Enhancement of Antitumor Activity of Ascorbate against Ehrlich Ascites Tumor Cells

Ascorbate in an aqueous solution is easily oxidized by molecular oxygen in the presence of cupric ion, thus producing reactive oxygen species and exhibiting cytotoxicity. In order to increase the antitumor activity of ascorbate, we used the innocuous form of cupric ion complexed with glycylglycylhistidine, a tripeptide designed to mimic the specific Cu(ll) transport site of albumin molecule. Although this square planar copper:glycylglycylhistidine complex did not significantly oxidize ascorbate at pH 7.4, it killed Ehrlich ascites tumor cells in v i t ro in a high concentration of ascorbate. The injections of large doses of ascorbate together with copper: glycylglycylhistidine prolonged the life span of mice inoculated i.p. with Ehrlich tumor cells. The target specificity against tumor cells was primarily attributable to their high peptide-cleaving activity. I N T R O D U C T I O N Ascorbate in an aqueous solution is easily oxidized by oxygen in the presence of transition metal ions and produces reactive oxygen species (5). The reaction path for the metalcatalyzed oxidation of ascorbate by oxygen was proposed by Taqui Khan and Martell (22) to involve molecular oxygen bound to the metal:ascorbate complex causing an electron transfer within the complex from ascorbate anion to metal ion and generating the partially reduced oxygen species. Malignant cells of several different types have been shown to have lowered levels of reactive oxygen-scavenging enzymes which should be primary factors in the biological defense against oxidative stress (3, 12, 15). High-energy irradiation and some cytotoxic antitumor drugs (1 3, 20), which cause the formation of reactive oxygen species in biological systems, have injurious effects on the viability of malignant cells. Therefore, ascorbate in certain circumstances should be considered to exhibit potent cytotoxicity against malignant cells. Several investigators have already pointed out the cytotoxicity of a large intake of ascorbate, in the presence of cupric ion, against tumor cells. Yamafuji et al . (23) disclosed that ascorbate, in cooperation with cupric ion, had a strong inhibitory potency against the growth of Sarcoma 1 80 implanted i.p. in mice. Stich e t al. (19) pointed out the enhancement of chromosome-damaging action of ascorbate by transition metal ions. Bram e t al. (4) reported that ascorbate possessed a preferential toxicity against malignant melanoma cells due to their elevated copper concentration. This study was supported by research grants from Central Research Institute of Fukuoka University, Fukuoka, Japan. 2 To whom requests for reprints should be addressed. 3 Nonresident Fellow of Linus Pauling Institute of Science and Medicine. Received May 14, 1982; accepted November 5, 1982. Cupric ion is, however, protein avid. An excess of copper has toxic effects in a wide variety of animals and in humans (1 7). In the presence of ascorbate, naked cupric ion may exert damaging action to malignant cells as well as normal cells. Serum albumin is a physiologically important Cu(ll) transport protein. GGH 4 is a tripeptide designed to mimic the specific Cu(ll) transport site of the albumin molecule (8). Accordingly, the copper:GGH complex may be considered as an innocuous form of cupric ion. The present paper deals with the in v i t ro and in v ivo antitumor activity of ascorbate and copper:GGH complex against Ehrlich ascites tumor cells and with the mechanism of its target specificity against malignant cells. M A T E R I A L S A N D M E T H O D S Chemical Reagents. GGH was synthesized according to the method of Lau et al. (8). The purity was checked by thin-layer chromatography. Catalase (2400 units/mg) from bovine liver was purchased from Sigma Chemical Co., St. Louis, Mo. All other chemicals were of analytical reagent grade. The 1:1 mixture (molar ratio) of cupric chloride and GGH was dissolved in Krebs-Ringer phosphate-buffered saline and adjusted to pH 7.4 by adding a few drops of concentrated sodium carbonate solution. The absorption maximum and extinction coefficient of the complex was 526 nm and 88 M -1 cm -1, respectively. ESR Studies. ESR spectra were obtained using a JEL-FE 1X spectrometer, JEOL, Ltd., Tokyo, Japan. The spectra of the copper complexes were measured at liquid nitrogen temperature (-196~ and those of ascorbate radicals and spin-trapped radicals were measured at room temperature. ESR signals were obtained as the first derivative of the absorption. The g-values and hyperfine splitting constant were determined by comparison with the standard of Mn(ll) in MgO. Oxidation of Ascorbate and Epinephrine and the Spin-trapping of Short-lived Radical Intermediates. The rate of oxidation of ascorbate in the presence of cupric ion or copper:GGH complex was measured by the decrease in UV absorption at 265 nm. Hydrogen peroxide was determined using leuko crystal violet and horseradish peroxidase as catalyst, according to the method of Mottola et al. (10). The formation of oxygen-centered radicals, superoxide radical and hydroxyl radical, was observed by the spin-trapping technique using BPN as a spin adduct, as adapted for the Fe(ll):bleomycin:O2 system (20). The oxidation of epinephrine in the presence of cupric ion or copper:GGH was examined in terms of the production of adrenochrome (9), which had an absorption maximum at 480 nm. Cytotoxicity Test in Vitro. Ascites of ddY mice, 10 to 12 days after i.p. inoculation with Ehrlich tumor cells, was used for the cytotoxicity test. Tumor cells (106 cells/ml) in Krebs-Ringer medium at pH 7.4 were incubated at 37 ~ with varied concentrations of ascorbate and cupric ion or copper:GGH for varying periods of time and assayed for their 4 The abbreviations used are: GGH, glycylglycylhistidine; ESR, electron spin resonance; BPN, N-tert-butyl-a-phenylnitrone. 824 CANCER RESEARCH VOL. 43 Research. on September 23, 2017. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from viability by trypan blue dye exclusion test. The inhibitory effect of catalase upon the cytotoxicity was investigated. Spleen cell suspension of ddY mouse was prepared by passing the minced spleen suspension in cold Eagle's minimal essential medium through a 100 mesh stainless sieve. It was used for the cytotoxicity test as a reference. Analysis of the Change of the Copper:GGH Complex After Incubation in Tumor Cell Suspension. Tumor cell suspension in KrebsRinger medium (108 cells/ml) in the presence or absence of 10 -4 M ascorbate was added with copper:GGH at the final concentration, 10 -4 M, and incubated at 37 ~ for 30 rain. It was deproteinized with picric acid, passed through the Dowex 1-X8 column, and analyzed for ninhydrin-positive compounds with an amino acid analyzer. ESR spectra of copper:GGH (10 -4 M) were measured after incubation in tumor cell suspension in the presence of 10 -4 M ascorbate. Antitumor Activity in Vivo. Two-tenths ml of ascites fluid containing 5 x 105 Ehrlich tumor cells was inoculated i.p. into ddY mice weighing about 25 g. Forty-eight hr after this inoculation, 0.2 ml of sodium ascorbate and/or copper:GGH was injected i.p. each day for 12 days. Increase in life span calculated on the basis of the mean survival time of the treated relative to the control animals and the number of 60-day survivors were used for the expression of antitumor activity of compounds.