L-Cycloserine Amplifies Anti-Tumor Activity of Glutamine Antagonist

J. Clin. Biochem. Nutr., 14, 53-60, 1993

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 transaminase (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 subcutaneously 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-cycloserine. **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. * <0.05 vs. DON-treated group.

Table 5. Amino acid concentrations in tumor tissue after treatment with DON or DON plus L-cYcloserine.

Dosage of DON: 1 mg/kg, L-cYcloserine: 10 mgkg. The abbreviations used are the same as for Table 4, and the values are the mean±SD umol g. *p<0.05 vs. DON-treated group. 58 A. OKADA et al. DON-treated group (0.45±0.19 g). Tumor weights in this study were lower than the data in Table 1, because the duration of tumor burden was shorter than that in Table 1 in order to check the early effects after these treatments. The GOT level in the group treated with DON plus 10 mg/kg of L-cycloserine was higher than that in the DON-treated group, while the glucose and NEFA levels were significantly higher after DON treatment than after treatment with DON plus 10 mg/kg of L-cycloserine (Table 3). The plasma levels of isoleucine and leucine in the group treated with DON plus L-cycloserine were significantly higher, while asparagine was significantly lower, than those in the DON-treated group (Table 4). In the tumor tissue, the glutamate level in the group treated with DON plus L-cycloserine was 2.01 ± 0.27 ji mol/g, which was significantly low compared with the level for the DON-treated group (2.78 ±0.41 1umol/g). The threonine, tryptophan, lysine, histidine, asparagine, proline, alanine, taurine and total amino acid levels in the group treated with DON plus L-cycloserine were also significantly lower than those in the DON-treated group (Table 5).

DISCUSSION

Our recent study showed an increase in the glutamate level in the tumor tissue after DON treatment of tumor-bearing rats [14]. However, glutamate should decrease in the tumor tissue after treatment with DON, since DON is thought to inhibit the catabolism of glutamine to ammonium and glutamate [3]. We hypoth- esized that the glutamate in the tumor tissue was either pooled in the tumor tissue or newly synthesized in the tumor for tumor survival when glutamine catabolism was blocked by DON. In this study, we showed a dose-dependent effect of L-cycloserine on the tumor weight when it was used together with a glutamine antagonist (DON), although it did not show a perceptible effect when administered alone. Furthermore, the increased glutamate level in the tumor tissue after treatment with DON alone was suppressed by simultaneous treatment with DON and L-cycloserine. These data suggest that pooled glutamate in the tumor tissue plays some role in the resistance to tumor reduction by treatment with a glutamine antagonist and that a glutamine antagonist may show better efficacy in clinical cases by combination with a transaminase inhibitor. Nevertheless, the decreases detected in other amino acids, i.e., threonine, tryptophan, lysine, histidine, asparagine, proline, alanine, and taurine, by administration of DON plus L-cycloserine cannot be explained by the action of L-cycloserine. More recently, we demonstrated the GOT mRNA and activity levels to be increased in rats with cancer cachexia [17]. If the increased GOT activity is related to the initiation or promotion of cachexia, it is possible that L-cycloserine exerts an anti-cachectic activity in the tumor-bearing state. However, Sundaram et al. did not find normal weight gain after administration of high-dose L-cycloserine to

J. Clin. Biochem. Nutr. GLUTAMATE AND TUMOR SURVIVAL 59 tumor-free rats in an attempt to inhibit 3-ketodihydrosphingosine synthetase, an enzyme of the sphingolipid pathway [18]. While they did not mention any decrease in food intake, they surmised that suppressed movement and response due to the treatment had diminished the weight gain. They also examined bacterial growth after L-cycloserine treatment using Bacteroides levii and suggested the application of L-cycloserine in chemotherapy for sphingolipid-synthesizing bacte- ria [19]. L-Cycloserine is also a potent inhibitor of y-aminobutyrate-transaminase (GABA-T) [EC 2.6.1.19], and it was tested for anticonvulsant action [20]. Thus, L-cycloserine has received attention as a metabolic inhibitor in experimental and clinical studies, but our literature survey did not find any studies concerning reduction of tumor growth by L-cycloserine administration. Moreover, Sundaram and Lev reported the effects of long-term administration of L-cycloserine. They subcutaneously injected 25 mg/kg of L-cycloserine to mice on alternate days for 2 months and found no neurological symptoms [21]. In conclusion, in the present study, we examined the effects of simultaneous administration of DON and L-cycloserine to tumor-bearing rats, aiming at inhibit- ing glutamine catabolism and glutamate synthesis. We were able to demonstrate a dose-dependent effect of L-cycloserine on the tumor weight when it was given with DON, a glutamine inhibitor.

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