0013-7227/90/126l-0666$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
SURAMIN PREVENTS ACTH-STIMULATED CORTICOSTERONE RELEASE BY DISPERSED ADRENOCORTICAL CELLS. Hamdy F.A.I. Marzouk , Joke Zuyderwijk, Piet Uitterlinden, Frank H. de Jong and Steven W.J. Lamberts Department of Medicine, trasmus University, Rotterdam, The Netherlands. cal Pathology, Mansoura University, Egypt.
On leave from the ..
-tment of Clini-
ABSTRACT: Suramin, a polyanionic compound which has been used in the treatment of trypanosomiasis and oncocerciasis, has recently been used in treatment of AIDS, while preliminary success has been reported in the treatment of cancer. However, suramin also causes adrenal insufficiency. The present study was undertaken in order to investigate the effect of suramin on ACTH-stimulated steroid production by dispersed rat adrenal cells. It was shown that suramin at concentrations of 10.-4-10-3M inhibits ACTH-stimulated corticosterone release in a dose-dependent manner IC5Q (2.10~ 4 M). In addition, suramin caused a parallel decrease in ACTH-stimulated pregnenolone, progesterone and corticosterone release, suggesting that suramin does not affect corticosteroidogenesis via an inhibition of its regulatory enzymes. Suramin at 10"^M did not inhibit cholera toxin (10 mg/1)-, forskol in (5 pM)- and dbcAMP (5 mM)-stirculated corticosterone release, while cholera toxin completely overcame the inhibitory effects of very high concentrations of suramin (up till iO~3M),on ACTH-stimulated corticosterone release. Finally, chromatographic studies with a matrex gel showed that suramin directly interacted with the ACTH molecule. In conclusion, suramin at "therapeutic" concentrations (10~^M and higher) prevents ACTH-stimulated corticosterone release probably via a direct interaction with the ACTH molecule. Suramin, the hexasodium salt of 8,8'(carbonylbis (imino-3,1 phenylene carbonylimino (4-methyl-3,1-phenylene) carbonylimino) bis-1,3,5-naphthalene trisulfonic acid, has been previously used in the treatment of African trypanosomiasis (sleeping sickness) and onchocerciasis (1). In 1979 De Clercq (2) reported that suramin is a potent inhibitor of the reverse transcriptase of a number of animal retroviruses. This observation was confirmed by several investigators (3,4). Because of its effect on HTLV-III/LAV, clinical investigators have used suramin in the treatment of patients with the acquired immunodeficiency syndrome (AIDS) (5). Thereafter it was found that suramin inhibits the binding to its receptor of platelet derived growth factor, which is encoded in oncogene Vsis of simian sarcoma (6,7). More recently the drug was described as a relatively selective competitive antagonist at several other growth factor receptors (8). These observations suggest that suramin might be successful in the treatment of cancer (8). Mahoney and Barrie (9) reported on the occurrence of adrenal insufficiency during suramin therapy of a patient with pemphigus. This was followed by several reports of other cases of adrenal insufficiency during suramin treatment (10). Cynomolgus monkeys treated with suramin showed decreased plasma cortisol responses to ACTH, elevated basal plasma ACTH levels and eventually developed a disruption of the adrenocortical architecture (11). In the present study we further evaluated the effejt of suramin on corticosteroidogenesis by cultured rat adrenal cells. MATERIALS AND METHODS Female RP rats, with body weights of 180-200 g, were kept in an artificially illuminated room with food and water till decapitation time at 8 a.m. The adrenals were cut into tiny pieces, and were incubated with 3 mg/ml collagenase (type I, Sigma, St. Louis, M0) at 37°C for 60 minutes in a Dubnoff shaker in an atmosphere of 95% 02/ 5% CO2 as described before (12). The investigational compounds (ACTH^_24, Synactben 0.25 mg/ml, Ciba-Geigy AG, Basel, Switserland; dbcAMP and cholera toxin, Sigma, St. Louis, M0; forskolin, Calbiochem, La Jolla, CA; Suramin, Bayer Leverkusen, Federal Republic of Germany) were prepared prior to
Received in Iowa City September 1, 1989
incubation. The dispersed cells (5.10^ per tube) were suspended in a Krebs-Ringer bicarbonate buffer containing 5.4 pmol of calcium. All studies were done in quadriplicate. Incubation time was 150 minutes; 0.5 ml distelled water was added to each tube, at the end of the incubation. Corticosterone concentrations in the media were measured according to a procedure described before (13). In addition pregnenolone and progesterone were measured by specific radioimmunoassay (14,15), intra-assay variations amounted to 20% and 10%, respectively. 125 I-ACTH (IRE, Fleurus, Belgium) was chromatographed on a 1 ml column of matrex gel red A (Amicon, Lexington,MA) in a buffer containing phosphate buffer saline (PBS). It has a chemical structure which is closely similar to that of suramin. Bound ACTH was eluted with suramin (10"^M) in the same buffer. Each experiment described was repeated twice. Results are expressed as means ± SEM and were evaluated using one way analysis of variance (ANOVA). Log transformation of data was used to stabilize variance. For the comparison of means the Newman Keuls method was applied (16). P values
Vol. 126, No. 1 Printed in U.S.A.
SURAMIN PREVENTS ACTH-STIMULATED CORTICOSTERONE RELEASE BY DISPERSED ADRENOCORTICAL CELLS. Hamdy F.A.I. Marzouk , Joke Zuyderwijk, Piet Uitterlinden, Frank H. de Jong and Steven W.J. Lamberts Department of Medicine, trasmus University, Rotterdam, The Netherlands. cal Pathology, Mansoura University, Egypt.
On leave from the ..
-tment of Clini-
ABSTRACT: Suramin, a polyanionic compound which has been used in the treatment of trypanosomiasis and oncocerciasis, has recently been used in treatment of AIDS, while preliminary success has been reported in the treatment of cancer. However, suramin also causes adrenal insufficiency. The present study was undertaken in order to investigate the effect of suramin on ACTH-stimulated steroid production by dispersed rat adrenal cells. It was shown that suramin at concentrations of 10.-4-10-3M inhibits ACTH-stimulated corticosterone release in a dose-dependent manner IC5Q (2.10~ 4 M). In addition, suramin caused a parallel decrease in ACTH-stimulated pregnenolone, progesterone and corticosterone release, suggesting that suramin does not affect corticosteroidogenesis via an inhibition of its regulatory enzymes. Suramin at 10"^M did not inhibit cholera toxin (10 mg/1)-, forskol in (5 pM)- and dbcAMP (5 mM)-stirculated corticosterone release, while cholera toxin completely overcame the inhibitory effects of very high concentrations of suramin (up till iO~3M),on ACTH-stimulated corticosterone release. Finally, chromatographic studies with a matrex gel showed that suramin directly interacted with the ACTH molecule. In conclusion, suramin at "therapeutic" concentrations (10~^M and higher) prevents ACTH-stimulated corticosterone release probably via a direct interaction with the ACTH molecule. Suramin, the hexasodium salt of 8,8'(carbonylbis (imino-3,1 phenylene carbonylimino (4-methyl-3,1-phenylene) carbonylimino) bis-1,3,5-naphthalene trisulfonic acid, has been previously used in the treatment of African trypanosomiasis (sleeping sickness) and onchocerciasis (1). In 1979 De Clercq (2) reported that suramin is a potent inhibitor of the reverse transcriptase of a number of animal retroviruses. This observation was confirmed by several investigators (3,4). Because of its effect on HTLV-III/LAV, clinical investigators have used suramin in the treatment of patients with the acquired immunodeficiency syndrome (AIDS) (5). Thereafter it was found that suramin inhibits the binding to its receptor of platelet derived growth factor, which is encoded in oncogene Vsis of simian sarcoma (6,7). More recently the drug was described as a relatively selective competitive antagonist at several other growth factor receptors (8). These observations suggest that suramin might be successful in the treatment of cancer (8). Mahoney and Barrie (9) reported on the occurrence of adrenal insufficiency during suramin therapy of a patient with pemphigus. This was followed by several reports of other cases of adrenal insufficiency during suramin treatment (10). Cynomolgus monkeys treated with suramin showed decreased plasma cortisol responses to ACTH, elevated basal plasma ACTH levels and eventually developed a disruption of the adrenocortical architecture (11). In the present study we further evaluated the effejt of suramin on corticosteroidogenesis by cultured rat adrenal cells. MATERIALS AND METHODS Female RP rats, with body weights of 180-200 g, were kept in an artificially illuminated room with food and water till decapitation time at 8 a.m. The adrenals were cut into tiny pieces, and were incubated with 3 mg/ml collagenase (type I, Sigma, St. Louis, M0) at 37°C for 60 minutes in a Dubnoff shaker in an atmosphere of 95% 02/ 5% CO2 as described before (12). The investigational compounds (ACTH^_24, Synactben 0.25 mg/ml, Ciba-Geigy AG, Basel, Switserland; dbcAMP and cholera toxin, Sigma, St. Louis, M0; forskolin, Calbiochem, La Jolla, CA; Suramin, Bayer Leverkusen, Federal Republic of Germany) were prepared prior to
Received in Iowa City September 1, 1989
incubation. The dispersed cells (5.10^ per tube) were suspended in a Krebs-Ringer bicarbonate buffer containing 5.4 pmol of calcium. All studies were done in quadriplicate. Incubation time was 150 minutes; 0.5 ml distelled water was added to each tube, at the end of the incubation. Corticosterone concentrations in the media were measured according to a procedure described before (13). In addition pregnenolone and progesterone were measured by specific radioimmunoassay (14,15), intra-assay variations amounted to 20% and 10%, respectively. 125 I-ACTH (IRE, Fleurus, Belgium) was chromatographed on a 1 ml column of matrex gel red A (Amicon, Lexington,MA) in a buffer containing phosphate buffer saline (PBS). It has a chemical structure which is closely similar to that of suramin. Bound ACTH was eluted with suramin (10"^M) in the same buffer. Each experiment described was repeated twice. Results are expressed as means ± SEM and were evaluated using one way analysis of variance (ANOVA). Log transformation of data was used to stabilize variance. For the comparison of means the Newman Keuls method was applied (16). P values
