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Regulation of endometrial cancer cell growth by insulin-like growth factors and the luteinizing hormone-releasing hormone antagonist SB-75
Regulatory Peptides, 48 (1993) 91-98
91
Elsevier Science Publishers B.V. REGPEP 01556
Regulation of endometrial cancer cell growth by insulin-like growth factors and the luteinizing hormone-releasing hormone antagonist SB-75 Dita Kleinman a, Charles T. Roberts, Jr. b, Derek LeRoith b, Andrew V. Schally c, Joseph Levy a and Yoav Sharoni a "Clinical Biochemistry Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Soroka Medical Center of Kupat Holim, Beer-Sheva (Israel), b Diabetes Branch, NIDDK, N1H, Bethesda, MD (USA) and ~Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center and Department of Medicine, Tulane University School of Medicine, New Orleans, LA (USA)
(Received 22 March 1993; revised version received 10 April 1993; accepted 6 May 1993)
Key words: Luteinizing hormone-releasing hormone; Insulin-like growth factor; Antagonist; Cancer cell growth
Summary The involvement of IGFs in growth regulation of the Ishikawa endometrial tumor cell line and the possible interference of LH-RH analogues with a potential autocrine or paracrine loop involving IGFs was evaluated. The mitogenic effects of IGF-I, IGF-II, and insulin were compared. IGF-I was found to be 3-fold more potent than IGF-II and 30-fold more potent than insulin, suggesting that the effects of these growth factors are mediated by the IGF-I receptor. Ishikawa endometrial cancer cells secrete IGF-II, but not IGF-I, and insulin (1/~M) stimulates IGF-II release. The LH-RH antagonist [Ac-D-Nal(2) l, D-Phe(4C1) 2, D-Pal(3) 3, D-Cit 6, D-Alal°]-GnRH (SB-75, CETRORELIX) inhibited basal and IGF-induced growth. Moreover, this antagonist almost completely inhibited IGF-II release from Ishikawa cells, while having no significant effect on the number or affinity of IGF-I binding sites. Inhibition of IGF-II release occurred at a lower SB-75 concentration than that needed for a reduction in cell number. The EDs0 of SB-75 for IGF-II release was 0.3 #M as compared to 1.5 #m concentration which is required for reduction in cell number, suggesting that inhibition of growth factor release precedes cell growth inhibition. We conclude that the LH-RH antagonist SB-75 can inhibit the growth of endometrial cancer cells by interfering with the autocrine action of IGF-II and also by directly inhibiting the growth-stimulatory effects of IGFs, probably through effects on a post-receptor mechanism.
Correspondence to: Y. Sharoni, Clinical Biochemistry Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Abbreviations: LH-RH, luteinizing hormone-releasing hormone; SB-30, [Ac-D-NaI(2)x, D-Phe(4CI) 2, D-Trp3, D-Cit6, D-AIaI°]-LH-RH; SB-75 (CETRORELIX), [Ae-D-Nal(2)1, D-Phe(4C1)2, D-Pal(3)3, D-Cit6, D-AIaI°]-Lh-RH; Buserelin, [ D-Ser(t-Bu)6, des-Glyl°-ethylamideLH-RH; Cit, citrulline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazofium bromide; IGF, insulin-like growth factor; IGF-BP, IGF-binding protein.
92 Introduction
Insulin-like growth factors I and II (IGF-I and IGF-II) constitute a family of related peptides with mitogenic actions. The mitogenic actions of both IGF-I and IGF-II appear to be initiated by ligand interaction with the IGF-I receptor which has a high affinity for IGF-I, a slightly lower affinity for IGF-II, and a low affinity for insulin [1,2]. Normal human endometrial cells have the capacity to synthesize and secrete a variety of growth factors [3] including IGF-I [4]. IGF-I and IGF-II mRNA have been detected in myometrial and endometrial cells [5-7]. Binding sites for IGF-I have been characterized in normal endometrial tissue [8,9]. The fact that these cells express IGF-I and IGF-II mRNA, secrete IGF-I, and possess IGF-I receptors supports the notion that these growth factors have autocrine growth-regulatory functions in normal endometrium. This raises the possibility that IGFs may also play a role in the growth of endometrial tumors. However, except for studies characterizing IGF-I binding sites in neoplastic endometrium [8,9], there is little information concerning the presence of these growth factors and their potential autocrine actions in malignant endometrial tissue. Various clinical studies have suggested that synthetic analogues of LH-RH inhibit the growth of sexsteroid dependent tumors such as carcinoma of the prostate [10], the breast [11,12], and the endometrium [ 13]. The rationale for this treatment is that continuous administration of LH-RH agonists causes inhibition of gonadotropin release from the pituitary and thus steroid production by the ovaries or testes. Treatment with agonistic analogues of LHRH, however, initially stimulates the release of gonadotropins and sex steroids which may cause a temporary flare-up of the disease [14,15]. The use of antagonistic analogues of LH-RH [ 14-16] causes an immediate inhibition of pituitary and gonadal function and thus should prevent the initial stimulatory effects on tumor growth. In vitro studies with breast [17] and ovarian [18]
cancer cell lines suggest that LH-RH antagonists may directly inhibit cell proliferation. We have recently shown a direct inhibitory effect of the LH-RH antagonist SB-75 on the human hormone-dependent Ishikawa endometrial cancer cell line [19]. The purpose of the present study was to determine whether IGFs play an autocrine role in the growth of endometrial cancer ceils and whether LH-RH antagonists interfere with these pathways. Specifically, we wished to determine whether inhibition of the growth of endometrial cancer cells by the LH-RH antagonist SB-75 involves inhibition of the release or of the activity of IGFs.
Materials and Methods
Peptides The antagonist SB-75 (CETRORELIX) was synthesized and purified as an acetate salt in the laboratory of one of us (A.V.S.) as previously reported [ 14,15 ]. Some batches were kindly provided by Asta Medica AG (Frankfurt, Germany). Human recombinant IGF-I and IGF-II were purchased from UBI (Lake Placid, NY). Porcine insulin was from EliLilly (Indianapolis, IN). Cell culture The hormone-dependent Ishikawa endometrial cell line originating from a well-differentiated tumor [20] was kindly supplied by H. Rochefort (Institute National de la Sante et de la Recherche Medicale, Montpellier, France). These cells were grown in Dulbecco's modified Eagle's medium (Biological Industries, Beth Haemek, Israel) containing penicillin (100 U/ml), streptomycin (0.1 mg/ml), nystatin (12.5 /~/ml), 0.6/~g/ml insulin, and 15~o fetal calf serum. A week before using cells for experiments, cells were stripped of endogenous steroids according to Vignon et al. [21] by successive passages in phenol red free medium containing 10~o and then 3~o of charcoal-stripped fetal calf serum (FCS/DCC).
93 Cell growth For growth studies, cells were seeded into 96-well plates (10,000-20,000 cells/well)in medium containing 3~o FCS/DCC. 1 day later, the medium was changed to 1~o FCS/DCC containing the various compounds, indicated in the figure legends. Cell number was estimated by the cellular reduction of MTT (Sigma, St. Louis, MO), by mitochondrial dehydrogenases of viable cells to a blue formazan product. When this product is dissolved in DMSO, its absorbance, as measured spectrophotometrically by an ELISA reader, is proportional to the number of cells [22]. To validate this procedure, we compared it, under various experimental conditions, to results obtained by cell counting in a hemocytometer after trypsinization (Fig. 1). The results highly correlated (r2 = 971) to those obtained by the MTT method. IGF-I and IGF-H radioimmunoassay Before estimation of cell number, conditioned medium was harvested from replicate wells and stored at -20 °C until assayed for IGF-I and IGF-II. IGF-I was measured with a commercial kit (lncstar, Still-
30-
25o 20-
150) 10
10
15
20
2'5
3'0
Cell number x 10"3 (cell counts) Fig. 1. Correlation between direct cell counts and the M T T assay in estimation of cell number. Cell number was measured by the two methods as described in Materials and Methods under various experimental conditions as shown in the graph; filled circles represent increasing concentrations of IGF-I (0.3-100 nM). This graph represents one of two similar experiments with five replicates of each condition.
water, MN). The assay for IGF-II was performed with a monoclonal antibody against rat IGF-II, purchased from Amono (Troy, VA). Cross-reaction of this antibody with human IGF-I and bovine insulin is 10~o and 0.01~o, respectively. The peptide was iodinated by mild chloramine-T treatment and was separated from free radioactive iodide on Sephadex G-25M (PD-10 column, Pharmacia, Uppsala, Sweden). Conditioned media were acid-extracted and IGF-binding proteins (IGF-BPs) were removed using ODS silica columns prior to the assay o f l G F s . The sensitivity of both assays was ~ 20 ng/ml. The inter-assay and intra-assay variance was 7.2 and 4.5%, respectively, for the IGF-I assay and 8.2 and 6.5~o, respectively, for the IGF-II assay. IGF-I receptor assay Binding experiments were performed essentially as described by Stewart et al. [23] but with labeled des(1-3)IGF-I (GroPep, Adelaide, Australia) instead of [ 125I]IGF-I. As suggested previously by others [24] and confirmed by us (data not shown), the truncated analogue exhibits markedly reduced binding to IGFBPs, and thus is more suitable for the measurement of IGF-I receptors. Des(1-3)IGF-I was iodinated as described above for IGF-II. Binding of this IGF-I analogue was measured on monolayers of Ishikawa cells in 24-well plates at 150,000 cells/well. Prior to the binding assay, the cells were washed with PBS and then incubated with 100,000 cpm of [125I]IGF-I (specific activity 104 cpm/fmol) in 0.2 ml PBS containing 1 mg/ml bovine serum albumin (globulin-free) for 2.5 h at 4°C. Background absorption to the wells was assessed by incubating the labeled peptide without cells. This was less than 3~o of total binding and was subtracted from all values. Nonspecific binding was assessed in the presence of 100 nM unlabeled IGF-I. The bound [~25I]des(l-3)IGF-I was displaced equally well by unlabeled des(l-3)IGF-I and by IGF-I, but less effectively by insulin (data not shown). After the incubation, the cells were washed three times with cold PBS, the cell layer was dissolved in 0.5 M NaOH,
94
and radioactively was measured in a 7 counter. The K d values and the number of binding sites were analyzed using L I G A N D program for the final Scatchard analysis.
2 r~
"~ l
[]
no addition
[]
buserelin
~.,
[]
SB-75
Results
Effects of lGFs on the growth of lshikawa cells The mitogenic action of IGF-I, IGF-II, and insulin was measured in Ishikawa cells (Fig. 2). The dose-response curve for cell growth after 4 days of treatment indicated that IGF-I was the most potent mitogen. The EDs0 were 3 nM, 10 nM, and 100 nM for IGF-I, IGF-II, and insulin, respectively. A small difference in the maximal effect of IGF-II was observed. The significance of this is unclear. The relative potencies suggest that, in these cells, the effects of IGF-I, IGF-II, and insulin are mediated by the IGF-I receptor. Effect of LH-RH analogues on IGF-induced cell growth We further studied whether proliferation induced by IGFs was affected by the L H - R H antagonist SB-75 and the L H - R H agonist buserelin (Fig. 3).
IGF-I
14-
insulin
¢9,
12-
O
"~ 1 L) control
IGF-I
IGF-II
insulin
Fig. 3. Effect of SB-75 and buserelin on IGF-I (100 nM), IGF-I1 (100 nM), and insulin (1/~M) induced Ishikawa cell proliferation. Results are expressed as percentage of the number of cells (10,000 to 20,000 cells) in the beginning of the treatment with peptides. Cells were grown for 3 days in the presence of the peptides. SB-75 and buserelin were used at 10/tM. Cell number was measured as described in the legend to Fig. 2. The results are the mean + S.E.M. of five different experiments each done in ten replicates.
SB-75 reduced the basal growth of Ishikawa cells grown in the absence of growth factors. The reduction in cell number in the presence of 100 nM IGF-I or IGF-II or 1 #M insulin was larger than that which occurred in the absence of the growth factors. Thus, the growth-stimulatory effect of IGF-I, IGF-II, and insulin was also affected by the L H - R H antagonist. The L H - R H agonist buserelin did not affect basal or IGF-induced cell growth. The inhibition of cell growth by SB-75 was evident over a wide range of IGF-I concentrations, with a larger effect at high IGF-I concentrations (Fig. 4).
10-
8
6
i
0
0.1
1 Hormone
lO
100
1000
[n_M]
Fig. 2. Dose-dependent effect of I G F s and insulin on Ishikawa cell growth. Cells were grown for 3 days in the presence of the peptides at the indicated concentrations. The number of cells was evaluated by the MTT method. The results are the mean _+S.E.M. of three different experiments each done in ten replicates.
Effect of the LH-RH antagonist SB-75 on the secretion of lGFs Since IGFs appear to be autocrine growth factors secreted by benign endometrial cells, therefore, we studied whether they are secreted from Ishikawa cancer cells, and whether the antagonist SB-75 affected their secretion. IGF-I was not detected in conditioned medium of the Ishikawa cancer cells. IGF-II was released from the cells, and its amount was increased by treatment with 1/~M insulin for two days (Fig. 5). Treatment with SB-75 (10/~M) alone, or in
95 20-
48 2 - 3o.o
36
¢,?, O r-I
•
15-
25.0
-
24
1,4 10 -~
• 20.0
12
0
r,.)
0
15.o
.1
1
lO
lOO
q
SB-75 [~tM] 0' 0.1
l
10
100
[IGF-I] nM Fig. 4. Effect of SB-75 on Ishikawa cell growth induced by increasing concentration of IGF-I. Cells were grown for 3 days with IGF-I at the indicated concentrations ( - O - ) . SB-75 was used at 10/~M ( - O - ) . Cell number was measured as described in the legend to Fig. 2. The results are the mean _+S.E.M. of three different experiments each done in ten replicates.
combination with insulin reduced the secretion of IGF-II to much lower levels, below those found in untreated cells. The stimulatory effect of insulin on IGF-II secretion was only partially abolished by SB75. The reduction in the secretion o f l G F - I I by SB-75 was dose-dependent (Fig. 6). The inhibition of IGF-II release occurred at a lower concentration of SB-75 than that needed for reduction of cell number.
100
Fig. 6. Dose-dependent effect of SB-75 on IGF-II release and on cell number in Ishikawa cells. Cells were grown for 3 days in the presence of SB-75 at the indicated concentrations. Cell number and IGF-II concentration were measured as described in Materials and Methods. The graph is representative of three similar experiments.
The ED50 of SB-75 for IGF-II release was 0.3 #M for cell inhibition.
[12SI]des(1-3)IGF-I binding to IGF-I receptor The inhibitory effect of SB-75 on IGF-induced growth could be explained by reduction of the number or affinity of IGF-I receptors. Scatchard plot 0.08
].
0.06-~•
0.040.02 -
~ O
o
control
•
SB-75
Ak~I
buserelin
80 60 40
0 2
20
4
6
8
bound DES(l-3) IGF-I (.fmole)
0 control SB-75 insulin insulin+SB-75
Fig. 5. Effect of insulin and SB-75 on IGF-II release from Ishikawa cells. Cells were grown for 2 days in the presence of insulin (1 /aM) and SB-75 (10/aM). The concentration of IGF-II in conditioned medium was determined as described in Materials and Methods, and is the mean + S.E.M. of three experiments, each done in duplicate.
Fig. 7. Scatchard analysis of [tZSl]des(1-3)IGF-I binding to Ishikawa endometrial cancer cells. Cell were incubated under control conditions O; with 10 #M SB-75 A; or buserelin O. Binding assays were performed as described in Materials and Methods with increasing concentrations of unlabeled IGF-I. The results are adjusted for 100,000 cells. The graph is representative of five similar experiments, each point done in duplicate.
96 analysis of [125]des(1-3)IGF-I binding was linear, indicating the presence of one population of highaffinity binding sites with a K d of 0.7 nM (Fig. 7). The affinity was not affected by SB-75. There was a slight but insignificant increase in the number of the binding sites in SB-75-treated cells as compared to untreated cells (5 vs. 7 fmol/100,000 cells, respectively).
Discussion Our study demonstrates that IGFs play an autocrine role in the growth of Ishikawa endometrial cancer cells and that the L H - R H antagonist SB-75 interferes with this pathway. These cells secrete IGFII, possess IGF-I receptors, and their growth is stimulated by these growth factors. The ability of Ishikawa cells to secrete IGF-II is at variance with a recent report [25], in which the Var-1 variant of the original I shikawa cell line did not express IGF-I or IGF-II mRNA. However, IGF-I and IGF-II mRNA have been detected in normal endometrial cells. Using labeled des(1-3)IGF-I, high-affinity IGF-I receptors were found on Ishikawa cells. This ligand is preferred to IGF-I and IGF-II since IGF-BPs which occur in excess of the receptors interact to a lesser degree with des(1-3)IGF-I and, therefore, will not interfere with the measurement. Receptors for IGF-I were previously demonstrated in human endometrial cancer biopsies [8,9]. IGF-I, IGF-II, and insulin probably stimulate Ishikawa endometrial cancer cell growth by interacting with the IGF-I receptor. A number of studies have shown that IGF-I is able to elicit a mitogenic response in vitro and in vivo in a variety of neoplastic cells such as carcinoma of the colon [26,27] and carcinoma of the breast [28]. According to the present results, SB-75 inhibits both the growth-stimulatory effect of both IGF-I and IGF-II and the autocrine secretion of IGF-II. Previous studies in our laboratory have shown that SB-75 inhibited cell growth of the estrogen-dependent MCF-7 mammary [ 17] and Ishikawa endome-
trial [19] cancer cell lines and of estrogen-independent MDA-231 mammary cancer cells [29]. The possibility that the inhibitory effect of SB-75 on IGFinduced growth involves changes in the IGF-I receptor level was studied. Scatchard plots of the binding to IGF-I receptors showed that the antagonist SB-75 did not significantly change the number or the affinity of the IGF-I receptors. These in vitro results are in agreement with in vivo studies in which treatment with SB-75 did not influence the level of IGF-I receptors in hamster pancreatic tumors [30] or in mouse MXT mammary tumors [31]. Thus, an effect on the IGF-I receptor cannot explain the inhibition of IGF-induced growth by the L H - R H antagonist. Other possible explanations include inhibition of IGF signal transduction mechanisms and alterations of IGF-BPs which are secreted from these cells [25] and are important modulators of IGF activity. We are currently investigating these possibilities by measuring tyrosine phosphorylation of the IGF-I receptor and the levels of IGF-BPs. The inhibition of IGF-II release induced by SB-75 occurred at a lower concentration than that needed for the inhibition of cell growth. We have already shown that treatment of MCF-7 cells with SB-75 reduced IGF-II secretion prior to inhibiting cell growth [32]. The results obtained in Ishikawa and MCF-7 cells support that inhibition of IGF-II release is one of the earliest events induced by the L H - R H antagonist. A recent study showing that explant cultures of myometrium obtained from women treated with an L H - R H agonist secreted significantly less IGF-I and IGF-II as compared with tissues obtained from placebo-treated controls [33] supports the view that the antagonist influences I G F secretion. In addition to IGFs, other autocrine factors such as P D G F and E G F were shown to be involved in the regulation of endometrial tumor growth [3,34]. Therefore, the possibility that SB-75 also interferes with the function of these factors should be investigated. L H - R H agonists are used for the treatment of en-
97 d o m e t r i a l c a n c e r . N e w L H - R H a n t a g o n i s t s m i g h t be m o r e suitable t h a n the a g o n i s t s as they inhibit the growth of estrogen-dependent endometrial tumors by t w o m e c h a n i s m s : e s t r o g e n d e p r i v a t i o n a n d a direct effect o n I G F a u t o c r i n e / p a r a c r i n e p a t h w a y s in t h e s e cells. S u c h direct effects m a y h a v e e v e n greater significance in e s t r o g e n - i n d e p e n d e n t t u m o r s w h e r e the c o n s t i t u t i v e s e c r e t i o n o f a u t o c r i n e g r o w t h factors m a y be higher. T h u s , the p o s s i b l e direct effect o f S B - 7 5 o n the action o f I G F s and o t h e r g r o w t h factors m a y o p e n n e w m o d a l i t i e s for the t r e a t m e n t o f these tumors.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by the Israeli M i n i s t r y o f H e a l t h . D e r e k L e R o i t h w a s the E r n a a n d J a c o b M i c h a e l Visiting P r o f e s s o r at the W e i z m a n n I n s t i t u t e of Science.
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33
34
like growth factor messenger RNA in human colon carcinomas and liposarcomas, Cancer Res., 46 (1986) 6169-6173. Chandrasekhar, Y., Xy, Z., Yallampalli, U. and Dia, B., Insulin-like growth factor (IGF) binding proteins and growth effects of IGFs on human colon cancers, 74th Annual Meeting of the American Endocrine Society, 1992, p. 342. Osborne, C.K., Clemmons, D.R. and Arteaga, C.L., Regulation of Breast Cancer Growth by Insulin-Like Growth Factors, J. Steroid Biochem. Mol. Biol., 37 (1990) 805-809. Sharoni, Y., Bosin, E., Miinster, A., Levy, J. and Schally, A.V., Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone, Proc. Natl. Acad. Sci. USA, 86 (1989) 1648-1651. Szende, B., Srkalovic, G., Groot, K., Lapis, K. and Schally, A.V., Regression of Nitrosamine-Induced Pancreatic Cancers in Hamsters Treated with Luteinizing Hormone-Releasing Hormone Antagonists or Agonists, Cancer Res., 50 (1990) 3716-3721. Szende, B., Srkalovic, G., Groot, K., Lapis, K. and Schally, A.V., Growth Inhibition of Mouse MXT Mammary Tumor by the Luteinizing Hormone-Releasing Hormone Antagonist SB75, J. Natl. Cancer Inst., 82 (1990) 513-517. Sharoni, Y., Kleinman, D., Hershkovitz, E., Marbach, M., Bosin, E., LeRoith, D. and Levy, J., Inhibition of breast and endometrial cancer cell growth by the GnRH antagonist, SB-75, involves interference in IGFs autocrine/paracrine pathways, Gynecol. Endocrinol., 7 (Suppl. 2) (1993) abstract 118. Rein, M.S., Friedman, A.J., Pandian, M.R. and Heffner, L.J., The secretion of insulin-like growth factors I and II by explant cultures offibroids and myometrium from women treated with a gonadotropin-releasing hormone agonist, Obstet. Gynecol., 76 (1990) 388-394. Huet-Hudson, Y.M., Chakraborty, C., De, S.K., Suzuki, Y., Andrews, G.K. and Dey, S.K., Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells, Mol. Endocrinol., 4 (1990) 510-523.
91
Elsevier Science Publishers B.V. REGPEP 01556
Regulation of endometrial cancer cell growth by insulin-like growth factors and the luteinizing hormone-releasing hormone antagonist SB-75 Dita Kleinman a, Charles T. Roberts, Jr. b, Derek LeRoith b, Andrew V. Schally c, Joseph Levy a and Yoav Sharoni a "Clinical Biochemistry Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Soroka Medical Center of Kupat Holim, Beer-Sheva (Israel), b Diabetes Branch, NIDDK, N1H, Bethesda, MD (USA) and ~Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center and Department of Medicine, Tulane University School of Medicine, New Orleans, LA (USA)
(Received 22 March 1993; revised version received 10 April 1993; accepted 6 May 1993)
Key words: Luteinizing hormone-releasing hormone; Insulin-like growth factor; Antagonist; Cancer cell growth
Summary The involvement of IGFs in growth regulation of the Ishikawa endometrial tumor cell line and the possible interference of LH-RH analogues with a potential autocrine or paracrine loop involving IGFs was evaluated. The mitogenic effects of IGF-I, IGF-II, and insulin were compared. IGF-I was found to be 3-fold more potent than IGF-II and 30-fold more potent than insulin, suggesting that the effects of these growth factors are mediated by the IGF-I receptor. Ishikawa endometrial cancer cells secrete IGF-II, but not IGF-I, and insulin (1/~M) stimulates IGF-II release. The LH-RH antagonist [Ac-D-Nal(2) l, D-Phe(4C1) 2, D-Pal(3) 3, D-Cit 6, D-Alal°]-GnRH (SB-75, CETRORELIX) inhibited basal and IGF-induced growth. Moreover, this antagonist almost completely inhibited IGF-II release from Ishikawa cells, while having no significant effect on the number or affinity of IGF-I binding sites. Inhibition of IGF-II release occurred at a lower SB-75 concentration than that needed for a reduction in cell number. The EDs0 of SB-75 for IGF-II release was 0.3 #M as compared to 1.5 #m concentration which is required for reduction in cell number, suggesting that inhibition of growth factor release precedes cell growth inhibition. We conclude that the LH-RH antagonist SB-75 can inhibit the growth of endometrial cancer cells by interfering with the autocrine action of IGF-II and also by directly inhibiting the growth-stimulatory effects of IGFs, probably through effects on a post-receptor mechanism.
Correspondence to: Y. Sharoni, Clinical Biochemistry Department, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Abbreviations: LH-RH, luteinizing hormone-releasing hormone; SB-30, [Ac-D-NaI(2)x, D-Phe(4CI) 2, D-Trp3, D-Cit6, D-AIaI°]-LH-RH; SB-75 (CETRORELIX), [Ae-D-Nal(2)1, D-Phe(4C1)2, D-Pal(3)3, D-Cit6, D-AIaI°]-Lh-RH; Buserelin, [ D-Ser(t-Bu)6, des-Glyl°-ethylamideLH-RH; Cit, citrulline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazofium bromide; IGF, insulin-like growth factor; IGF-BP, IGF-binding protein.
92 Introduction
Insulin-like growth factors I and II (IGF-I and IGF-II) constitute a family of related peptides with mitogenic actions. The mitogenic actions of both IGF-I and IGF-II appear to be initiated by ligand interaction with the IGF-I receptor which has a high affinity for IGF-I, a slightly lower affinity for IGF-II, and a low affinity for insulin [1,2]. Normal human endometrial cells have the capacity to synthesize and secrete a variety of growth factors [3] including IGF-I [4]. IGF-I and IGF-II mRNA have been detected in myometrial and endometrial cells [5-7]. Binding sites for IGF-I have been characterized in normal endometrial tissue [8,9]. The fact that these cells express IGF-I and IGF-II mRNA, secrete IGF-I, and possess IGF-I receptors supports the notion that these growth factors have autocrine growth-regulatory functions in normal endometrium. This raises the possibility that IGFs may also play a role in the growth of endometrial tumors. However, except for studies characterizing IGF-I binding sites in neoplastic endometrium [8,9], there is little information concerning the presence of these growth factors and their potential autocrine actions in malignant endometrial tissue. Various clinical studies have suggested that synthetic analogues of LH-RH inhibit the growth of sexsteroid dependent tumors such as carcinoma of the prostate [10], the breast [11,12], and the endometrium [ 13]. The rationale for this treatment is that continuous administration of LH-RH agonists causes inhibition of gonadotropin release from the pituitary and thus steroid production by the ovaries or testes. Treatment with agonistic analogues of LHRH, however, initially stimulates the release of gonadotropins and sex steroids which may cause a temporary flare-up of the disease [14,15]. The use of antagonistic analogues of LH-RH [ 14-16] causes an immediate inhibition of pituitary and gonadal function and thus should prevent the initial stimulatory effects on tumor growth. In vitro studies with breast [17] and ovarian [18]
cancer cell lines suggest that LH-RH antagonists may directly inhibit cell proliferation. We have recently shown a direct inhibitory effect of the LH-RH antagonist SB-75 on the human hormone-dependent Ishikawa endometrial cancer cell line [19]. The purpose of the present study was to determine whether IGFs play an autocrine role in the growth of endometrial cancer ceils and whether LH-RH antagonists interfere with these pathways. Specifically, we wished to determine whether inhibition of the growth of endometrial cancer cells by the LH-RH antagonist SB-75 involves inhibition of the release or of the activity of IGFs.
Materials and Methods
Peptides The antagonist SB-75 (CETRORELIX) was synthesized and purified as an acetate salt in the laboratory of one of us (A.V.S.) as previously reported [ 14,15 ]. Some batches were kindly provided by Asta Medica AG (Frankfurt, Germany). Human recombinant IGF-I and IGF-II were purchased from UBI (Lake Placid, NY). Porcine insulin was from EliLilly (Indianapolis, IN). Cell culture The hormone-dependent Ishikawa endometrial cell line originating from a well-differentiated tumor [20] was kindly supplied by H. Rochefort (Institute National de la Sante et de la Recherche Medicale, Montpellier, France). These cells were grown in Dulbecco's modified Eagle's medium (Biological Industries, Beth Haemek, Israel) containing penicillin (100 U/ml), streptomycin (0.1 mg/ml), nystatin (12.5 /~/ml), 0.6/~g/ml insulin, and 15~o fetal calf serum. A week before using cells for experiments, cells were stripped of endogenous steroids according to Vignon et al. [21] by successive passages in phenol red free medium containing 10~o and then 3~o of charcoal-stripped fetal calf serum (FCS/DCC).
93 Cell growth For growth studies, cells were seeded into 96-well plates (10,000-20,000 cells/well)in medium containing 3~o FCS/DCC. 1 day later, the medium was changed to 1~o FCS/DCC containing the various compounds, indicated in the figure legends. Cell number was estimated by the cellular reduction of MTT (Sigma, St. Louis, MO), by mitochondrial dehydrogenases of viable cells to a blue formazan product. When this product is dissolved in DMSO, its absorbance, as measured spectrophotometrically by an ELISA reader, is proportional to the number of cells [22]. To validate this procedure, we compared it, under various experimental conditions, to results obtained by cell counting in a hemocytometer after trypsinization (Fig. 1). The results highly correlated (r2 = 971) to those obtained by the MTT method. IGF-I and IGF-H radioimmunoassay Before estimation of cell number, conditioned medium was harvested from replicate wells and stored at -20 °C until assayed for IGF-I and IGF-II. IGF-I was measured with a commercial kit (lncstar, Still-
30-
25o 20-
150) 10
10
15
20
2'5
3'0
Cell number x 10"3 (cell counts) Fig. 1. Correlation between direct cell counts and the M T T assay in estimation of cell number. Cell number was measured by the two methods as described in Materials and Methods under various experimental conditions as shown in the graph; filled circles represent increasing concentrations of IGF-I (0.3-100 nM). This graph represents one of two similar experiments with five replicates of each condition.
water, MN). The assay for IGF-II was performed with a monoclonal antibody against rat IGF-II, purchased from Amono (Troy, VA). Cross-reaction of this antibody with human IGF-I and bovine insulin is 10~o and 0.01~o, respectively. The peptide was iodinated by mild chloramine-T treatment and was separated from free radioactive iodide on Sephadex G-25M (PD-10 column, Pharmacia, Uppsala, Sweden). Conditioned media were acid-extracted and IGF-binding proteins (IGF-BPs) were removed using ODS silica columns prior to the assay o f l G F s . The sensitivity of both assays was ~ 20 ng/ml. The inter-assay and intra-assay variance was 7.2 and 4.5%, respectively, for the IGF-I assay and 8.2 and 6.5~o, respectively, for the IGF-II assay. IGF-I receptor assay Binding experiments were performed essentially as described by Stewart et al. [23] but with labeled des(1-3)IGF-I (GroPep, Adelaide, Australia) instead of [ 125I]IGF-I. As suggested previously by others [24] and confirmed by us (data not shown), the truncated analogue exhibits markedly reduced binding to IGFBPs, and thus is more suitable for the measurement of IGF-I receptors. Des(1-3)IGF-I was iodinated as described above for IGF-II. Binding of this IGF-I analogue was measured on monolayers of Ishikawa cells in 24-well plates at 150,000 cells/well. Prior to the binding assay, the cells were washed with PBS and then incubated with 100,000 cpm of [125I]IGF-I (specific activity 104 cpm/fmol) in 0.2 ml PBS containing 1 mg/ml bovine serum albumin (globulin-free) for 2.5 h at 4°C. Background absorption to the wells was assessed by incubating the labeled peptide without cells. This was less than 3~o of total binding and was subtracted from all values. Nonspecific binding was assessed in the presence of 100 nM unlabeled IGF-I. The bound [~25I]des(l-3)IGF-I was displaced equally well by unlabeled des(l-3)IGF-I and by IGF-I, but less effectively by insulin (data not shown). After the incubation, the cells were washed three times with cold PBS, the cell layer was dissolved in 0.5 M NaOH,
94
and radioactively was measured in a 7 counter. The K d values and the number of binding sites were analyzed using L I G A N D program for the final Scatchard analysis.
2 r~
"~ l
[]
no addition
[]
buserelin
~.,
[]
SB-75
Results
Effects of lGFs on the growth of lshikawa cells The mitogenic action of IGF-I, IGF-II, and insulin was measured in Ishikawa cells (Fig. 2). The dose-response curve for cell growth after 4 days of treatment indicated that IGF-I was the most potent mitogen. The EDs0 were 3 nM, 10 nM, and 100 nM for IGF-I, IGF-II, and insulin, respectively. A small difference in the maximal effect of IGF-II was observed. The significance of this is unclear. The relative potencies suggest that, in these cells, the effects of IGF-I, IGF-II, and insulin are mediated by the IGF-I receptor. Effect of LH-RH analogues on IGF-induced cell growth We further studied whether proliferation induced by IGFs was affected by the L H - R H antagonist SB-75 and the L H - R H agonist buserelin (Fig. 3).
IGF-I
14-
insulin
¢9,
12-
O
"~ 1 L) control
IGF-I
IGF-II
insulin
Fig. 3. Effect of SB-75 and buserelin on IGF-I (100 nM), IGF-I1 (100 nM), and insulin (1/~M) induced Ishikawa cell proliferation. Results are expressed as percentage of the number of cells (10,000 to 20,000 cells) in the beginning of the treatment with peptides. Cells were grown for 3 days in the presence of the peptides. SB-75 and buserelin were used at 10/tM. Cell number was measured as described in the legend to Fig. 2. The results are the mean + S.E.M. of five different experiments each done in ten replicates.
SB-75 reduced the basal growth of Ishikawa cells grown in the absence of growth factors. The reduction in cell number in the presence of 100 nM IGF-I or IGF-II or 1 #M insulin was larger than that which occurred in the absence of the growth factors. Thus, the growth-stimulatory effect of IGF-I, IGF-II, and insulin was also affected by the L H - R H antagonist. The L H - R H agonist buserelin did not affect basal or IGF-induced cell growth. The inhibition of cell growth by SB-75 was evident over a wide range of IGF-I concentrations, with a larger effect at high IGF-I concentrations (Fig. 4).
10-
8
6
i
0
0.1
1 Hormone
lO
100
1000
[n_M]
Fig. 2. Dose-dependent effect of I G F s and insulin on Ishikawa cell growth. Cells were grown for 3 days in the presence of the peptides at the indicated concentrations. The number of cells was evaluated by the MTT method. The results are the mean _+S.E.M. of three different experiments each done in ten replicates.
Effect of the LH-RH antagonist SB-75 on the secretion of lGFs Since IGFs appear to be autocrine growth factors secreted by benign endometrial cells, therefore, we studied whether they are secreted from Ishikawa cancer cells, and whether the antagonist SB-75 affected their secretion. IGF-I was not detected in conditioned medium of the Ishikawa cancer cells. IGF-II was released from the cells, and its amount was increased by treatment with 1/~M insulin for two days (Fig. 5). Treatment with SB-75 (10/~M) alone, or in
95 20-
48 2 - 3o.o
36
¢,?, O r-I
•
15-
25.0
-
24
1,4 10 -~
• 20.0
12
0
r,.)
0
15.o
.1
1
lO
lOO
q
SB-75 [~tM] 0' 0.1
l
10
100
[IGF-I] nM Fig. 4. Effect of SB-75 on Ishikawa cell growth induced by increasing concentration of IGF-I. Cells were grown for 3 days with IGF-I at the indicated concentrations ( - O - ) . SB-75 was used at 10/~M ( - O - ) . Cell number was measured as described in the legend to Fig. 2. The results are the mean _+S.E.M. of three different experiments each done in ten replicates.
combination with insulin reduced the secretion of IGF-II to much lower levels, below those found in untreated cells. The stimulatory effect of insulin on IGF-II secretion was only partially abolished by SB75. The reduction in the secretion o f l G F - I I by SB-75 was dose-dependent (Fig. 6). The inhibition of IGF-II release occurred at a lower concentration of SB-75 than that needed for reduction of cell number.
100
Fig. 6. Dose-dependent effect of SB-75 on IGF-II release and on cell number in Ishikawa cells. Cells were grown for 3 days in the presence of SB-75 at the indicated concentrations. Cell number and IGF-II concentration were measured as described in Materials and Methods. The graph is representative of three similar experiments.
The ED50 of SB-75 for IGF-II release was 0.3 #M for cell inhibition.
[12SI]des(1-3)IGF-I binding to IGF-I receptor The inhibitory effect of SB-75 on IGF-induced growth could be explained by reduction of the number or affinity of IGF-I receptors. Scatchard plot 0.08
].
0.06-~•
0.040.02 -
~ O
o
control
•
SB-75
Ak~I
buserelin
80 60 40
0 2
20
4
6
8
bound DES(l-3) IGF-I (.fmole)
0 control SB-75 insulin insulin+SB-75
Fig. 5. Effect of insulin and SB-75 on IGF-II release from Ishikawa cells. Cells were grown for 2 days in the presence of insulin (1 /aM) and SB-75 (10/aM). The concentration of IGF-II in conditioned medium was determined as described in Materials and Methods, and is the mean + S.E.M. of three experiments, each done in duplicate.
Fig. 7. Scatchard analysis of [tZSl]des(1-3)IGF-I binding to Ishikawa endometrial cancer cells. Cell were incubated under control conditions O; with 10 #M SB-75 A; or buserelin O. Binding assays were performed as described in Materials and Methods with increasing concentrations of unlabeled IGF-I. The results are adjusted for 100,000 cells. The graph is representative of five similar experiments, each point done in duplicate.
96 analysis of [125]des(1-3)IGF-I binding was linear, indicating the presence of one population of highaffinity binding sites with a K d of 0.7 nM (Fig. 7). The affinity was not affected by SB-75. There was a slight but insignificant increase in the number of the binding sites in SB-75-treated cells as compared to untreated cells (5 vs. 7 fmol/100,000 cells, respectively).
Discussion Our study demonstrates that IGFs play an autocrine role in the growth of Ishikawa endometrial cancer cells and that the L H - R H antagonist SB-75 interferes with this pathway. These cells secrete IGFII, possess IGF-I receptors, and their growth is stimulated by these growth factors. The ability of Ishikawa cells to secrete IGF-II is at variance with a recent report [25], in which the Var-1 variant of the original I shikawa cell line did not express IGF-I or IGF-II mRNA. However, IGF-I and IGF-II mRNA have been detected in normal endometrial cells. Using labeled des(1-3)IGF-I, high-affinity IGF-I receptors were found on Ishikawa cells. This ligand is preferred to IGF-I and IGF-II since IGF-BPs which occur in excess of the receptors interact to a lesser degree with des(1-3)IGF-I and, therefore, will not interfere with the measurement. Receptors for IGF-I were previously demonstrated in human endometrial cancer biopsies [8,9]. IGF-I, IGF-II, and insulin probably stimulate Ishikawa endometrial cancer cell growth by interacting with the IGF-I receptor. A number of studies have shown that IGF-I is able to elicit a mitogenic response in vitro and in vivo in a variety of neoplastic cells such as carcinoma of the colon [26,27] and carcinoma of the breast [28]. According to the present results, SB-75 inhibits both the growth-stimulatory effect of both IGF-I and IGF-II and the autocrine secretion of IGF-II. Previous studies in our laboratory have shown that SB-75 inhibited cell growth of the estrogen-dependent MCF-7 mammary [ 17] and Ishikawa endome-
trial [19] cancer cell lines and of estrogen-independent MDA-231 mammary cancer cells [29]. The possibility that the inhibitory effect of SB-75 on IGFinduced growth involves changes in the IGF-I receptor level was studied. Scatchard plots of the binding to IGF-I receptors showed that the antagonist SB-75 did not significantly change the number or the affinity of the IGF-I receptors. These in vitro results are in agreement with in vivo studies in which treatment with SB-75 did not influence the level of IGF-I receptors in hamster pancreatic tumors [30] or in mouse MXT mammary tumors [31]. Thus, an effect on the IGF-I receptor cannot explain the inhibition of IGF-induced growth by the L H - R H antagonist. Other possible explanations include inhibition of IGF signal transduction mechanisms and alterations of IGF-BPs which are secreted from these cells [25] and are important modulators of IGF activity. We are currently investigating these possibilities by measuring tyrosine phosphorylation of the IGF-I receptor and the levels of IGF-BPs. The inhibition of IGF-II release induced by SB-75 occurred at a lower concentration than that needed for the inhibition of cell growth. We have already shown that treatment of MCF-7 cells with SB-75 reduced IGF-II secretion prior to inhibiting cell growth [32]. The results obtained in Ishikawa and MCF-7 cells support that inhibition of IGF-II release is one of the earliest events induced by the L H - R H antagonist. A recent study showing that explant cultures of myometrium obtained from women treated with an L H - R H agonist secreted significantly less IGF-I and IGF-II as compared with tissues obtained from placebo-treated controls [33] supports the view that the antagonist influences I G F secretion. In addition to IGFs, other autocrine factors such as P D G F and E G F were shown to be involved in the regulation of endometrial tumor growth [3,34]. Therefore, the possibility that SB-75 also interferes with the function of these factors should be investigated. L H - R H agonists are used for the treatment of en-
97 d o m e t r i a l c a n c e r . N e w L H - R H a n t a g o n i s t s m i g h t be m o r e suitable t h a n the a g o n i s t s as they inhibit the growth of estrogen-dependent endometrial tumors by t w o m e c h a n i s m s : e s t r o g e n d e p r i v a t i o n a n d a direct effect o n I G F a u t o c r i n e / p a r a c r i n e p a t h w a y s in t h e s e cells. S u c h direct effects m a y h a v e e v e n greater significance in e s t r o g e n - i n d e p e n d e n t t u m o r s w h e r e the c o n s t i t u t i v e s e c r e t i o n o f a u t o c r i n e g r o w t h factors m a y be higher. T h u s , the p o s s i b l e direct effect o f S B - 7 5 o n the action o f I G F s and o t h e r g r o w t h factors m a y o p e n n e w m o d a l i t i e s for the t r e a t m e n t o f these tumors.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by the Israeli M i n i s t r y o f H e a l t h . D e r e k L e R o i t h w a s the E r n a a n d J a c o b M i c h a e l Visiting P r o f e s s o r at the W e i z m a n n I n s t i t u t e of Science.
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27
28
29
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31
32
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