Growth Inhibition of Physarum Polycephalum for the Evaluation of Chemotherapeutic Agents.

Using 50 percent growth inhibition of Physarum polycephalum (a coenocytic slime mold) as a screen for the selection of antitumor agents, 44 of the 195 compounds tested were effective at concentrations of@lOO @tg/m1. Results of inhibition of KB cell cul ture screen are given for comparison. INTRODUCTION Physanun polycephalum can be grown in a surface culture as a large, multinucleate plasmodium showing synchronous division, or in agitated, submerged culture as a sus pension of microplasmodia which resemble monoor polynucleate amoeba (1,2). In agi tated culture, under controlled incubation conditions, the microplasmodia proliferate rapidly and growth can be measured by protein or pigment content (1). MATERIALS AND METHODS Physarum @o1ycepha1um was grown in agitated submerged culture in a medium con taming 1 percenW Txyptone (Difco ), 1 percenÃ1⁄4/ dextrose, 0. 15 percent@/ yeast extract (Difco), salts, citrate buffer, and heniatin. The medium and conditions of growth were described previously (1) with the exception that citrate (0.0192 M) replaced the calcium carbonate (the pH of the medium was adjusted to 4.6 with potassium hydroxide) and hematin replaced the chick embryo extract. Hematin was prepared as a 500 p.g/ml solution of hemin (Si@na) in 1 percent sodium hythoxide. When this solution had been auto claved, 1 ml. was added to each 100 ml. of sterile medium. An 0.8 ml. inoculum of a 68-70 hour culture was added to 10 ml. of medium and 1 ml. of test compound solution in a 25 x 150 mm. test tube. In darkness the tubes were agitated on a rotary shaker (240 rpm) at 220C. Pigment and protein analyses were carried out after 69 hours to measure the ex tent of growth. The contents of duplicate tubes were added to 50 ml. graduated centri fligetubes containing 24 nil.of 8 percent trichioroacetic acid in acetone, mixed, and stored overnight in darkness at 40C. Before analysis the samples were warned to room temperature, stirred, and centrifuged for 5 minutes in an International Clinical Centri fuge at 350 g. The supernatant was retained for pigment analysis and the precipitate analysed for protein by the lowry modification of the Folin method (3 ) . Pigment content was determined in a Bausch and lomb Spectronic 20 colorimeter by measuring the optical density of trichioroacetic acid and acetone solutions at 415 m@t. and is expressed as optical density units per ml. Because of light sensitivity of the pigment, solutions were protected from prolonged exposure to light. Tests were conducted in duplicate. Four compounds were tested simultaneously at 1, 10, and 100 p.g,/ml of medium. A compound of known inhibitory value, purified pyronine B, served as a positive control. lvkximum normal growth was measured on drug-free cul tures. Immediately after inoculation, initial protein and pigment levels in two cul ture tubes were determined. After 69 hours of incubation protein, when necessary, and pigment were measured. The percent of normal growth was calculated and from this the ED@j (calculated @xg/mi of compound pennitting 50 percent of maximum normal growth) was determined. @/ Percent= w/v. on June 2, 2017. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Becker et al. Cancer Research ChemotherapyScreening Data 1911 Test compounds were prepared, when possible, as 1.1 percent aqueous solutions. If the compound were not water soluble, it was prepared as a 1.1 percent solution or suspension in one of the following solvents: 0.01 percent sodium hydroxide; 1 percent sodium bicarbonate; 1 percent sodium carbonate; 0.01 normal sulftric acid; 3 percent, 10 percent, or 95 percent ethanol; 10 percent steroid diluentV (Philadelphia Ampoule Laboratories ). Occasionally, warming was neces sary to obtain solution. Addition of 1 ml. of such a solution to 10 ml. of medium gave a final drug concentration of 100 kg/nil. Serial dilutions with water were made as required. All solvents used were tested for growth-inhibitory effect with none affecting growth at the maximum concen tration used. The particular solvent used for each compound is listed in Table II and III. RESULTS Pigment production was shown to be a satisfactory measure of growth, correlating well with protein content in normal and growthinhibited cultures (Figure 1; Table I). Table I shows typical results of growth inhibition measured both by pigment and protein content. Good agreement in tests such as these made protein determination unnecessary except when compounds under test absorbed at 415 rip. Interference by dnigs absorbing at this wavelength was a minor problem, as the highly sensitive pigment determination permitted great dilution of solutions before optical density was determined. Protein, as well as pigment, was always measured for positive and negative controls. The sim plicity of the screening system rests on the assumption of correspondence between protein and pigment. It is possible, however, that test compounds might stimulate or depress pigment formation without affecting protein, although compounds with this effect were not detected. This caution with regard to pi@nent development as a parameter of growth must be extended to protein level. Recent experiments have shown a level of actinomycin D that induces unbalanced growth in P. polycephalum, inhibiting RNA synthe sis, but not affecting protein or DNA synthesis during the first 48 hours. Of 195 compounds tested, 44 were effective inhibitors of growth at 100 iLg/mi or less; that is, their ED5Q values were less than 100. These compounds are listed in Table II with results given as ED@ for P. polycephalum. For comparative purposes, ED@j values as determined by the KB cell culture screen (4) have been included (unpublished CCNSC screening data ). Among the clinically known antitunior compounds which inhibited the growth of P. ppl@ycephalum to this extent were certain antifolic agents, e.g., 5fluorouracil and thioguanine. Aerobic inhibitors, such as dinitrophenol and sodium azide, were also active in the Pilysarumscreen. Of the 44 compounds listed in Table II, five did not inhibit the KB cells (NSC Nos. 30091, 30111, 31156, 31986, 39701; toward 16 of them P. polycephalum was more sensitive or equally so; and toward 23 of them ;@.polycephalumshowedless sensitivity. The differences fromthe KBscreen do not seem great by this measure. This test system missed a number of substances such as some of the alkylating agents, steroid hormones, and antibiotics, which have inhibited the growth of tumors in other test systeras. AU compounds that were non-inhibitory to growth of P. polycephalum or inhibited growth less than 50 percent at 100 @ig/mi are listed in Table III. ED@j values from the KB cell culture screen are also shown. From Table III one learns that the KB cell is much less selective than P. polycephalum. On the 151 compounds listed, 102 inhibited growth of the KB cells at levels which did not have this effect on @. polycephalum. Further study should be undertaken to determine if this difference means that P. polycephalum is sensitive to tumor-inhibitory compounds of potential value, but is less frequently affected by non-specific agents. If this were the case, the use of P. polycephalum as a routine screen for the selection of tumor-inhibitory compounds should receive serious consideration, because the procedure is simple, rapid, and inexpensive; and because in comparison to some other existing screens it shows a high degree of selectivity. .@/ Steroiddiluent : Aqueous solutionof sodiumchloride(0 9%)polysorbate80 (0 4%), carboxymethylcellulose (0.5%), and benzyl alcohol (0.9%). on June 2, 2017. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from 1912 CancerResearch Vol.23,No. 10,Nov. 1963 Figure 1 exhibits the growth of P. pqlyce@halum in agitated culture measured by pigment (optical density units/na) and protein (mg/rn].). The formation of protein and pi@nent occur in parallel for the first 72 hours of growth. REFERENCES 1. Daniel, J. W. , and Ruach, H. P. The Pure Culture of Physarum polycephalum on a Partially Defined Soluble Medium. J. Gen. Microbiol., 25:47-59, 1961. 2. Guttes, E.; Guttes, S.; and Rusch, H. P. Morphological Observations on Growth and Differentiation of Physarum polycephalum Grown in Pure Culture. Develop. Biol., 3:588-614, 1961. 3. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; and Randall, R. J. Protein Measure ment with the Folin Phenol Reagent. J. Biol, Chem., 193: 265275, 1951. 4. Eagle, H., and @bley, G. E. Cytotoxicity in Human Cell Cultures as a Primary Screen for the Detection of Anti-tumor Agents. Cancer Research, 18:1017-1025, 1958. on June 2, 2017. © 1963 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from ENTRY NO.NSC NO.COMPOUND NAMEFORNULATABLE REFERENCEIIIII 1913 TABLEA LIST OF COMPOUT@DS