effects of suramin-related and other clinically ...€¦ · ters such as the tumor promoter tpa....

Vol. 1. 1 13-122, January 1995 Clinical Cancer Research 113

Effects of Suramin-related and Other Clinically Therapeutic

Polyanions on Protein Kinase C Activity1

Zahangir Khaled, Darryl Rideout,2

Kevin R. O’Driscoll, Daniel Petrylak,

Angela Cacace, Rena Patel, L-C. Chiang,

Susan Rotenberg, and C. A. Stein3

Department of Medicine. Columbia University, New York. New York

10032 [Z. K.. K. R. O’D.. D. P.. A. C.. C. A. SI: Queens College-

CUNY, Queens, New York 1 1367 [S. RI; and The Scripps Research

Institute. La Jolla. California 92037 ID. R.. R. P., L-C. CJ

ABSTRACT

The mechanism of the antineoplastic effects of suramin

may involve interference with signal transduction, but in

general is not well understood. We examined several poly-

anions to determine their effects on the kinase activity of the

protein kinase C (PKC) �1 and other PKC isoforms. Similar

to suramin, a phosphorothioate oligodeoxynucleotide 28.

mer homopolymer of cytidine (SdC28) inhibited the phos-

phatidylserine and Ca2�-dependent phosphorylation of an

epidermal growth factor receptor octapeptide substrate. The

inhibition by suramin was mixed competitive/noncompeti-

tive with respect to ATP, but uncompetitive with respect to

substrate. In contrast, the inhibition by SdC28 was compet-

itive with respect to substrate (K1 5.4 p.st) and not corn-

petitive with respect to ATP. The PKC a and �31 isoforms

were inhibited to the same extent with SdC28, while PKC #{128}

was not inhibited. SdC28, in the absence of lipid cofactor,

stimulated substrate phosphorylation, and in the absence of

substrate induced PKC �1 autophosphorylation. Similar

behavior was seen with another polyanion, the polysulfated

carbohydrate pentosan polysulfate (polyxylyl hydrogen sul-

fate). H4, a bis-naphthalene disulfonate tetraanion structur-

ally related to suramin, also inhibited kinase activity but was

not competitive with respect to ATP. Dianions closely re-

lated to H4 failed to inhibit PKC �31, suggesting that multi-

pie ( >2) negative charges are required. The interactions of

polyanions with PKC are complex, and are dependent on the

molecular structure of the polyanion, the presence of cofac-

tors, and the PKC isoform.

INTRODUCTIONSuramin, a bis-polysulfonated naphthylurea hexaanion ( 1).

causes clinical remissions in patients with adrenocortical cancer

(2), hormone-refractory metastatic prostate cancer (3). and

heavily pretreated non-Hodgkin’s lymphoma (4). However. the

mechanism of the antineoplastic effect of suramin is probably

complex, and may involve the ability of suramin to block the

binding of a variety of growth factors (e.g. . bFGF�: Refs. 5. 6),

Kaposi’s FGF (7), platelet-derived growth factor (8, 9), trans-

ferrin ( 10) to their receptors, to inhibit DNA polymerases ( 1 1),

to interfere with the activity of topoisomerase II ( I 2), to affect

mitochondrial function (13), and to inhibit the lysosomal catab-

olism of glycosaminoglycans (14, 15). An additional intracel-

lular target for sumamin may be PKC ( 16, 17). PKC is a family

of at least 10 related protein serine/threonine kinases that are

activated allosterically by phospholipids and are implicated in

cellular growth control (18). The isoforms of PKC mediate

intracellular signal transduction initiated by transmembrane-

receptor-linked hydrolysis of phospholipids and by phorbol es-

ters such as the tumor promoter TPA. Based on sequence

homology and biochemical properties. these isoforms of PKC

can be divided into three functional classes: the Ca2�-sensitive

conventional isoforms (PKC a, �3I, �3II, and rny)’ the Ca2 � -

insensitive novel isoforms (PKC #{128},& q. and 0), and the Ca2�-

and phorbol ester-insensitive atypical isoforms (PKC � and X;

Ref. 19). Inhibitors of PKC activity such as the compound

dequalinium (20, 2 1) and the stauroporine analogue CGP 41251

(22) inhibit the growth of transplantable rodent tumors. Hensey

et a!. ( 16) have shown that inhibition of in vitro PKC activity bysuramin, and also by staurosporine, closely correlates with the

ability of these agents to differentiate the neuroblastoma NB2A

cell clone.

However, the effects of suramin on PKC activity are corn-

plex. For example, Mahoney ci a!. ( 17) have recently demon-

stnated that suramin, in the presence of the lipid cofactor phos-

phatidylserine. is an inhibitor of PKC activity (histone Ill-S

substrate) and that the inhibition is competitive with respect to

ATP. In the absence of phosphatidylsenine, surarnin is a stirnu-

lator of PKC activity.

We wanted to determine if this complex behavior was

specific to suramin or was a general property of polyanions.

Thus, we have examined the inhibitory effects of several struc-

turally distinct classes of polyanions (Fig. 1 ) on PKC � I activ-

ity. The molecules we have studied are either themselves, or

closely related to, compounds that are currently or will soon he

in clinical cancer trials. One of these classes is phosphorothioate

oligos, which were initially synthesized by Stec ci a!. (23).

Received 7/I 3/94: accepted I 0/I 0/94.

I This is manuscript 8007-MB from the Department of Molecular Bi-

ology. The Research Institute of Scripps Clinic.

2 Received partial support from PPG Industries and the National lnsti-

tute of Allergy and Infectious Disease Grant A128202.

3 The Irving Assistant Professor of Medicine and Pharmacology. Re-

ceived partial support from the Mathieson Foundation, the DuBose

Hayward Foundation. and National Cancer Institute Grant CA60639. To

whom requests for reprints should be addressed. at Department of Medi-

cine, Columbia University. 630 West 168th Street, New York. NY I(X)32.

4 The abbreviations used are: bFGF. basic tibroblast growth factor:PKC, protein kinase C: TPA. I 2-O-tetradecanoylphombol- I 3-acetate:oligos. oligodeoxynucleotides; SdC28. phosphophorothioate 28-men ho-

mopolymer of cytidine: LYCH, lucifer yellow CH; EGFR. epidermal

growth factor receptor: ps, phosphatidylserine.

Research. on March 29, 2021. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

114 Polyanions Inhibit Protein Kinase C Activity

These are analogues of normal, or phosphodiestem, oligos in

which one oxygen atom in a nonbnidging position at each

phosphorus is replaced by sulfur (reviewed in Ref. 24). Se-

quence-specific phosphorothioate oligodeoxynueleotides are

currently in Phase I clinical trials in a cohort of patients infected

with HIV (25-mer; target HIV-1 gag mRNA; Ref. 25) and in

another group suffering from chemotherapy-refractory acute

rnyelogenous leukemia (20-men; target = p53 mRNA; Ref. 26).

SdC28, the phosphomothioate oligo we have used in this study,

is a homopolymem of eytidine, 28 bases in length. It is, like suramin

(27), an in vitro anti-HIV-1 agent (28, 29) and may exert this effect

by multiple mechanisms (for a review see Ref. 30). SdC28 is also

an in vitro inhibitor of PKC �3 1 activity (1C3() = 5 �i.M); this activityseems to correlate with its ability to inhibit the internalization of

some fluid-phase markers in HL6O cells (31).

Another class of polyanion we examined is the sulfated

carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate).

This polyanion, like sumamin, can bind to hepamin-binding

growth factors (32) and has also entered recent, albeit less

successful clinical trials than sumamin.

In the present study, we examine the inhibition of activity

of several isoforms of PKC by a variety of polyanions, including

SdC28 and pentosan polysulfate. We also examine the effects

these and other polyanions have on the phosphomylating activity

of PKC 1� 1 in the absence of lipid cofactors. These data providesome important, early, structure-activity relationships that ex-

pand our understanding of the possible mechanisms of action of

polyanionic compounds with potential importance as clinical

antineoplastie agents.

MATERIALS AND METHODS

Reagents. LYCH was either purchased from Molecular

Probes (Eugene, OR) on Aldrich Chemical Company (Milwau-

kee, WI). H3 and H4 were synthesized and purified by previ-

ously described procedures (33). Pentosan polysulfate was a

generous gift from the Hoechst Corporation and was used with-

out further purification. SdC28 was synthesized on a ABI-380B

automated DNA synthesizer according to the standard phos-

phoramidite method using the manufacturer’s procedure and

substituting tetmaethylthiuram disulfide for the iodine oxidation

step (Applied Biosystems, Foster City, CA). A part of the

SdC2S was also kindly provided by Dr. G. Zon. After base

depmoteetion, the SdC28 was HPLC purified as described pme-

viously (34), 5’-detnitylated with 3% acetic acid, lyophilized,

and precipitated with NaC1 from aqueous ethanol. Sumamin was

obtained from the Mobay Chemical Corporation (New York,

NY). The chemical structures of the polyanions used in this

work are shown in Fig. 1.

Assay of PKC Activity. PKC �31 activity was assayed

according to the method of Rotenbemg et a!. (21). Briefly, this

isoform was isolated from mumine embryo C3H lOTl/2 fibro-

blasts that had been genetically engineered to overproduce mat

brain PKC �1 (35, 36). The initial phosphorylation of EGFR

substrate, either with or without SdC28, increased linearly for at

least 10 mm. The kinase activity slowed at -30 mm and

terminated at 60 mm of reaction time. PKC activity was taken as

the difference in the amount of 32P[P�] transferred from

[-y-32P]ATP to a synthetic peptide sequence ofthe EGFR, R-K-

R-T-L-R-R-L, in the absence and presence of phosphatidyl-

semine. The reaction medium (60 �i.l), placed in 13- X 100-mm

disposable glass test tubes, consisted of 20 mrvi Tris (pH 7.5),

10 mM MgC12, 1.0 m�i CaCl2, 2.0 �i.g synthetic peptide, 5 �i.g

phosphatidylsenine on 5 �i.l water, 5 p.1 polyanion inhibitor at the

desired concentration, and 5 �j.l PKC �31 preparation. The reac-

tion was initiated with the addition of 5 p.1 792 p.M [-y-32P]ATP

(-200 epm/pmol). Each (-) and (+) determination was carriedout in duplicate for each oligo concentration measured. There

was less than 10% variability between the duplicates. The me-

action was carried out in a 30#{176}Cwater bath for 10 mm and was

quenched by transferring 30 p.1 reaction medium to a 3- X 3-em

square of phosphocellulose paper and immediately immersing it

in a 1-liter beaker of tap water. The squares were washed with

water and counted. In control experiments, the presence of

phosphatidylsemine typically stimulated the kinase reaction 4- to

7-fold with the EGFR peptide as substrate.

To determine the extent of PKC �31 autophosphorylation,

the assay as described above with phosphatidylsenine and the

EGFR substrate omitted, was performed in the presence of the

appropriate concentrations of polyanion inhibitor. After 10 mm,

an equal volume of Laemmli’s buffer was added, and the mix-

tune was eleetrophomesed on a SDS-7.5% polyacrylamide gel.

The gel was dryed and exposed to Kodak film overnight, after

which the film was developed.

PKC a and PKC #{128}were partially purified from cell lines

engineered to overproduce them (37). PKC � was provided by

D. Fabbro (Department of Pharmaceutical Research, Ciba Geigy,

Basel, Switzerland) and purified as described previously (38) PKC

a activity was determined in the same manner as PKC �31 activity.

PKC �i activity was determined likewise, but in the presence of 2

mM EGTA. PKC s-specific activation was detected by inclusion of

phosphatidylserine (83 p.g/ml) or phosphatidylsemine and TPA (100

ng/ml). PKC #{128}activity was determined, in the absence of Ca2�,

using a synthetic peptide that is a 12-amino acid fragment of the

Aplysia PKC #{128}pseudosubstrate (Ac peptide), with serine substi-

tuted for alanme (39).

PKC Catalytic Fragment. This was isolated fromDEAE-Sephaeel (21) as phosphatidylsemine-independent aetiv-

ity from Sf9 insect cells that had been infected with baeulovimus

bearing the full-length eDNA of mat brain PKC �31. The phos-

phatidylsemine-independent activity arose by limited proteolysis

of intact PKC �31, presumably by endogenous proteases, after

the cell pellets were thawed. On SDS-8% PAGE, a major band

was observed that migrated at the appropriate rate for the

catalytic fragment (between 50 and 60 kDa).

RESULTS

Comparison of Kinetics of Inhibition of PKC �1 Kinase

Activity by SdC28 and Suramin Using EGFR Substrate.

The inhibition of the kinase activity of PKC a, �3, and �y by

suramin (17) has previously (17) been demonstrated to be sim-

ple linear competitive with respect to ATP when histone Ill-S

was used as the substrate. In the opinion of Mahoney et a!. (17),

kinase activity was also inhibited by direct interaction of

suramin with the substrate.

We reexamined the kinetics of inhibition of PKC �31 ac-

tivity by suramin. However, instead of histone 111-5, we used a



LYCH H3

[03s0 0S03j ,.�

PentosanPolysulfate

H4 Suramin

Clinical Cancer Research 115

Fig. I Chemical structures of polyanions. Numbers of negative charges per molecule: Sumamin, 6; H4, 4; LYCH, 2; H3, 2; SdC28, 27; pentosanpolysulfate (-18).

synthetic fragment of the EGFR in its place. Inhibition of PKC

131 kinase activity by suramin was noneompetitive with respectto EGFR at all suramin concentrations tested (data not shown).

On the other hand, inhibition of PKC �31 activity by suramin was

mixed competitive/noncompetitive with respect to ATP when

EGFR was the substrate.

We then wanted to determine if the kinetics of inhibition of

PKC 131 kinase activity by SdC28 were similar to that of

suramin. Similar to sumamin, the inhibitory activity of SdC28

appeared to reside in the constitutively active catalytic fragment

(ICc0 7.5 p.M for SdC28). Phosphorylation of EGFR by the

catalytic fragment, in the presence on absence of SdC28, was

independent of phosphatidylsenine and calcium.

We then examined the kinetics of inhibition of the holoen-

zyme by SdC28. As shown in Fig. 2 (top), the Lineweavem-

Burke plot of the PKC �31 substrate phosphomylation reactions,

conducted with varying concentrations of substrate (EGFR pep-

tide), indicates that SdC28 inhibited PKC �31 activity in a

simple, linear competitive fashion with regard to substrate.

Furthermore, a Hill plot was linear (r� = 0.99) with a slope of

unity. (The Hill plot was constructed using the observed inhi-

bition of kinase activity [log(V0 - V)/V is plotted versus

log(SdC28)J, where V0 and V are the reaction velocities in the

absence and presence of SdC28, respectively; Fig. 3, top.)

Following the example of Majumdam et a!. (40), these data

suggest that the mode of inhibition of PKC �31 activity is

unimolecular. These data further suggest (but do not prove) that

SdC28 may interact with PKC �31 at or near the substrate

binding site.

We also examined the kinetics of PKC 31 phosphomylation

of EGFR as a function of AlT concentration. In the mange of

ATP concentrations from 25 to 150 p.M. we observed no inhi-

bition of substrate phosphomylation as a function of ATP con-

centrations at constant oligo concentrations (i.e. , a Lineweavem-Burke plot as above was composed of a series of parallel,

horizontal lines). In contrast, as mentioned above, in this sys-

tem, using EGFR as substrate, the inhibition of PKC �31 phos-

phorylating activity by sunamin is mixed competitive/noncom-

petitive with respect to ATP. Therefore, even though both

SdC28 and sunamin can inhibit PKC �31 kinase activity, only

suramin is a competitive inhibitor with respect to ATP.

The meplot of the slopes of the Lineweavem-Bumke lines

from Fig. 2 (top) versus SdC28 concentration is shown in Fig. 2

(bottom). The individual data points vary in a random fashionfrom linearity (r� = 0.95). The value of K. - 5.4 jiM.

Determination of the apparent dissociation constant (Kd) of

SdC28 binding to PKC �31 was accomplished using the initialmate method of Mildvan and Leigh (41). This method has found



1000

800

600

400

200

0

‘l

>

0

0

0 200 400 600 800 1000-2

-2 -1 0 1

>

0

.2

-6 -4 -2 0 2 4 6

-1

-1 0 1 2

its greatest use in studies on irreversible enzyme inhibitors. In

our experience, the rate of dissociation of phosphonothioate

oligo/protein complexes is sufficiently slow so as to mender the

binding of phosphomothioate oligo to protein virtually imrevers-

ible (31). Under the conditions of our experiments (Ca2� 1

mM, phosphatidylsemine = 83.3 jig/mI, EGFR = 11.75 p.M.

ATP = 66 jiM), initial phosphorylation of EGFR substrate, either

with on without SdC28, increases linearly for at least 10 mm.

A plot of the log V/V0 versus time (where V� concen-

tration of phosphorylated EGFR at 60 mm and V = Va-V,, where

V, = concentration of phosphorylated EGFR at t 3, 5, and 7

mm) was created by expressing V/V0 as a percentage of maxi-

mum phosphomylation V0. When log V/V() versus time was

plotted in the presence of several concentrations of SdC2S (0.5,

2, 5, and 10 jaM) a series of straight lines was produced. The

slopes of these lines are equal to kap�,/2.3, where � is the

apparent pseudo-first order rate constant of the inhibition meac-

tion. (The inhibitor concentration was always in substantial

excess relative to the enzyme.) However, //kapp 1/k1 +

[I]/ktKd, where k1 is the true rate constant of the substratephosphorylation reaction and [I] is the concentration of SdC28.

A plot of � versus [SdC28j is a straight line (r� = 0.99; Fig.4) and the value of apparent Kd (as calculated from the X-inten-

cept) = 2.3 p.M.


V.

0V.

*

0

1ILSJ. huM xlO

(SdC2�J. uM

Fig. 2 Top, double reciprocal kinetic plot demonstrating competitive

inhibition with respect to substrate (EGFR) by SdC28. Assays were

performed at various concentrations of substrate in buffer containing

20 msi Tnt (pH 7.5), It) mM MgCl2, 1.0 msi CaC1,, 5 jig phosphati-dylsenine. 66 jiM AlP, and 5 �il PKC �3l preparation. The determinationwas carried out in duplicate (

E

V.

400

300

200

I 00

-4 -2 0 2 4 6 8 10 12

LSdC2SI, uM0 1 2 3 4 5 6

tSdC2eJ. uM

0 10 20 30

tPentosan polysulfatej. uM


Fig. 4 Determination of apparent 1�d of SdC28 binding to PKC �3laccording to the method of Mildvan and Leigh (41). Plotted is [SdC28]

versus l/kapp. where � represents the pseudo-first order mate constant

of the inhibition reaction. The assays of enzyme activity were performedas described in ‘ ‘ Materials and Methods. ‘ ‘ Values of kapp were deter-mined as described in ‘ ‘ Results. ‘ ‘ A straight line was fit to the datapoints using the method of linear least squares. Value of the apparent

Kd 2.3 p.M.

Length Dependence of Inhibition of PKC �1 KinaseActivity by Phosphorothioate Oligodeoxynucleotides. Be-

cause of the relatively high value of IC5() for SdC28 (-5 jiM),

we synthesized longer phosphorothioate homopolymems of cyt-

idine in an attempt to increase the potency of the inhibition of

PKC �31 kinase activity. However, the value of IC5() for both

SdC35 and SdC4O was approximately 1 jiM, which is only

slightly less than that for SdC28. Other, nonhomomenic oligos

were also tested as inhibitors of PKC �31 kinase activity in

the presence of phosphatidylsemine and EGFR. The IC5() for

the phosphomothioate 21-mer 5’-dC7(T7)C7-3’ was approxi-

mately 20 jiM, which was similar to that of SdC2I (31). A

similar IC5() value (20 jiM) was also obtained for the par-

tially self-complementary phosphorothioate 21-mer 5’-dCGC-

GCGC(T7)GCGCGCG-3’.

Stimulation of PKC �1 Kinase Activity by SdC28 in the

Absence of Lipid Cofactor. To further explore the effects of

SdC28 on the phosphomylating activity of PKC �31, we examined

its activity in the absence of lipid cofactor. These effects are

similar to that of sunamin under identical experimental condi-

tions. SdC28, at low concentrations (2-4 jiM) stimulated the

phosphorylation of the EGFR peptide by 300-400% (Fig. 5,

top). This effect was independent of the Ca24 concentration (up

to 1 mM tested). Furthermore, the dose response of the substrate

phosphomylation reaction with respect to the Mg2� coneentra-

tion was not affected by the presence of SdC28 (5 jiM), sug-

gesting that this ion did not directly interact with the oligo or

with the enzyme. Longer phosphorothioate oligos, including

both SdC3S and SdC4O, also stimulated EGFR phosphomylation

in the absence of phosphatidylserine (250-325% increase at 2.5

jiM concentration versus control in the absence of oligo).

When the substrate phosphorylation reaction was per-

formed in the absence of substrate, but in the presence of

phosphatidylsenine, SdC28 markedly stimulated PKC autophos-

300

.�

8 200

0>

100

0

Fig. 5 Effects of polyanions on PKC �31 kinase activity in the presence

or absence of phosphatidylsemine. Assays were performed as described

in ‘ ‘ Materials and Methods’ ‘ and compared to enzyme velocity with no

added polyanion. Top. SdC28, E�J; + phosphatidylsenine (83 p.g/ml); #{149},absent phosphatidylsemine. Bottom. Pentosan polysulfate, �; + phos-

phatidylsemine (83 pg/mI); #{149}, absent phosphatidylserine.

phorylation (Fig. 6). At a concentration of 2.5 p.M SdC28, the

increment was as much as 300-400% greaten, as determined by

laser scanning densitometmy, than that of control in the absence

of SdC28. The enhancement is dose dependent (only twice that

of control at 0.5 and 1.0 jiM).

Effects of SdC28 on the Kinase Activity of PKC a, �,

and #{128}. To determine if the effects of SdC28 on PKC kinaseactivity were isoform specific, we examined PKC phosphory-

lating activity with the a, �, and #{128}isoforms. Under our reaction

conditions, we did not observe a differential inhibitory effect of

SdC28 on PKC a versus PKC �31 (IC5() for both -5 jiM). On theother hand, calcium-independent PKC #{128}kinase activity (A #{128}

peptide substrate, assayed after addition of 100 ng/ml TPA) was

neither stimulated nor inhibited by SdC28 at concentrations up

to 20 p.M.



. SdC28, 0.5

. SdC28, 1

S SdC28, 2.5 uM� Pentosan, 1 uM

0 Pentosan, 2.5 uM. Pentosan 5


p80

I 1

.�

0 800

l000f______ -.

�

�

b� .

.� 600

C’)

.� 400IPolyanion

Fig. 6 Top. autoradiogram of the 80-kDa PKC �l protein after stimulated autophosphorylation by polyanions. Phosphatidylserine and EGFR

substrate were omitted. as described in “Materials and Methods.�’ The reaction time was 10 mm. Lane 1, control (no added polyanion); Lane 2,

SdC28. 0.5 jiM: Lane 3. SdC28, 1 jiM: Lane 4, SdC28, 2.5 jiM; Lane 5, pentosan polysulfate. 1 jiM: Lane 6, pentosan polysulfate. 2.5 p.M: Lane 7,pentosan polysulfate, S p.si. Bottom. band intensity. as determined by scanning laser densitometry. of the bands in Fig. 6. top. Intensity is expressed

as percentage of control (no added polyanion) after subtraction of background.

The effects of surarnin on PKC � kinase activity have not

previously been examined. These assays were performed in the

presence of 2 mM EGTA to remove free Ca2� ions: we used

either the EGFR octapeptide or histone Ill-S as the substrate.

Under these conditions activation of PKC � by phoshatidyl-

serine produced less than a 2-fold stimulation of kinase activity

relative to the constitutive activity. However, PKC � activity

was stimulated by greater than 9-fold in the presence of both

phosphatidylserine and TPA using either substrate. Suramin

(5-125 p.M. histone Ill-S substrate) could not cause PKC �

activation in the presence of phosphatidylserine alone: it could

only do so in the presence of both phosphatidylserine and TPA

(Fig. 7). In contrast, with EGFR as substrate, 250 p.M suramin

markedly inhibited PKC �i activation by phosphatidylserine plus

TPA. In the absence ofcofactors, suramin (5-125 jiM) enhanced

constitutive phosphorylation of the EGFR peptide by -3-5-fold

(Fig. 7).

SdC28, similar to suramin, enhanced the phosphorylation

of histone Ill-S by PKC � in the presence of phosphatidylserine

and TPA, and also enhanced the phosphorylation of the EGFR

octapeptide in the presence of phosphatidylserine alone or in

the absence of cofactors (data not shown). However, also with

the EGFR peptide as the substrate, the effects of SdC28 on the

activity of PKC � (in presence of both phosphatidylserine and

TPA) are complex (Table 1). Based on three independent de-

terminations of the specific activity of PKC �i calculated by

subtraction of constitutive activity (in the absence of cofactors)

from activity in the presence of both phosphatidylserine and

TPA, we determined that the ICS() for suramin was -28 p.M.

From the data in Table 1 , the ICS() for SdC28 - 10 p.M. How-

ever, concentrations of SdC28 of 2 p.M or less could actually

slightly enhance PKC � activation by phosphatidylserine and

TPA (Table I).Kinetics of Inhibition of PKC �1 Kinase Activity by

Pentosan Polysulfate Using EGFR Substrate. To determine

if the kinetic data obtained in our experiments with SdC28 were

of a general nature, we examined the inhibition of PKC �3 I

kinase activity by a structurally unrelated polyanion. Pentosan



uM Suramin


2000

1800

1600>‘

:� 1400

� 1200

� 1000

C 800

600

400

200

00 50 100 150 200 250 300

Fig. 7 Differential effects of suramin on PKC � activation by histone

Ill-S and EGFR octapeptide substrates in the presence of phosphatidyl-serine and TPA. PKC f� enzyme activity was assayed as described in“Materials and Methods’ ‘ utilizing either histone Ill-S (667 p.g/ml) oralternatively the EGFR octapeptide (33.3 jig/mI) as the phosphoaccep-tom. Kinase activity was measured in the presence of phosphatidylserine

or phosphatidylserine plus TPA. and in the absence or presence of

suramin. The results are expressed as the increase of activity detected in

the absence of these cofactors. �, control (no added histone); #{149},+histone III-S, +ps; #{149},+histone III-S, +ps, +TPA; K�, control, noEGFR; #{149},+EGFR, +ps; LI, +EGFR, +ps, +TPA.

polysulfate is a polymer of xylose hydrogen sulfate, has a Mr

-3 100, and contains two sulfate groups per carbohydrate mono-

men. One way in which it differs from phosphorothioate oligo-

nueleotides is that the negative charge is localized on the oxygen

atoms of the sulfate groups, in contrast to the phosphorothioate

oligos where the negative charge is localized predominantly on

the sulfur atom of the phosphorothioate groups (42). Pentosan

polysulfate also inhibits the PKC �l catalyzed phosphorylation

of the EGFR substrate (ICS() = 5 jiM). The kinetics of inhibition

at low concentrations are shown in Fig. 8 (top), in which is

displayed the Lineweaver-Burke plot of the substrate phosphor-

ylation reaction performed in the presence of variable amounts

of EGFR substrate. At low pentosan polysulfate concentrations

(1-10 jiM), the mode of inhibition is simple linear competitive.

At higher concentrations (20-50 jiM), the mode of inhibitionchanges to uncompetitive (data not shown). The replot of the

low-concentration Lineweaver-Burke data gave the value of

K. = 2.3 p.M. Similar to SdC28, the inhibition of the phosphor-

ylation reaction in the presence of pentosan polysulfate was not

competitive with respect to ATP concentration. The Hill plot,

performed in a manner identical to that for SdC28, was linear

(,2 0.99) with a slope of 0.92 (Fig. 3, bottom), suggesting a

unimolecular order of inhibition. In addition, pentosan polysul-

fate resembled SdC28 in stimulating EGFR phosphorylation in

the absence of lipid cofactor (300% relative to control at 2.5 jisi;

Fig. 5, bottom) and was an inducer of autophosphorylation in the

absence of lipid eofactor and substrate (Fig. 6).

We extended some of these observations to a polyanion

structurally related to pentosan polysulfate. Dextran sulfate (500

Table I Effects of SdC28 on PKC � activity”

SdC28 (jiM)

PKC S activity (c/c control)

A B

0 100 100

0.08 106 108

0.4 92 117

2 119 10310 55 53

50 22 9

‘, PKC t� activity was determined in triplicate in the presence ofTPA and phosphatidylsenine using the EGFR substrate (A. 16 p.M; B. 8

riM) as described in ‘ ‘Materials and Methods.’ ‘ (SEM for each triplicateis -�-- I 5%). Specific PKC activity detected in the presence of SdC28 wascalculated and expressed as a percentage relative to the control. ICS) is

-10 p.M.

kDa), in the presence of phosphatidylserine, was also an inhib-

itor of PKC �l activity, with ICS() �-50 p.g/ml.

Inhibition of PKC �1 Kinase Activity by 114 and Other

Suramin “Analogues”. Because the kinetics of inhibition of

PKC �l were different for suramin versus the other polyanions

tested, we examined other polyanions structurally related to

suramin. Although H4 is a tetraanion, it resembles the hexaan-

ion suramin in that both molecules consist of two sulfonated

naphthylamine moieties separated by a bridge containing hy-

drophobic aromatic groups (Fig. 1). H4 also inhibited PKC � I

kinase activity in the presence of phosphatidylserine (EGFR

fragment as substrate, ICS() = 25 jiM). Inhibitable activity me-

sided in the PKC �l catalytic fragment (ICS() �-35 jiM). We

examined the substrate phosphorylation reaction in the presence

of variable concentrations of EGFR fragment and ATP and

found that the inhibition was simple linear competitive with

respect to EGFR; like SdC28 and pentosan polysulfate, there

was no competition with respect to ATP concentration. The

replot of the slopes of the Lineweaver-Burke lines versus H4

concentration is linear (r� = 0.94, K = 8.3 jiM). However, in

contrast to both SdC28 and pentosan polysulfate, the slope of

the Hill plot is not close to 1 .0 at the concentrations of EGFR

fragment used, indicating that the molecular order of the inhi-

bition is not unity. This suggests that the H4 might be interacting

with multiple sites on the enzyme and perhaps with the substrate

as well. In the absence of phosphatidylsenine, H4 resembled

suramin in that it stimulated PKC �3l activity (350-450% at

10-20 jiM). H4 also induced PKC �3l autophosphorylation in

the absence of both substrate and lipid cofactor, but the effect

was not as dramatic as that which is seen with SdC28. No

stimulation was present at an H4 concentration of > 10 p.M.

H3, a dianion comprising one half of H4, (Fig. 1 ), not only

did not inhibit PKC �3 1 activity in the presence of lipid cofactor,

but was actually slightly stimulatory at all concentrations as-

sayed (e.g., 144% of control, 35 p.tvi; 139% of control, 50 jiM).

On the other hand, the dianion LYCH, which is similar to H3

except that it lacks an appended benzylidene group (Fig. 1 ), was

neither stimulatory nor inhibitory in the absence or presence of

lipid cofactor at concentrations up to 50 p.M. The behavior of

these dianions contrasts sharply with that of the tetraanion H4,

which is equipotent with suramin.



3000

2000

=

x>V.

0

100 300 500 700 900

illS!. huM X 10

2

.2

-2 0 2 4 6 8 10 12

fPentosan polysulfatej. uM


5 C. Stein, W. Stec. J. Tonkinson. and A. Wilk, unpublished results.

3;�,.

l�ig. S Top. double reciprocal kinetic plot demonstrating competitive

inhibition with respect to substrate (EGFR) by pentosan polysulfate.

Assays were performed at various concentrations of substrate in buffer

containing 20 mM Tris (pH 7.5). 10 nmi MgCl,, 1.0 m�i CaCI,, 5 jigphosphatidylserine (83 jig/mI). 66 p.M ATP. and 5 p.l PKC �3l prepa-ration. The determination was carried out in duplicate (


(51). Presumably, binding of the polyanion (or lipid cofactom) to

the enzyme induces a conformational change that results in

dislocation of the pseudosubstrate from its binding site (52). On

the basis of charge considerations alone, it seems unlikely that

any polyanion can bind to the same site as the lipid cofactom.

More subtle differences in chemical structure can also lead

to significant changes in the kinetics of inhibition of PKC �31

activity. H4 resembles sumamin in that it contains two aminon-

aphthalene sulfonic acid groups. Nevertheless, it differs from

suramin in that H4 does not inhibit PKC �31 activity competi-

tively with respect to ATP. Surprisingly, H4 resembles SdC28

in being competitive only with respect to substrate. However,

the mode of inhibition of PKC �31 activity by H4 is not unimo-

leculan. The lack of inhibition of PKC �31 activity by LYCH and

H3 suggests that more than two negative charges may be me-

quimed for the inhibition. The ability of H3 and H4 to stimulate

PKC �31 activity may be due to the presence of their hydropho-

bic aromatic rings, which are lacking in the inactive dianion

LYCH.

Both suramin and pentosan polysulfate (32, 53) are capable

of binding hepamin-binding autoemine and paracrine growth fac-

tors, including bFGF and Kaposi’s FGF. It has been proposed

that this mechanism may be responsible for the clinical antine-

oplastic effects of these compounds (54). However, as indicated

above, these polyanionic compounds may have a multiplicity of

effects on the signal transduction pathway. These effects include

both interference with the hepanin-binding growth factom-recep-

tom interaction and possibly, with secondary, PKC-mediated

events, and all may all contribute to the observed in vivo

antineoplastic effects. The ability to affect multiple targets si-

multaneously, if it occurs, may help to explain the relatively

large number of responses seen clinically against chemotherapy-

refractory tumors when suramin has been used as monotherapy.

Furthermore, the dependence of potency and perhaps mecha-

nism of PKC inhibition on the chemical structure of polyanions

suggests that it may eventually be possible to synthetically

design other polyanionic compounds with augmented PKC in-

hibitory properties.

ACKNOWLEDGMENTS

Helpful discussions with I. B. Weinstein are greatly appreciated.

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1995;1:113-122. Clin Cancer Res Z Khaled, D Rideout, K R O'Driscoll, et al. polyanions on protein kinase C activity.Effects of suramin-related and other clinically therapeutic

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