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L-Cycloserine Amplifies Anti-Tumor Activity of Glutamine Antagonist

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L-Cycloserine Amplifies Anti-Tumor Activity of Glutamine Antagonist J. Clin. Biochem. Nutr., 14, 53-60, 1993 L-Cycloserine Amplifies Anti-Tumor Activity of Glutamine Antagonist Akira OKADA, Hiroo TAKEHARA, Kanehiro YOSHIDA, Masaharu NISHI, Hidenori MIYAKE, and Nobuhiko KOMI* The First Department of Surgery, School of Medicine, The University of Tokushima, Tokushima 770, Japan (Received September 22, 1992) Summary We examined the effect of simultaneous treatment with 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, and L- cycloserine, a transaminase inhibitor, on Yoshida sarcoma-bearing rats, with the aim of achieving inhibition of both glutamine catabolism and glutamate synthesis. L-Cycloserine, in a dosage of 1-30 mg/kg, was intraperitoneally administered to Yoshida sarcoma-bearing rats, but the administration of L-cycloserine alone did not show any significant effect on the tumor weight. However, combined treatment with DON (1 mg/kg) caused a dose-dependent reduction in the tumor weight. Amino acid analysis of the tumor tissue, performed 24 h after these treatments in order to confirm their influences on amino acid metabolism in the tumor, showed that the increased glutamate level seen after treatment with DON in a previous study was suppressed by simultaneous administration of DON and 10 mg/kg of L-cycloserine. These results suggest that pooled glutamate in the tumor tissue plays some role in resistance to the tumor- reducing activity of this glutamine antagonist. This, in turn, suggests that concurrent inhibition of glutamate production and blockage of glutamine utilization might result in enhanced tumor reduction in a clinical study. Key Words: glutamine antagonist, transaminase inhibitor, glutamate, Yoshida sarcoma, tumor reduction The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) evoked tumor reduction in experimental tumors [1-3], but it did not show adequate efficacy in clinical studies [4, 5]. We have been using DON to study the effects of glutamine * To whom correspondence should be addressed . 53 54 A. OKADA et alL antagonists on tumor amino acid metabolism since our previous study suggested that glutamine plays an important role in tumor growth [6-8], a finding also reported by others [9-13]. In a recent paper, we reported that glutamate increases in the tumor tissue after administration of DON to Yoshida sarcoma-bearing rats , with an increase in glutamine [ 14] . The increased glutamine was, of course , due to the blockage of glutamine utilization by DON. Nevertheless, the increased glutamate was hard to explain because glutamate is the product of glutamine hydrolysis, and DON is considered to block this pathway. Therefore , this increased glutamate in the tumor tissue was of particular interest and suggested that it was produced through a different pathway or that glutamate is actively taken up by the tumor tissue. Besides the pathway of glutamine hydrolysis , glutamate is also produced by the pathway of transamination, and glutamate oxaloacetate trans- aminase (GOT) [EC 2.6.1.1] and glutamate pyruvate transaminase (GPT) [EC 2. 6.1.2] are the most common transaminases in the liver . Therefore, we initially tried to inhibit glutamate synthesis through the pathway of aspartate aminotransferase by treatment with L-cycloserine, which has potential as an inhibitor of aspartate and alanine aminotransferases, as described elsewhere [15, 16]. In this study, we performed simultaneous administration of L-cycloserine and DON to tumor-bearing rats and examined their effects on tumor growth and the glutamate concentration in the tumor tissue. MATERIALS AND METHODS Tumor and animals Donryu rats carrying Yoshida sarcoma ascites tumors implanted by intraper- itoneal injection were kindly provided by Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan) and have been maintained by serial transplantation for over one year. For each experiment, the ascites tumor cells were collected and resuspended in physiological saline to a concentration of 1 x 108 viable cells/ml (by trypan blue exclusion). Then 0.1 ml of this tumor suspension was sub- cutaneously inoculated into the right flank. All animals were provided a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo) and tap water. Experimental design Determination of effects of L-cycloserine treatment on tumor growth. Twenty-three male Donryu rats (Japan SLC, Inc.; Shizuoka, Japan) weighing around 150 g were inoculated with 1 x 107 cells of Yoshida sarcoma. The rats were separated into four groups and injected intraperitoneally with 1 mg/kg (n = 5), 10 mg/kg (n = 6), or 30 mg/kg (n = 6) of L-cycloserine (Sigma Chemical Co., St. Louis, MO), or saline (n=6) on the 5th day after tumor cell inoculation. The rats were sacrificed under light ether anesthesia on the 8th day after tumor inoculation, and the tumor was removed and weighed. Determination of effects of simultaneous treatment with L-cycloserine and DON on tumor growth. In this experiment, 26 male Donryu rats weighing .I. Clin. Biochem. Nutr. GLUTAMATE AND TUMOR SURVIVAL 55 around 150 g were inoculated with Yoshida sarcoma cells. The rats were separated into four groups and injected intraperitoneally with 1 mg/kg of DON (Sigma Chemical Co., St. Louis) (n = 7), 1 mg/kg of DON and 1 mg/kg of L-cycloserine (n=6), 1 mg/kg of DON and 5 mg/kg of L-cycloserine (n = 7), or 1 mg/kg of DON and 10 mg/kg of L-cycloserine (n = 7) on the 5th day after tumor inoculation. The rats were sacrificed on the 8th day after injection of the tumor cells. Analysis of amino acids in tumor tissue after treatment with L-cycloserine and DON. In this experiment, male Donryu rats weighing around 150 g were inoculated with Yoshida sarcoma cells. Four rats were injected with 1 mg/kg of DON (n=7) and seven rats were injected with 1 mg/kg of DON and 10 mg/kg of L-cycloserine on the 5th day after tumor cell inoculation. The rats were sacrificed 24 h after the treatment, and the tumor and blood were collected for analysis. Blood chemical examination and amino acid analysis Blood samples were immediately centrifuged at 1,300 X g for 10 min at 4°C. Plasma was collected for biochemical examination by the standard laboratory methods, or for determination of the amino acid concentration after deproteiniza- tion with 5% sulfosalicylic acid. The tumor tissues were homogenized with tri- chloroacetic acid for amino acid analysis, as described elsewhere [8]. Amino acid analysis was performed with an Amino Acid Analyzer Model A-3300 (IRICA Instrument Inc., Kyoto). All reagents for the analysis were purchased from Wako Pure Chemical Industries, Ltd., Osaka. Statistics All results are given as the mean + SD. The data were statistically analyzed by one-factor ANOVA using StatViewII (Abacus Concept; Berkeley, CA), and significant differences were defined as p < 0.05. The relationship between the tumor weight and the dose of L-cycloserine was also analyzed by StatViewII, and the correlation coefficients and their significance were calculated. RESULTS Effects of L-cycloserine treatment on tumor growth The treatment with L-cycloserine alone showed no detectable effect in terms of either tumor reduction or body weight change (Table 1). The blood chemical examinations also did not reveal any particular abnormalities caused by the L-cycloserine treatment (Table 2). Effects of simultaneous treatment with L-cycloserine and DON on tumor growth By the treatment with DON or DON and L-cycloserine, the tumor weight was significantly reduced compared with that after saline or L-cycloserine treatment, while the body weight was slightly reduced by the treatment with DON and DON plus L-cycloserine (Table 1). The tumor weight in the group treated with DON plus 10 mg/kg of L-cycloserine was significantly lower than the weight in the group treated with DON plus 1 mg/kg of L-cycloserine, although the difference between Vol. 14, No. 1, 1993 56 A. OKADA et al. Table 1. Body weight and tumor weight after treatment with DON and L-cycloserine. Values are the mean ± SD. *p < 0.05 vs. the groups treated with DON or DON plus L-cyclo- serine. **p <0.05 vs. the group treated with DON and 1 mg/kg of L-cycloserine. Table 2. Blood chemical findings after treatment with saline or L-cycloserine. The abbreviations used are as follows: GOT, glutamate oxaloacetate aminotransferase; GPT, glutamate pyruvate aminotransferase; TP, total protein; ALB, albumin; GLU, glucose; TCHO, total cholesterol; TG, triglyceride; NEFA, non-esterified fatty acid. Values are the mean± SD. Table 3. Blood chemical findings after treatment with DON or DON plus L-cycloserine. Dosage of DON: 1 mg/kg, L-cycloserine: 10 mg/kg. The abbreviations are the same as for Table 2, and the values are the mean ± SD. *p <0.05 vs. DON-treated group. the groups treated with DON plus 10 mg/kg of L-cycloserine and DON plus 5 mg/ kg of L-cycloserine did not reach significance. Changes in amino acid concentration in tumor tissue after treatments with L-cycloserine and DON The tumor weight in the group treated with DON plus 10 mg/kg of L- cycloserine was 0.23 ±0.06 g, which was significantly lower than the weight in the J. Clin. Biochem. Nutr. Table 4. Plasma amino acid concentrations after treatment with DON or DON Plus L-cYcloserine. Dosage of DON: 1 mg/kg, L-c closerine: 10 mg/kg. The abbrevations used are as follows: Thr, threonine; Val valine; Met, methionine; lie, isoleucine; Leu7 leucine; Phes PhenY lalanine; Trp, trYPtoPhan; LYs, lysine; His histidine; Arg, arg inine; Asp, asPartate; Ser, serine; Asn, asParagine; Glu, glutamate; Gin, glutamine; Pro, p roline; G1Y, g1Ycine; Ala alanine; CYs, cYstine; TYr, tyrosine; Orn ornithine; Tau, taurine; EAA, essential amino acids NEAA non-essential amino acids TAA total amino acids. Values are the mean+SD mol dl.
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