desirable to compare doublings and were used at their 16th to 30th passage. their cytotoxic action on malignant cells with that on normal Cells were renewed from original stock maintained at -80°. cells. Systematic studies by Eagle and Foley (3-5) reported Cells were monitored to assure the absence of mycoplasmic that there was little difference between malignant and contamination. The doubling times of HAIN-6 and PC-1 normal cells in growth inhibition by antitumor agents. cells were similar, about 24 hr, and their saturation densi However, Biesele (1) showed some differences in the sus ties were 8 x 10s and 2.5 x 10" cells/35-mm plastic dish, ceptibility of tumor and liver cells to cellular morphological respectively. damage by antitumor agents; the results suggested that Determination of Cell Lethality. Suspended cells were tumor cells might be more sensitive in vitro to some antitu- seeded at about 1 x 105 cells/35-mm plastic dish (Lux mor agents. Scientific Corporation, Newbury Park, Calif.) with 2 ml of Several human malignant cell lines derived from cancer growth medium and cultured at 37°in 5% CO2-air. The tissues have been established recently; most of these cell agents were added after 2 days to the cultured monolayer lines produce tumors when transplanted into nude mice. cells at final concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, Some normal human diploid cell lines derived from embry 1.0, 2.0, 5.0,10, 20, and 50 /¿g/ml.Their action on the cells onic lung tissues have also been established recently. When was determined every 12 hr for 6 days by counting viable the drug action on malignant cells was tested in vitro, cell numbers with a hemocytometer after the cells were normal cells to be used as controls should be taken from stained with 0.2% nigrosin (8). Morphological comparisons those derived from the corresponding tissue. From this between treated and untreated cells were made by phase- point of view, we selected normal and malignant human contrast microscopy. Each experiment was repeated at lung cells for target cells. least 3 times. In this paper we report a difference in the sensitivity of Reagents. Adriamycin, bleomycin, carboquone (Esqui- non), and neocarzinostatin were purchased from Kyowa Hakko, Nippon Kayaku, Sankyo, and Kayaku Antibiotics 1This work was supported by Grants for Cancer Research from the Research, Tokyo, Japan, respectively. Actinomycin D, o- Japanese Ministries of Health and Welfare and of Education, Science, and amanitin, 5-fluorouracil, and mitomycin C were obtained Culture. Received October 31, 1977; accepted April 24, 1978. from Boehringer-Mannheim, Yamanouchi, Tokyo, Japan. 1- AUGUST 1978 2529 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. H. Tsukeda et al. ß-D-Arabinofuranosylcytosine and 5-fluorodeoxyuridine after shorter Adriamycin treatment (Table 1). were from Sigma Chemical Co., St. Louis, Mo. Amphoteri- The 2 cell lines were also studied for their susceptibility cin B and G-strophanthin (ouabain) were purchased from to 8 other known antitumor agents and 3 non-antitumor Calbiochem, Los Angeles Calif, and Merck & Co., Inc., toxic substances. The results were summarized in Chart 2 Rahway, N.J., respectively. Actinomycin D and amphoteri- together with the data for Adriamycin; the drug concentra cin B were dissolved first in a small amount of ethanol and tions that caused 50% cell death were plotted with the time dimethyl sulfoxide, respectively, and thereafter diluted with of drug treatment. The results showed that PC-1 cells were water. All other agents were dissolved in distilled water . preferentially killed by antitumor agents at lower concentra tions and in shorter times of drug treatment (Chart 2, a to /'). RESULTS This different sensitivity of the 2 cell lines was evident with every antitumor agent tested; it was especially remarkable Chart 1 shows the kinetics of growth inhibition of the 2 with Adriamycin in the early days of treatment and with cell lines by various concentrations of the antitumor anti biotic Adriamycin. The growth of HAIN-6 was almost com neocarzinostatin and actinomycin D in the later days. Only a slightly different sensitivity was noted with non-antitumor pletely inhibited by Adriamycin (0.05 ¿tg/ml)after 2 days; agents such as amphotericin B, G-strophanthin, and a- the decrease in viable cell number was gradual after 2 days at 0.1 MQ/ml (Chart 1a). The growth of PC-1 cells was amanitin (Chart 2,y to /). Chart 3 clearly shows the different sensitivity of the 2 cell inhibited similarly, but the reduction in viable cell number lines at higher doses and after longer treatment with bleo- was more rapid than with HAIN-6 cells (Chart 1b), suggest mycin, 5-fluorouracil, and 5-fluorodeoxyuridine. 5-Fluoro- ing that PC-1 cells were killed more rapidly than were HAIN- deoxyuridine did not show the 50% lethality to HAIN-6 cells 6 cells after cell growth was inhibited. Morphological ex even at 200 ¿¿g/ml,whereasit was obtained in PC-1 cells at amination confirmed that PC-1 cells were damaged more severely than were HAIN-6 cells at lower concentrations and 0.1 ¿¿g/mlafter6 days of treatment (Chart 3c). Chart 4 shows the dose-response relationship of the 2 cell lines to Adriamycin, neocarzinostatin, and amphoteri cin B. When the concentration of Adriamycin was in creased, the difference in sensitivity became larger. Acti nomycin D also showed this type of dose response (not shown). On the other hand, when the concentration of neocarzinostatin was increased, the difference in sensitivity became smaller. Mitomycin C and 1-/3-D-arabinofuranosyl- cytosine showed this type of dose response (not shown). A minor difference in sensitivity was demonstrated to ampho tericin B only at lower concentrations; G-strophanthin and «-amanitinalso exhibited this type of dose response. 01 2345 Days after addition DISCUSSION Chart 1. Growth profiles of HAIN-6 and PC-1 cells at various concentra tions of Adriamycin. Adriamycin was added at Day 0 (arrows), a, HAIN-6; b, Nine clinically available antitumor agents were tested for PC-1; O, control; A. 0.01 M9/ml; •¿0.05M9/rnl; •¿,0.1jig/ml. differential cytocidal action in vitro on normal and malig- Table 1 Cytotoxic effects of Adriamycin on HAIN-6 and PC-1 cells The cells were examined morphologically every 12 hr for 4 days at 12 serial concentrations, as described in "Materials and Methods." The extent of cellular damage was graded in 5 categories according to cellular morphological observation and growth recovery, as determined by subculture in drug-free medium. HAIN-6 PC-1 after addition at following day after addition at following day (M9/ml)40.10 0.51 1.5 2 2.5 3 3.5 4 0.5 1 1.5 2 2.5 3 3.5 _______ ____±± + + + 0.05 ± ± + 0.02 0.01 ________________ " -, No damaged cells, almost full recovery; ±,0 to 30% cells damaged, moderately impaired recovery; +, 30 to 60% cells damaged, extensively impaired recovery; + + , 60 to 90% cells damaged, almost no recovery; + + + , more than 90% cells damaged, no viable cells.