[CANCER RESEARCH 51,6025-6030, November 15. I991| Cytotoxic Effect of as-Parinaric Acid in Cultured Malignant Cells1 Albert S. Cornelius,2 Negender R. Yerram, Deborah A. Kratz, and Arthur A. Spector Division of Hematology/Oncology, Department of Pediatrics [A. S. C, D. A. K.J, and Department of Biochemistry ¡N.R. Y., A. A. S.J, University of Iowa College of Medicine, Iowa City, Iowa 52242 ABSTRACT Parinaric acid, a naturally occurring 18-carbon fatty acid containing 4 conjugated double bonds, is toxic to human monocytic leukemia cells at concentrations of 5 MMor less. Conditioning of the medium reduces the cytotoxic effect, suggesting that parinaric acid and not a metabolite is the active agent. The mechanism of parinaric acid toxicity appears to involve lipid peroxidation because the toxic action can be blocked by the addition of butylated hydroxytoluene. When U-937 cells are differentiated to the monocytic form, they become resistant to as much as 30 MMparinaric acid. This difference in sensitivity may be explained in part by the fact that the undifferentiated cells take up 3 to 4 times more parinaric acid. Concentrations of parinaric acid less than 5 MMare also toxic to human 11IP-1 monocytic leukemia, III -00 human promyelocytic leukemia, and Y-79 human retinoblastoma cells. Measurements of protein synthesis indicate that differentiated U-937 cells, confluent cultures of human fibroblasts, bovine aortic endothelial cells, and CaCo-2 colonie mucosa! cells are much less sensitive to parinaric acid than the malignant cell lines tested, suggesting that the cytotoxic action may be selective for rapidly growing malignant tumors. Thus, parinaric acid may be the prototype of a new class of lipid chemotherapeutic agents that contain a conjugated system of double bonds and act by sensitizing tumor cells to peroxidation. INTRODUCTION Polyunsaturated fatty acids can have important influences on cellular proliferation (1,2). Essential fatty acids such as linoleic acid (18:2 <<j-6) stimulate the proliferation of fibroblasts during wound healing (3), suggesting that they may act as growth- stimulatory factors under certain conditions. In addition, lino- lenic acid, arachidonic acid (20:4 u-6), and the u-3 polyunsat- urated fatty acids are cytotoxic to cultured cells at concentra tions between 60 and 100 /UM(4, 5). The cytotoxic potential of these fatty acids appears to be related but not directly propor tional to the number of double bonds that they contain. Based on these observations, it has been suggested that polyunsatu- rated fatty acids containing 3 to 5 double bonds may be useful as antitumor drugs (5, 6). Although the mechanism through which these fatty acids produce cytotoxicity in malignant cells has not been fully elu cidated, it appears to correlate with Superoxide production (5) and is reduced by the presence of antioxidants (7). The poly- unsaturated fatty acids previously tested have had the usual unconjugated configuration in which each pair of double bonds is separated by a méthylène carbon. A conjugated system of double bonds, however, is known to be more effective in trap ping electrons. To determine whether fatty acids containing conjugated double bonds may be more cytotoxic for malignant cells, we compared the effect of as-parinaric acid with corre sponding unconjugated polyunsaturated fatty acids. c/s-Parinaric acid (9C,11,,13,,15C-18:4) is an 18-carbon u-3 Received 6/5/91; accepted 9/9/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely Io indicate this fact. 1This work was supported by NIH Grant HL39308. 2To whom requests for reprints should be addressed, at Department of Pediatrics, Division of Hematology and Oncology, 2514 JCP University of Iowa Hospitals and Clinics, University of Iowa, Iowa City, IA 52242. polyunsaturated fatty acid that contains four conjugated double bonds (8). Parinaric acid absorbs light between 280 and 320 nm and has been used previously as a fluorescent probe for studies of membrane fluidity (9-12) and lipid peroxidation (13- 15). The findings we present indicate that parinaric acid is cytotoxic to cultured human leukemia and retinoblastoma cells at concentrations less than 5 /¿M. The cytotoxicity of these low concentrations is specific for the malignant cells tested and is prevented by the addition of an antioxidant. Concomitant stud ies with unconjugated fatty acids emphasize the critical role that double-bond configuration can exert in enhancing the cytotoxic effect of polyunsaturated fatty acid against malignant cells. MATERIALS AND METHODS Cell Culture Conditions. U-937 human monocytic leukemia, HL-60 human promyelocytic, and Y79 human retinoblastoma cells were ob tained from the American Type Culture Collection and maintained in RPMI-1640 supplemented with 15 MMHEPES,3 2 mM L-glutamine, and 10% FBS. THP-1 human monoblastic leukemia cells were cultured in RPMI-1640, described above, which was additionally supplemented with MEM vitamins (Sigma Chemical Co., St. Louis, MO), nonessen- tial amino acids without L-glutamine (Sigma Chemical Co.), and 2.2 mg/ml 2-mercaptoethanol. Human skin fibroblasts, bovine aortic endo thelial cells, and CaCo-2 human colonie mucosal cells were grown to confluence in Dulbecco's modified Eagle's medium containing 10% FBS, 2 HIM L-glutamine, 15 MMHEPES, and 0.4 Mg/ml gentamicin supplemented with nonessential amino acids and MEM vitamins. The culture medium during conditioning or cytotoxicity testing contained 5% FBS. Cells were free of Mycoplasma contamination on periodic testing. U-937 cells were differentiated to their macrophage form by exposure to 0.2 MMTPA in 1 mg/ml dimethyl sulfoxide for 48 h (15-17). The medium containing TPA then was replaced with regular culture me dium containing 10% FBS. Differentiation was confirmed by cell adhe sion to the surface of the flasks and reduction of nitroblue tetrazolium 96 h after the initial TPA exposure. Viability was assessed microscop ically by trypan blue exclusion in a hemacytometer. The differentiated cells were physically dissociated from the culture dish prior to testing for viability. Fatty Acids. The fatty acids tested were oleic acid (18:1 w-9), linolenic acid (18:3 u-3), moroctic acid (18:4 unconjugated w-3; 6c,9c,12<:,15c- 18:4), and rá-parinaric acid (18:4 conjugated o>-3;9,,11C,I3C,15,-18:4). In order to evaluate the reproducibility of parinaric acid cytotoxicity, this fatty acid was obtained from three independent commercial sources. Parinaric acid, obtained from Calbiochem (San Diego, ÇA),ICN Bio- chemicals (Irvine, CA), or Molecular Probes (Eugene, OR), was stored in the shipped container at -70°C. Prior to use, the parinaric acid was dissolved in 95% ethanol to a final concentration of 3 mM and stored in an oxygen-free, light-protected vial at -70°C. The parinaric acid concentration of this solution was determined spectrophotometrically prior to use (18). To further ascertain the reproducibility of our data, parinaric acid also was purified from Impatiens blasamina seeds (19). Two g of the seeds were ground in a mortar, and the oil was extracted overnight with 100 ml of hexane at room temperature in a light- 'The abbreviations used are: HEPES 4-(2 hydroxyethyl)-l-piperazineethane- sulfonic acid; FBS, fetal bovine serum; MEM, minimal essential medium; TPA, 12-O-tetradecanoyl phorbol-13 acetate; PBS, phosphate-buffered saline; BHT, butylated hydroxytoluene; LDSO,50% lethal dose. 6025 Research. on December 9, 2021. © 1991 American Association for Cancer cancerres.aacrjournals.org Downloaded from