107

Cancer Letters, 49 (1989) 107-113 Elsevier Scientific Publishers Ireland Ltd.

Suramin blocks intracellular Ca2+ release and growth factorinduced increases in cytoplasmic free Ca*+ concentration M.J. Seewald, Mayo

R.A. Olsen

and G. Powis

Clinic and Foundation, Department ofPharmacology, 2OOFirstStreet, S. W. Rochester, MN 55905 (U.S.A.)

(Received 10 July 1989) (Revision received 31 August 1989) (Accepted 5 September 1989)

Summary

Introduction

Suramin, a polysulfonated naphthylurea with antitumor actioity, has been shown to be an inhibitor of the release of Ca2+ from nonmitochondrial stores induced by the putative intracellular second messengers inositol 1,4,5GTP in saponin trisphosphate and permeabilieed Swiss 3T3 fibroblasts. The lCm for the effect of suramin was about 40 @4 in both cases. Suramin did not block Ca2+release induced by the Ca2+ ionophore 4-bromo A231 87 or by the membrane perturbing agent halothane. Suramin, 7 x lo-5 M, caused a 49% decrease in the elevation of intracellular free Ca2+ concentration ([Ca2+]$ caused by platelet deriued growth factor (PDGF) in intact Swiss 3T3 fibroblasts but did not block the increases in [Ca2+jicaused by bradykinin or uasopressin. Suramin decreased PDGF binding to its receptor on intact Swiss 3T3 fibroblasts but had no effect on the binding of bradykinin and uasopressin. The results show that the effect of suramin in decreasing the [Ca2+], response to growth factors may be mediated by a block of growth factor-receptor binding, but an effect on intracellular Caz+ release cannot be ruled out.

Suramin is a polysulfonated naphthylurea that has been used for a number of years to treat African trypanosomiasis [37]. Recent studies have shown suramin to be an inhibitor of retroviral reverse transcriptase [ll] and to inhibit human immunodeficiency virus (HIV) in vitro [24]. Clinical trials have been conducted to test the value of suramin in the treatment of the acquired immunodeficiency syndrome (AIDS) [23]. Suramin has also been found to exhibit growth inhibitory activity against a number of tumor cell lines in vitro [2,5,8,14,33,35] and clinical trials of suramin as an anticancer agent have been initiated, with initially promising results [7,34]. An increase in intraceliular free Ca2+ concentration ([Ca*+]i)produced by inositol 1,4,5trisphosphate [IP,(1,4,5)] and other intracelluar second messengers is important for mediating cell proliferation caused by growth factors, mitogens [3,29] as well as, probably, some oncogenes (36,381. Heparin has been reported to be an inhibitor of the IP,(1,4,5)mediated release of Ca*+ from non-mitochondrial intracellular stores (16,191. Because of the structural similarity of suramin to heparin and suramin’s reported anticoagulant properties [13] we examined the effects of suramin on the release of intracellular Ca2+produced by IP,(1,4,5) and other agents in permeabilized Swiss 3T3 fibroblasts, and the effect of sur-

Keywords: suramin; calcium; fibroblasts. Correspondence

to: G. Powis.

0304~3835/90/$03.50 0 1990 Eisevier Scientific Publishers Ireland Ltd Published and Printed in Ireland

108

amin on increases in [Ca2+],caused by growth factors and mitogens in intact Swiss 3T3 fibroblasts. Materials and methods

Suramin sodium (NSC 34936; the symmetrical 3”-urea of the sodium salt of 8-(3benzamido-4-methylbenzamido)-naphthalene 1,3,5-trisulfonic acid, mol. wt. 1429 Da) was supplied by the Division of Cancer Treatment, National Cancer Institute (Bethesda, MD, U.S.A.). PDGF, human recombinant B chain dimer, was purchased from Bachem (Torrance, CA, U.S.A.). Bradykinin, A$-vasopressin, GTP and 4-bromo A23187 were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.), IP,(1,4,5) from Calbiochem, (San Diego, CA, U.S.A.) and arachidonic acid from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). 45CA2+(2.5 mCi/mg) and (c-cis)-*251-PDGF (780 Ci/mmol) were obtained from Amersham Corp. (Arlington Heights, IL. U.S.A.), and [2,3-propyl-3,4-3H (N)Jbradykinin (78.7 Ci/mmol) and [phenyl alanyl-3,4,5-3H(N)]8-L-arginine vasopressin (68.5 Ci/mmol) from NEN DuPont (Boston, MA, U.S.A.). The uptake and release of Ya2+ by saponin permeabilized Swiss 3T3 fibroblasts was measured by a modification of the method of Gill and Cheuh [17]. Briefly, a suspension of 2 x 106/ml Swiss 3T3 fibroblasts was permeabilized with medium containing 0.005% saponin for 20 min at 37OC. After washing the cells to remove saponin @Ca2+uptake was measured from medium containing 4 x lo6 cells/ml, 1 mM ATP, 3% polyethylene glycol, 50 PM 45Ca2+ (160 Ci/mol) and sufficient EGTA to buffer the free Ca2+ concentration to low7 M. 2,4-Dinitrophenol, 0.5 mM, antimycin A, 10 PM, and oligomycin, 2 pg/ml, were included in the incubation as inhibitors of mitochondrial function. Aliquots, 0.1 ml, of cell suspension were collected on glass microfiber filters (GF/ A, Whatman International, Maidstone, Kent, U.K.) and washed with medium containing LaCl, before solubilization and liquid

scintillation counting. 45Ca2+ uptake was measured between 0 and 6 min of incubation when preliminary studies showed that ‘Ya2+ levels had reached a plateau value. 45Ca2+ release was studied by adding the releasing agent at 6.25 min and measuring 45Ca2+ remaining in the cells at 7 min. Suramin was added to incubations at 0 min or, when investigated as a releasing agent, at 6.25 min. Halothane was added to the incubations as aliquots of a saturated solution and final concentrations in the incubation medium confirmed by gas chromatography [ZO] . Measurement of changes in [Ca2+],in intact Swiss 3T3 fibroblasts employed the Ca2+-sensitive photoprotein aequorin [l]. The cells were loaded with aequorin using a reversible hyperpermeabilization technique based on centrifugation of the cells in low Ca2+ medium as previously described [28]. Changes in [Ca2+],caused by growth factors and mitogens were measured 20 h later with cells that had been deprived of serum for at least 2 h [28]. An estimate of [Ca2+],was obtained from the

100

75

5

60

2

60

0’

s $! 40 d 20 0

0

50

100

Suramin

150

200

(@i)

Fig. 1. Inhibition by suramin of ‘Ya*+ uptake and 45Ca2+release in saponin-permeabilized Swiss 3T3 fibroblasts. (0) ATP-dependent Ya2+ uptake, (0) 45Ca2+ release by 10 PM IP,( 1,4,5), and (A) 45Ca2+release by 10 PM GTP, measured as described in Materials and methods. Values are expressed as the percent of a control value without suramin and are the mean of 5 observations. Bars are S.D. The control value for 45Ca2+uptake was 219 -C 29 pmol/l06 cell. IP,, 10 pM, alone caused the release of 36.5 + 3.8% of the 45CaZ+and GTP, 10 FM, alone 51.4 + 3.4% of the 45CaZ+.

109

Table 1.

Inhibition by suramin of 45Ca2+release in permeabilized Swiss 3T3 fibroblasts. The release of 45Ca2+ by various agents was measured in permeabiiiied Swiss 3T3 fibroblasts as described in materials and methods. Values are the mean of at least 5 determinations f S.D. Suramin, 7 x lO+M, was added at 0 min and releasing agents at 6.25 min. 45CaZ+release is expressed as the percent release of total cellular ‘YaZ+ at 6 min, measured at 7 min. 7 x 10-5M Suramin

45Ca2’

Release (a)

45Ca2+

Release (% ) Control IP,(L4,5), 10 PM GTP, 10pM Arachidonic Acid, 100 FM 4-Bromo A23187, 10 FM Halothane, 0.5 mM

2.3 31.9 51.8 32.2

f 1.3 f 4.8 f 3.1 + 12.7

3.1 12.5 19.8 34.5

* zt -c -c

2.9 9.2 4.4 8.9

67.7 f

2.6

64.4 * 2.0

24.5 f

6.7

19.6 f 4.6

“P < 0.01 compared with value without suramin

light emitted by the cells using the calibration method for aequorin of Allen and Blinks [ 11. Binding of growth factors and mitogens to Swiss 3T3 fibroblasts was measured by the method of Collins et al. [6] using concentrations of [1251]PDGF of 3.3 x lo-*’ M, bradykinin of 9 x 10e9 M and vasopressin of 5 x 10m9 M, at 4OC for 3 h. Non-specific binding was determined using lOOO-fold higher concentrations of non-radiolabeled bradykinin and Non-specific binding was not vasopressin, determined for PDGF which exhibits minimal non-specific binding to Swiss 3T3 fibroblasts

WI. Groups of data were analyzed using Student’s t-test [32] and a P < 0.05 was considered significant. Results Suramin produced a concentration dependent inhibition of the uptake of 45Ca2+ into a non-mitochondrial compartment of saponin-

permeabilized Swiss 3T3 fibroblasts. The maximum inhibition measured was 475 at 214 FM suramin (Fig. 1). Suramin also produced a concentration-dependent inhibition of the release of 45Ca2+ caused by IP,(1,4,5) with an IC,, of 36 FM, and by GTP with an IC of 40 PM. Suramin itself did not cause the re 5ease of 45Ca2+ (results not shown) and did not block the release of 45Ca2+ caused by arachidonic acid, the calcium ionophore 4-bromo A23187 or halothane (Table 1). When added to intact Swiss 3T3 fibroblasts suramin reduced the increase in [Ca2+], caused by PDGF but did not significantly alter the increase in [Ca2+], caused by bradykinin or vasopressin (Fig. 2). The mean peak [Ca2+li values for several celf preparations showing the effects of 7 x 10m5 M suramin are given in Table 2. Binding studies with intact Swiss 3T3 fibroblasts showed that suramin decreased the binding of PDGF but had no effect on the specific binding of bradykinin or vasopressin (Table 3). Discussion Suramin has been shown to block the release of Ca*+ induced by IP,(1,4,5) and GTP from the non-mitochondrial stores of permeabilized Swiss 3T3 fibroblasts. Suramin, thus, differs from heparin which blocks only the release of Ca*+ induced by IP,(1,4,5) from non-mitochondrial stores but not the release induced by GTP [16,22,30]. Heparin has been suggested to inhibit IP,(1,4,5)-induced Ca*+ release by binding to a receptor for IP,(1,4,5) on the endoplasmic reticulum that modulates the endoplasmic reticulum Ca2+ release channel [16,39]. Suramin may act in a similar way to heparin and inhibit the receptor for IP,(1,4,5), thus, blocking Ca*+ release. Such a mechanism is consistent with the failure of suramin to block Ca*+ release by the membrane perturbing agents halothane 1201, 4bromo A23187 [lo] and arachidonic acid [31] that do not act through specific receptors on the endoplasmic reticulum. The block of the

Fig. 2.

Effect of suramin on the increase in [Ca*+li caused by growth factors and mitogens in Swiss 3T3 fibroblasts. The panels show the increases in [Ca”], produced by A, PDGF 3.3 x 10W9M; B, vasopressin (VP) lo+ M; and C, bradykinin (BK) lo-’ M added at the arrows. Left panels, without suramin, right panels, with 7 x lo+ M suramin.

release of Ca*+ by GTP is more difficult to explain because of our limited knowledge of GTPs action. The GTP releasable pool of Ca*+ Table 2.

Effect of suramin on peak [Ca’+], responses to growth factors and mitogens in Swiss 3T3 fibroblasts. Peak increases in [Caz+], were measured as described in Materials and methods. Suramin, 7 x 10T5 M, was added 5 min before the addition of the growth factor or mitogen. Values are mean f S.D. of at least 4 determinations.

lCa’+l,

PDGF, 3.3 x lO+M Bradykinin , lo-’ M Vasopressin, lo-’ M “P< 0.01 compared

PM

7 x lO+M Suramin [Caz+li PM

0.78 f 0.13 1.23 f 0.16 0.84 + 0.22

0.40 + 0.03 1.25 rt 0.06 0.86 f 0.04

with value without suramin.

appears to be different to that released by IP,(1,4,5) [9,18] and GTP has been suggested to induce the translocation of Ca2+ from an IP,(1,4,5)-insensitive to an IP,(1,4,5)-sensitive pool [25]. However, a specific GTP receptor site has not been identified so that a mechanism for a block of GTPs action by suramin must remain speculative. Suramin was a weak inhibitor of Ca*+ uptake into non-mitochondrial stores of permeabilized cells. It should be noted that the IC,, for the block of Ca*+ release by suramin of around 40 FM is within the achievable range for suramin in cancer patients, although lower than levels associated with tumor response [7]. Suramin blocked the increase in [Ca*+], in intact Swiss 3T3 fibroblasts caused by PDGF but not the increases in [Ca2+li caused by bradykinin or vasopressin. The simplest explanation for this effect is that suramin prevents the

111

Table 3. Effect of suramin on growth factor and mitogen receptor binding in Swiss 3T3 fibroblasts. The binding of 9 X lo+’ M PHlbradvkinin. 5 X lo9 M (3H]vasopressin and 3.3 x lo-” M [12s1]PDGFto Swiss 3T3 fibroblasts was measured as described in materials and methods. Incubations were for 3 h at 4%. Non-specific binding was measured using a lOOO-fold excess of non-radioactively labeled bradykinin and vasopressin. Non-specific binding was not measured for PDGF. Values are mean f S.D. of 4 determinations. Suramin

PDGF Bradykinin Vasopressin

7ccM

22.0 + 0.6 6.9 f 0.4 47.3 -c 4.6

22.5 f 0.5 7.4 + 1.2 50.5 f 2.7

(fmol/106

71yM

36 pM

OctM (fmol/106 cell)

(fmol/106

cell)

(fmol/106

cell)

cell)

6.6 + 0.4” 9.5 f 4.2 47.7 + 3.5

10.9 + 0.9 9.9 f 4.0 49.18 z!I 3.8

9 < 0.01 compared with value with no suramin.

binding of PDGF to the PDGF receptor on Swiss 3T3 fibroblasts, thus, preventing initiation of the intracellular signalling cascade that results in an increase in [Ca2+],.We found that suramin prevented [rz51]PDGF binding to Swiss 3T3 fibroblasts, in agreement with a previous report [ 151, but did not block the specific binding of bradykinin and vasopressin. Suramin might also exert an intracellular effect to block increases in [Ca2+li. Suramin is known to be taken up into cells and accumulated in the lysosomes and nucleus [21,35]. PDGF, vasopressin and bradykinin increase [Ca2+],by different mechanisms in Swiss 3T3 fibroblasts [4,26,27] which might explain the different effect of suramin on [Ca*+],changes by these agents, assuming an intracellular action for suramin. In summary, we have shown that suramin is a weak inhibitor of Ca*+ uptake by the endoplasmic reticulum of permeabilized cells and a stronger inhibitor of Ca2+ release induced by IP,(1,4,5) and GTP. Suramin did not block 45Ca2+release caused by membrane perturbing agents. Suramin blocked the increase in [Ca*+], in intact cells caused by PDGF but not by vasopressin or bradykinin. These effects are consistent with the observed inhibition of the binding of PDGF to its receptor by suramin, while the binding of vasopressin and bradykinin was not affected. However, an effect of suramin on

changes in [Ca*+],mediated through inhibition of intracellular Ca*+ release cannot be ruled out. Acknowledgements

The excellent secretarial assistance of Ms. Wanda Rhodes is gratefully acknowledged. The work was supported by NIH Grant CA 42286. References Allen, D.G.

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