Life Sciences, Vol. Printed in the USA

51, pp. 1355-1361

Pergamon Press

REGULATION OF ARGINASE PRODUCTION BY GLUCOCORTICOID IN THREE HUMAN GASTRIC CANCER CELL LINES

Chew-Wun Wu, Soo-Ray Wang l, Shu-Lin Chien, Tsuey-Hwa Yeh, Shi-Long Lian 2, Nobuyoshi Shimizu3, Wing-Yiu Lui, Fang-Ku P'eng, and Chin-Wen Chi4. Departments of Surgery, Medicine I and Medical Research4,Veterans General Hospital-Taipei, National Yang-Ming Medical College,and Department of Radiology, Kaohsiung Medical College2, Kaohsiung, Republic of China. Department of Biochemistry, Keio University, Tokyo, Japan 3. (Received in final form August 25, 1992)

Summary Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and arginase, three human gastric cancer cell lines (AZ521, NUGC-3, KATO-III) were treated with hydrocortisone in the presence or absence of a glucocorticoid antagonist RU38486. GR were found to be present in all three lines, and hydrocortisone significantly increased the production of total arginase in all 3 lines. The induction of arginase production by hydrocortisone was inhibited by RU38486. These findings suggest that the regulation of arginase production by hydrocortisone in gastric cancer cells is mediated through GR. Arginase is an enzyme in urea cycle. It has been found to inhibit the proliferation of both T and B lymphocytes. The inhibition was dose-related, reversible and non-cytotoxic (1,2). Arginase activity was found to be markedly elevated in prostatic cancer tissues as compared to adjacent normal tissues (3,4). Previous reports from our laboratory found that gastric cancer tissues also contained higher arginase levels than that of the normal gastric mucosa (5,6). These observations suggest that arginase may be involved in immune response in tumors. Glucocorticoids, synthesized in the adrenal gland, are important hormones which regulate many physiologic functions in human body. In addition, glucocorticoids have major anti-inflammatory effects at pharmacological Corresponding author: Chew-Wun Wu, M.D., Department of Surgery Veterans General Hospital-Taipei, Taiwan, 11217, Republic of China 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.

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doses, and are used in the therapy of lymphomas and breast cancer (7). Glucocorticoids act through their specific intracellular receptors (GR) (8). The levels of GR in gastric cancer tissues were significantly higher than that of the normal gastric mucosas (6).

Recent study by Haggerty et al. (9) reported that arginase activity is regulated by glucocorticoids in a time- and dose- dependent fashion in cultured rat hepatoma cell line. Since both GR and arginase were found to be present in gastric cancer tissues, it is likely that the arginase activity may be regulated by glucocorticoid via GR. The objective of the present study was to examine the interrelation of glucocorticoid, GR and arginase in gastric cancer.

Materials and Methods

Tumor cell lines: Three human gastric cancer cell lines AZ-521 (10), NUGC-3 (11) and KATO-III (12) were used. KATO-III was cultured from metastasis in pleural effusion of a signet-ring cell carcinoma. NUGC-3 was established from a metastatic tumor in the brachial muscle of a poorly differentiated adenocarcinoma. AZ-521 was adenocarcinoma. For KATO-III cells, RPMI-1640 was used, and RPMI-1640 plus DMEM, mixed in equal volume was used for AZ-521 & NUGC-3 cells. The culture media contained 10% fetal calf serum, kanamycin 100 gg/ml and amphotericin 1 lag/ml.

Reagents: R U 3 8 4 8 6 was a gift from Roussel Uclaf, France. Hydrocortisone, RNase, bovine serum albumin (BSA) and other analytical grade reagents were purchased from Sigma Chemical Co., St Louis, Mo, USA. Trypsin was purchased from DIFCO Laboratory, Detroit, Michigan, USA. RPMI-1640 and DMEM media were purchased from GIBCO, Grand Island, NY, USA. Monoclonal anti-GR antibody was a generous gift from Dr. Okret (13). Fluorescein isothiocyanate-conjugated goat antimouse IgG antibody was purchased from Jackson Immunological Laboratory, CA, USA,

Glucocorticoid receptor assay: GR was assayed according to the method of Remvikos et al. (14) with some modifications. Single cell suspensions ( l x l 0 6 cells) were washed in phosphate buffered saline (PBS) and added 0.1 ml normal saline, then fixed in 70% cold ethanol for 30 min. at 0°C. Cells were resuspended in 2 ml of 4 N HCI at room temperature for 30 rain., centrifuged and washed with 1 ml 0.1 M N a 2 B 4 0 7 - 1 0 H 2 0 . After centrifugation, the pellet was incubated with 0.1 ml PBS buffer containing 1:100 monoclonal anti-GR antibody, 0.5% Tween 20 and 0.5% BSA for 30 min. at room temperature. For negative control, PBS was used without GR antibody. The cells were washed with PBS twice and the resuspended

Vol. 51, No. 17, 1992

GR and Arginase in Gastric Cancer

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Regulation of arginase production by glucocorticoid in three human gastric cancer cell lines.

Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and argi...
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