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Suramin Interference with Transforming Growth Factor-P Inhibition of Human Renal Cell Carcinoma in Culture’ TERENCE P. WADE, M.D., ATTAN KASID, PH.D., C. A. STEIN, M.D.,* RENATO V. LAROCCA, M.D.,* ERIC R. SARGENT, M.D., LEONARD G. GOMELLA, M.D., CHARLES E. MYERS, M.D.,* AND W. MARSTON LINEHAN, M.D. National

Cancer Institute, Surgery and *Medicine Branches, National Institutes of Health, Bldg. 10, Room 2B42, 9000 Rockville Pike, Bethesda, Maryland 20892 Submitted

for publication

Suramin is a polyanionic compound used clinically for the treatment of trypanosomiasis, which is known to inhibit the action of many protein factors in vitro. Transforming growth factor-j3 (TGF-8) is a multifunctional regulatory protein which inhibits the growth of renal cell carcinoma in culture. While suramin at 50500 fig/ml had no significant effect on the growth of renal cell carcinoma in culture in our experiments, it did partially reverse the growth inhibition induced by TGF-j3 in the two cell lines tested. This effect apparently is caused by suramin’s direct interference with 12’1-labeled TGF+‘s ability to bind to the cell, and not by any effect of suramin on the TGF-fi receptor. Furthermore, suramin dissociates TGF-,5 bound to the cell with a t f of less than 30 min. These results are consistent with those previously reported regarding suramin’s interaction with other protein growth factors, and suggest that suramin may interact with the TGF-/3 protein itself to inactivate it. INTRODUCTION

TGF-P is a polypeptide (MW = 25,000) which is expressed and secreted by many cell types, and was originally described as a component of sarcoma growth factor [l]. It exists as a disulfide-linked homodimer and was first purified from platelets, placenta, and kidney. TGFp was first described as a transforming growth factor because it reversibly inducedphenotypic transformation of normal rat fibroblastic cells in culture. However, as its properties were studied in depth, TGF-/3 was shown to be a multifunctional regulatory protein, stimulating mitogenesis of a variety of mesenchymal cell types but inhibiting the growth of epithelial [2] and endothelial [3] cells. We have previously described inhibition of renal cell carcinoma (RCC) cultures by TGF-P [4]. In the present study we investigate the effect of suramin when added to these cultures with and without TGF-P. 1 The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.

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Suramin is a polyanionic drug used clinically for decades in the treatment of African trypanosomiasis and onchocerciasis. Suramin has been shown to inhibit some protein enzymes, specifically in the complement system [5], and to inhibit ATPase [6], GTPase [7], and reverse transcriptase [8]. The inhibition of reverse transcriptase led to clinical trials of suramin in the treatment of patients with the acquired immunodeficiency syndrome [9]. Studies of the interaction of suramin with peptide growth factors have demonstrated that suramin inhibits platelet-derived growth factor (PDGF) binding to its receptor and growth stimulation, and also dissociates bound PDGF from its receptor [lo]. Suramin has been shown to reverse the u-sis transformation of fibroblasts [ 111 and interacts with the intracellular PDGF receptor [12]. Coffey et al. [13] have shown that suramin inhibits the effects of a number of growth factors on murine cells in culture, including TGF-/3. The present study was performed in order to evaluate the effect of suramin on proliferation of human kidney cancer in uitro, to assess the effect of suramin on TGF-6 mediated inhibition of these cells, and to investigate the effect of suramin on TGF-P binding to these malignant cells. MATERIALS

AND

METHODS

Cell lines. SKRC-7 is an established human RCC line provided by Dr. Neil Bander (Memorial Sloan-Kettering Cancer Center, New York, NY) and was used in passage >lOO for all experiments. UOK-39 is a primary renal tumor-derived cell line produced by enzyme dispersion and mechanical disaggregation of fresh tumor from a surgical specimen from a patient with metastatic RCC. The cells were separated by centrifugation on a Ficoll-Hypaque gradient (LSM, Litton Bionetics, Kensington, MD) and dispersed in complete medium (Dulbecco’s Modified Eagles Medium (DMEM) with glutamine 2 m&f, Hepes buffer 10 mM, penicillin 100 U/ml, streptomycin 100 pg/ml, fungizone 25 rig/ml, and gentamicin 50 Fg/ml) with 10% fetal calf serum (FCS), insulin 5 pg/ml, epidermal growth factor 10 rig/ml, and cholera toxin 10 rig/ml. After the third passage, UOK-39 was 195

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maintained in complete medium with 10% FCS for >30 passages. SKRC-7 was carried throughout in complete medium with 5% FCS. Both lines were incubated in 5% CO, at 37°C and were transferred to complete medium with 2% FCS for at least 5 days prior to plating and throughout the following studies. Reagents. TGF-0 derived from porcine platelets was obtained from R&D Systems, Inc. (Minneapolis, MN), reconstituted in 5 mM HCl + 1% bovine serum albumin, and diluted for use in serum-free DMEM. Porcine TGF0, labeled with lz51 by the chloramine-T method (2.5 /IC/ pmol), was a kind gift of Dr. L. Wakefield. Suramin was obtained from Bayer (Leverkusen, FRG), lyophilized, and stored at -2O”C, then reconstituted with serum-free DMEM prior to use. Cell counts. Cells were plated at a density of 7 X 104/ ml of medium in 24-well plates in triplicate on Day 0 of each experiment. Plating efficiency, which was calculated on Day 1 (prior to the addition of reagents) averaged nearly 90% for SKRC-7, 50% for UOK-39. Fresh media with additions were added to cultures on Days 1, 4, and 7, and triplicate wells were counted to determine proliferation. Cells were resuspended for counting in 1 ml of Hanks’ balanced salt solution (HBSS) (Biofluids, Inc., Rockville, MD) with 0.02 mg/lOO ml EDTA at 37°C for 20 min, and then washed with 1 ml of HBSS. Aliquots were diluted and counted on a Zsi Coulter Counter (Hialeah, FL). Binding experiments. SKRC-7 cells were used for the binding experiments. SKRC-7 cells were plated as described above, medium was aspirated at 24 hr and plating efficiency calculated. Cells were then washed three times with 1 ml binding buffer (BB), consisting of DMEM with Hepes buffer (pH, 7.4) 25 n-&f and 0.1% bovine serum albumin. TGF-p binding. Total binding of TGF-/3 was determined by exposing cells to 0.25-5 rig/ml of lz51-labeled TGF-6 for 4 hr in 0.2 ml BB at 25°C. Cells were then washed three times with ice-cold HBSS, bound counts were solubilized with 0.75 ml solubilization buffer (1 mg/lOO ml Triton X-100,10 mg/lOO ml glycerol, Hepes buffer 20 mM, pH, 7.4) and an aliquot counted on a Beckman 4000 gamma counter. Nonspecific binding was calculated and a curve compiled with the same concentrations of lz51-labeled TGF-P described above in the presence of a greater than lo-fold excess of cold TGF-P. Nonspecific binding was subtracted from total counts in order to calculate specific binding. Suramin on TGF-p receptor. The effect of suramin on the TGF-0 binding was assessed by incubation of the cells for 3 hr in 0.2 ml of BB containing 1000 pg/ml of suramin, then washing the cells three times with BB and exposing the cells to various concentrations (250 pg to 5 rig/ml) of lz51-labeled TGF-fl for 1 hr in 0.2 ml BB. Cells were solubilized and counted as above after the 1 hr incubation and the binding was compared to that at 4 hr of ‘251-labeled TGF-/3 binding without suramin exposure.

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FIG. 1. Effect of suramin alone on renal cell carcinoma (SKRC7) proliferation. Increasing concentrations of suramin: 50 rg/ml (triangles), 250 pg/ml (open squares), and 500 rg/ml (closed squares), had no significant effect on cell proliferation from control without suramin (circles). Data points represent the mean of three cell cultures counted, and the bars represent 1 standard deviation.

Coincubation. The ability of suramin to affect 1251labeled TGF-/3 binding directly was studied by the coincubation of 2 rig/ml ‘251-labeled TGF-P with suramin in concentrations from l-1000 pg/ml for 4 hr in 0.2 ml BB. Specific counts were calculated as described above and expressed as a percentage of the control count of 2 rig/ml of ‘251-labeled TGF-/3 without suramin. Dissociation. To assess suramin’s ability to dissociate bound ‘251-labeled TGF-/3 cells were exposed to 2 ng/ ml of 1251-labeled TGF-P for 3 hr, and then washed with BB three times and exposed to 1000 pg/ml of suramin for 10,30,90, and 240 min. Cell layers were then washed with HBSS and counted as described above, with counts expressed as a percentage of the control count of 2 rig/ml of ‘251-labeled TGF-/3 without exposure to suramin. Statistics. Cells were plated and counted in triplicate for each experimental condition at each time point, and all points graphed were means and standard deviations of three samples. Statistical analysis was performed using a standard t test, and significance was computed for a one-tailed P value. RESULTS Proliferation assays. Suramin alone, added in concentrations ranging from 50 to 500 pg, had no significant effect on the cell growth of either SKRC-7 or UOK-39 (P > 0.05) (Fig. 1). Coincident culture with 300 pg/ml of suramin also had no effect on proliferation, while treatment with 3 rig/ml of TGF-P significantly inhibited proliferation of SKRC-7 at Days 7 and 11 with SKRC-7 and UOK-39 (P < 0.0002) (Fig. 2). The addition of 50-500 pg/ml of suramin to cultures containing 3 rig/ml of TGF-/3 significantly reversed the effect of TGF-P alone on cell growth (P < 0.02); however, all proliferation remained less than that of control (or 300 pg/ml of suramin alone), P < 0.01. In two later experiments with SKRC-7 (data not shown), these results were repeated

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FIG. 2.

Suramin inhibits growth retardation by TGF-fl. SKRC-7 and UOK-39 cells grown in suramin 300 pg/ml (closed circles) had the same growth rate as control (open circles). TGF-P (3 rig/ml) significantly inhibited proliferation of both cell lines (open triangles), with partial reversal of the TGF-fl inhibition by suramin at 50 pg/ml (closed triangles), 300 pg/ml (open squares), and 500 fig/ml (closed squares).

and, in addition, growth with TGF-P addition alone at Day 8 was significantly less than with TGF-6 plus suramin (P < 0.05). TGF-/3 binding. Specific lz51-labeled TGF-/3 binding to SKRC-7 cells was nearly linear with increasing concentration. This binding was not significantly changed when the cells were preincubated with 1000 pg/ml of suramin except at one measurement with a low (250 pg/ ml) concentration of ‘251-labeled TGF-P (Fig. 3). Coincubation. When cells were incubated for 4 hr with 2 rig/ml of ‘251-labeled TGF-P plus suramin at l-1000 pug/ml, suramin as low as 50 pg/ml inhibited binding of ‘251-labeled TGF-& Fifty-percent inhibition of 1251-labeled TGF-/3 binding occurred at a concentration of approximately 80 pg/ml (60 PM) of suramin (Fig. 4). Counts were done in triplicate and are expressed as a

FIG. 4. Suramin inhibits TGF-P binding. Addition of increasing concentrations of suramin with 2 rig/ml of ‘251-labeled TGF-/3 results in inhibition of binding to the cells with 50% inhibition (IC,,) at about 80 pg/ml(60 pcM). Counts were done in triplicate and are expressed as a percentage of the count obtained with 2 ng of ?-labeled TGF-fl without suramin. The TGF-0 concentration was approximately 2 ng (0.08 pmol) in 0.2 ml, or 400 pM.

percentage of the counts obtained with 2 rig/ml of 1251labeled TGF-P alone. The TGF-/3 concentration in this experiment was approximately 2 ng in 0.2 ml, or 400 PM; the 50% inhibition at 60 PM therefore represents an approximately 1OO:l molar ratio of suramin to TGF-/3. TGF-/3 dissociation. Cells with bound ‘251-labeled TGF-P that were exposed to a lo-fold excess of cold TGF-0 had a drop off in ‘251-labeled TGF-P binding over time which was not significantly different from spontaneous dissociation. However, exposure of cells with bound ‘251-labeled TGF-/3 to 1000 pug/ml of suramin resulted in rapid, increased dissociation of bound 1251-labeled TGF-P from its receptor, with a t f of approximately 20 min (P < 0.0005) (Fig. 5). DISCUSSION

1

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pg/ml

of ’ 251 TGF-p

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FIG. 3. Specific binding of ‘251-labeled TGF-fl to cultured renal cell carcinoma is not effected by preincubation of cells with suramin. Control (open circles) and cells after preincubation with 1000 pg/ml of suramin for 3 hr (closed circles) are shown. Data points are the mean of three cell cultures counted and the bars represent 1 standard deviation.

Suramin is a polyanionic compound used clinically as an antiparasitic agent since the 1920’s. Its possible role as an anti-neoplastic agent is currently under investigation. The mechanism of suramin’s effect on in vitro growth of neoplastic cells is incompletely understood; however, a number of investigators have demonstrated that suramin inhibits the effect of various mitogenic factors on cellular proliferation. Hosang [lo] demonstrated in 1985 that suramin is an inhibitor of PDGF function, and stated that the inhibition is likely due to a direct interaction between the anionic suramin and the polycationic protein growth factor. In that study, although suramin inhibited PDGF-induced cellular proliferation and DNA synthesis, it had no apparent effect on the membrane PDGF receptor. In 1987 Coffey et al. showed that suramin inhibited the in vitro effects of fetal bovine serum, TGF-fi heparin-binding growth factor type-2, and epidermal growth factor on

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cytotoxicity on RCC line in vitro. As such, suramin appears to be effective in the in vitro inhibition of growth factor activity. Its potential effect on tumor growth in vivo is currently under investigation. REFERENCES 1.

2. 0.00

I

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0

30

60

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120

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180

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time in minutes

3.

FIG. 5.

Suramin removes TGF-fl bound to SKRC-7 renal cell carcinoma cells. Suramin (1000 @g/ml) added to cells with bound iz51labeled TGF-fi (closed circles) dissociates the TGF-P with a t i of approximately 20 min, while lo-fold excess of cold TGF-fl (triangles) has no difference from control (open circles).

4.

5.

murine AKR-2B cells in culture [ 131. In addition, Huang et al. demonstrated that in v-.&-transformed cells, the suramin is transported intracellularly and can inhibit the activity of the intracellular v-sis protein product, which shares structural and functional homology with the P-chain of PDGF [12]. We have previously demonstrated a consistent increase in the expression of TGF-P mRNA in human renal cell carcinoma tissue compared to the adjacent, histologically normal kidney [17], and that TGF-P inhibits proliferation of RCC in vitro [4]. The present study demonstrates that suramin in a dose of 50-500 pg/ml does not have a significant effect on proliferation of RCC in culture. However, this agent does alter the inhibitory effect of TGF-P on RCC in vitro. In fact, suramin inhibits the binding of lz51-labeled TGF-/3 to RCC and also induces a rapid dissociation of lz51-labeled TGF-(3 bound to RCC. It does not appear to affect the TGF-P receptor, as preincubation of RCC with suramin had no significant effect on lz51-labeled TGF-/3 binding. The results of this series of experiments complement those of Hosang, confirming that the most likely mechanism of suramin’s inhibition of protein growth factors is through direct interaction with the protein itself, and not through a receptor-dependent mechanism. Suramin interference with growth factor action is consistent, reversing both the proliferative effect of PDGF and the inhibitory effect of TGF-P each with 50% inhibition at 50-100 pg/ml of suramin. These inhibitory concentrations were similar to those found with ATPase (70 pg/ ml), GTPase (30 pg/ml), and complement (100-300 pg/ ml), but higher than that for reverse transcriptase [8]. The rate of dissociation of bound growth factor is similar as well, as both PDGF and, in our study, TGF-/3 are removed with a t + of about 20 min. Suramin is an effective inhibitor of the activity of a number of peptide growth factors, with little if any direct

6.

7.

Massague, J. The TGF-beta family of growth and differentiation factors. Cell 49: 437, 1987. Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kasid, A., Derynck, R., and Dickson, R. B. Evidence that transforming growth factor beta is a hormonally regulated negative growth factor in human breast cancer cells. Cell 48: 417, 1987. Heimark, R. L., Twardzik, D. R., and Schwartz, S. M. Inhibition of endothelial regeneration by type-beta transforming factor from platelets. Science 233: 1078, 1986. Gomella, L. G., Sargent, E. R., Linehan, W. M., and Kasid, A. Transforming growth factor-beta inhibits the growth of renal cell carcinoma in vitro. J. Ural. 141: 1240-1244, 1989. Fong, J. S. C., and Good, R. A. Suramin-a potent reversible and competitive inhibitor of complement systems. Clin. Exp. Zmmunol. 10:127,1972. Fortes, P. A. G., Ellory, J. C., and Lew, V. L. Suramin: A potent ATPase inhibitor which acts on the inside surface of the sodium pump. Biochim. Biophys. Actu 318: 262, 1973. Butler, S. J., Kelly, E. C. H., McKenzie, F. R., Guild, S. B., Wakelam, M. J. O., and Milligan, G. Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxinsensitive quanine-nucleotid-binding proteins. Biochem. J. 251:

201,1988. 8. DeClercq, E. Suramin: 9.

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15. 16.

A potent inhibitor of the reverse transcriptase of RNA tumor viruses. Cancer Lett. 8: 9, 1979. Levin, A. M., Gill, P. S., Cohen, J., Hawkins, J. G., Formenti, S. C., Aguilar, S., Meyer, P. R., Krailo, M., Parker, J., and Rasheed, S. Suramin antiviral therapy in the acquired immunodeficiency syndrome. Ann. Intern. Med. 105: 32, 1986. Hosang, M. Suramin binds to platelet-derived growth factor and inhibits its biological activity. J. Cell. Biochem. 29: 265, 1985. Betsholtz, C., Johnsson, A., Heldin, C., and Westermark, B. Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc. Natl. Acad. Sci. USA 83: 6440, 1986. Huang, S. S., and Huang, J. S. Rapid turnover of the plateletderived growth factor receptor in sis-transformed cells and reversal by suramin. J. Biol. Chem. 263: 12,608, 1988. Coffey, R. J., Leaf, E. B., Shipley, G. D., and Moses, H. L. Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J. Cell. Physiol. 132: 143, 1987. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294, 1979. Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 7’7(9): 5201, 1980. Kasid, A., Bell, G. I., and Director, E. P. Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors. Autocrine inhibition of cellular proliferation and differentiation to immune killer cells. J. Zmmunol. 141(2): 690,

1988. 17. Gomella, L. G., Sargent, E. R., Wade, T. P., Ewing, M. W., Kasid, A. K., and Linehan, W. M. mRNA expression of transforming growth factor beta (TGF-beta) in normal kidney and renal cell carcinoma tissues. J. Ural. 139: 214A, 1988.