Dual action of hydroxylated diphenylethylene estrogens on protein kinase C

Vol. 166, No. 3, 1990 February 14, 1990

DUAL

BIOCHEMICAL

ACTION

OF HYDROXYLATED

ESTROGENS Eric Bignon*,

AND BIOPHYSICAL

Jacques Gilbert#,

DIPHENYLETHYLENE

ON PROTEIN

Akira Kishimoto*,

RESEARCH COMMUNICATIONS Pages 1471-1478

KINASE

Michel Ponsg, Andrt

C’ Crastes de Paulets,

Jean-Franqois Miquel# and Yasutomi Nishizuka*

* DepartmentofBiochemistry, Kobe University School of Medicine, Kobe 650, Japan I INSERM, Unite’58, 60 me deNavacelles,34090Montpelier, Face # CNRS-CERCOA, 2 rue H. Dunant, 94320 Thiaik, Face Received

January

4,

1990

SUMMARY : Protein kinase C (PKC) I (v), 11 (p) and III (a) subspeciesare all activated by 1,1-di-(p-hydroxyphenyl)ethylene derivatives (DPE) at micromolar concentrations. This PKC activation dependson the presenceof both caZ+and phosphatidylserine(PS) but doesnot require diacylglycerol (DG). DPEs enhancePKC activity at low PS concentrations,but not at saturating PS concentrations. Like DG, DPEs increasethe apparent affinity of PKC for PS as well as for Ca2+,but lead to a decreasein the catalytic activity (Vmax). In the presenceof saturating DG concentrations, DPEs exhibit an inhibitory action. The derivatives alsoinhibit the activity of the proteolytic fragment of PKC, protein kinase M. It is concluded that DPEs are mixed-type inhibitors, probably interacting with the catalytic domainof the enzyme. 01990 Academic Press, Inc. The physiological

importance

recognized and well documented (1).

of protein Diacylglycerol

phospholipids in response to extracellular

kinase

C (PKC)

is widely

(DG) produced from inositol

signals greatly increases the affinity

of

PKC for Ca2+and phospholipids (2). Phorbol esters, known to be potent tumor promoters, bind and activate the enzyme (3). Molecular cloning and biochemical analysis have revealed that the enzyme exists as a family

of multiple

subspecies

with closely related structures (4). 1Eric Bignon is supportedby a fellowship (BourseLavoisier) from the Minis&e Fran@s des Affaires Etrangires and by a grant from Roussel-Uclaf. J.-P. Raynaud and T. Ojasoo (RousselUclaf, Paris) aregratefully acknowledgedfor critically readingthe manuscript. The Departmentof Biochemistry of the Kobe University School of Medicine was supported by researchgrantsfrom the Special Scientific ResearchFund of the Ministry of Education, Science and Culture, Japan; the Muscular Dystrophy Association; the Juvenile DiabetesFoundation; and the YamanouchiFoundation for Researchon Metabolic Disorders. Abbreviations used: PKC, protein kinase C; DG, diacylglycerol; DPE, l,ldiphenylethylene derivatives; ER, estrogenreceptor; PKM, ca2+-and phospholipid-independent protein kinase; PKA, c-AMP dependentprotein kinase; EGTA, ethylene glycol bis(&aminoethyl ether)- N, I& X, N’ -tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PS, phosphatidylserine;DMSO, dimethylsulfoxide.

1471

0006-291XBO $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The 1,1-diphenylethylenes (DPEs), a chemical class of compounds related to the well-known triphenylethylenes (5), bind to the estrogen receptor (ER), and exert partial agonist/antagonist cell proliferation

(5-10).

and tri-phenylethylene parameters involved

estrogenic activity

Pharmacological

on various responses including

studies on several members of the di-

series have been performed in ER binding,

progesterone receptor induction,

control of human breast cancer cell proliferation implicated in cell proliferation and differentiation action of non-steroidal

antiestrogens

to determine the structural and in the

(11-13). Since PKC may be (14) and in the antitumoral

(15), we have investigated

systematically

structure-activity relationships with respect to PKC kinase activity. Only inhibitory effects have been reported for classical triphenylethylenes (15- 18), whereas the present report shows that the 1,I-di(p-hydroxyphenyl)ethylenes

have

a dual effect on PKC : they are stimulatory at low concentrations of phospholipid and DG, but inhibitory at high concentrations of these lipid cofactors.

EXPERIMENTAL

PROCEDURES

Chemicals and Materials : The preparationof theseDPE derivatives and their analytical characteristicshave beendescribedin previous papers(6,19). Their purity was checkedbefore use by HPLC analysisas describedpreviously ( 12) using chloroform / methanol/ triethylamine in the proportion 9 1/ 7 / 2. Relative Binding Affinities (RBA) of DPEs for Calf Uterus Cytosol Estrogen Receptor : RBAs were measuredafter incubation for 5 h at 25°C by a competition methodusing [6,7-3H] 17fl-estradiol(RBA of estradiol = 100%) as previously described(12). Cell Growth Experiments : The effects of DPEs on MCF-7 and BT-20 human breast cancer cell lines were studied in 24-well tissueculture cluster platesaspreviously described( 12) using culture mediumwithout phenol red containing 5% dexttan coatedcharcoal-treatedfoetal calf serum. After an g-day growth period, triplicate wells of cellswere assayedby measuringthe DNA content according to the method of Kissaneand Robins (20). Enzyme Purification : PKC was purified from the rat brain soluble fraction and separated into three types, type I, II and III as previously described (21). Each PKC was apparently homogeneousupon sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically active fragment (PKM) waspreparedby cleavageof PKC by a Ca2+-dependent neutral protease (calpain) followed by its isolation by DEAE-column chromatography as previously described(22). The catalytic subunitof PKA waspurified from rabbit skeletalmuscleaccordingto a publishedprocedure(23). Assay of PKC : PKC was assayedas previously described (16). Unless otherwise indicated, the standardreaction mixture (0.125 ml) contained 20 p&J Tris-HCl at pH 7.5, 10@J ]Y~~PIATP(400-500 cpm/pmol), 5 I,&$ magnesiumacetate,0.0 1 p.rhJEGTA and 0.0 1 mM EDTA (from enzyme fraction), each type of PKC (approximately 0.05 ug), 200 un/ml calf thymus Hl histone, 0.1 fi CaC12and 2 ug/ml phosphatidylserine(PS) or 2 &ml PS plus 0.2 ug/ml 1,2diolein. DPE derivatives or DMSO [final DMSO concentration in the incubation, 5% (v/v)] were added directly to the reaction mixture (Caz+- and PS- independentassay)or mixed (vortex) for 1.5 min with the sonicated PS solution (similar results were obtained without mixing). The incubation was carried out for 2 min at 30°C and the reaction was terminated by the addition of trichloroacetic acid 25% (w/v). The radioactivity of the acid precipitable materialwas determined by liquid scintillation spectrometry.All points were performedonce, except for control (duplicate). All experimentswere carried out at leasttwice. 1472

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AND BIOPHYSICAL

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Assay of Cal+- and PS-independent Activity of PKC : The Ca2’- and PSindependentprotein kinase activity was determinedasdescribedabove, in the presenceof EGTA (0.5 mM final concentration) insteadof Ca2’ and PS, using 200 us/ml Hl histone (for PKM and catalytic subunit of PKA) or 400 ug/ml protaminesulfate (for PKC). RESULTS The ethylene bonds of DPEs are substituted by a hydrophobic isopropyl group (DPEs r and 2, or a hydrophobic cycloalkylidene moiety (DPEs 3 and &) (Fig. 1). These DPEs bind to ER with various affinities, stimulate the proliferation of ER-positive MCF-7 human breast cancer cells at nanomolar concentrations to the level equivalent to that obtained with estradiol, and exhibit no cytotoxicity for both ER-positive (11) and ER-negative (BT-20) human breast cancer cells at micromolar concentrations (Table 1). The DYE derivatives

stimulated PKC activity

in the presence of PS (Fig. 2).

The stimulation was approximately 2-fold over control, but only about half of that obtained with DG (0.2 pg/ml 1,2-diolein). Similar results were obtained with three different PKC with

PKC subspecies [I (y), II (p), 111(a)] (Table 2). DPE 3 stimulated

an EC50 of 8-16 pM,

(EC50 of 30-60 PM). Although (“desacetylated

cyclofenyl”)

whereas

DPEs were less active

bis-(p-hydroxyphenyI)cyclohexylidene-methane

is structurally

had no effect OII PKC at these concentrations The nature of this activation

the other

similar to these DPEs (6, 11, 12), it (data not shown).

was studied further with PKC III

(a), which is

co~nn~only present in a variety of tissues (24). PKC 111 activity was enhanced by increasing the pbospbolipid concentration (Fig. 3). DYES and DC increased the apparent resulting

affinity of PKC for PS (by about 3- and lo-times, in a stimulation of the enzyme activity

concentrations

(Fig. 3).

in the presence of saturating

respectively) at low PS

amounts

of PS, DG

increased the maximum velocity (Vmax) by approximately lo%, whereas DPEs, depending on their structure, reduced it by up to 20% (Fig. 3). DPE & showed the most significant inhibitory action. PKC activity (Fig. 4).

was normally

Like l,2-diolein,

DPE 1 Finure

enhanced by increasing

the Ca2+ concentration

DPEs decreased the Caz+ concentration

DPE 2

DPE 2

DPE 2

1 : Structures of diphenylethylene derivatives(DPE). 1473

needed for

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 : Relative binding affinity (WA) of DPE derivatives for the calf uterus cytosol estrogen receptor (RBA of estradiol = 100%) and their effect on human breast cancer cell proliferation. Their stimulatory effect on ER-positive MCF-7 human breast cancer cell line was expressed both as the percentage of their agonistic action (percent of the maximum response recorded with estradiol) and as EC50 (nM). Their inhibitory effect on the ER-negative BT-20 human breast cancer cell line was expressed as a percentage of inhibition at 10 PM. Biochemical

DPE 1

parameters

DPE 2

4.2 + 0.8

RBA for ER (%)

MCF-7 cell proliferation EC50 (nM) Agonist activity (%)

18 f 10 65

BT-20 cell proliferation % of inhibition at 10 PM

40 210

DPE 2

DPE 4

2.9 f 0.4

0.84 zk 0.05

2.65 + 0.17

30 +15 96

36 +6 0.16 f 0.08 96

100

45 *1

20 +2

10 +1

Resultsarethemeans(k range)of 2-4 independent experiments.

of the enzyme. The concentration of added calcium required for halfmaximum activation in the presence DPEs or 1,2-diolein was 0.08 and 0.04 mM, activation

respectively,

and with PS alone was 0.2 mM.

In addition, the maximum activity of PKC obtained with DPEs was 60-80% of that obtained with DC. The

stimulatory

concentrations saturating

effect

of DG,

of

DYES was observed

whereas these compounds

concentrations

of this lipid

(Fig.

only

were rather

5). The extent

remains constant over a wide range of DG concentrations DPEs and DG can interact displayed inhibited

PKC only slightly

T

150

ii

(about 60% inhibition

B

C

100

Y a

50

a

1

100

sites.

at 200 PM).

DPE 4

DPEs L-2 in the

.D - 200 8 88

-150

,TTI

J

E

LOO

,I-;

-50

I

10

100

Diphenylethylene

I

10

concentration

100

1

10

100

(pIb4)

Fkure 2 : Stimulation of type Ill (a) protein kinase. C activity by diphenylethylene derivatives in the presence of 2 ug/ml phosphatidylserine as described under experimental procedures. Dose response curves of DPE derivatives [DPE 1 (panel A), DPE 2. (panel B), DPE 2 (panel C), DPE 4 (panel D)] in the presence (m) or absence (Cl) of 2 us/ml PS. Results are the mean (* range) of 2 to 4 independent experiments. Results are expressed as a percentage of the activity obtained with 2 &ml PS. The activity of PKC was about 400% in the presence of 2 @ml PS plus 0.2 ug/ml 1,2-diolein. Similar results were obtained with type I and ll enzyme (see Table 2). 1474

at

(Fig. 5) suggesting that

(less than 30% at 200 PM). This is reflected

A

A .=: .g

inhibitory

of this inhibition

with the enzyme possibly at different

the greatest inhibition

200

at submaximal

Vol.

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166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 2 : Effect of diphenylethylene derivatives (DPE) on PKC I (y), II (l3) and III (a) subspecies, PKM and on the catalytic subunit of PKA. Stimulation and inhibition by DPEs of PKC subspecies were determined in the presence of either 2 u#ml phosphatidylserine (PS), 2 us/ml phosphatidylserine plus 0.2 ug/ml 1,2-diolein (PS + DO), or protamine sulfate (Prot. sulf.). PKA and PKM were assayed as described under experimental procedures. Results are expressed as EC50 [concentration (PM) of the DPE giving 50% of its maximum stimulatory effect] and IC5, [concentration (PM) of the DPE giving 50% inhibition of the enzyme activity in its

absence]. Protein EC50

PS

IC50

PS+DO

Kinase

DPE

1

DPE

2

DPE

2

DPE

4

40 f 8 43 f 5 39*4

32 *4 35 *4 35 f 7

8fl 8&l 16k4

56 f 4 46 % 9 41 f9

III

> 200 > 200 > 200

> 200 ’ 200 > 200

> 200 > 200 > 200

210 f 10 140 4 10 147 % 20

II III lk

ICSO

Prot. snlf.

I II III

> 200 > 200 > 200

’ 200 > 200 ’ 200

> 200 > 200 > 200

220 % 10 140 % 10 130*20

IC50

Hl

histone

M

250 * 50

155 % 5

> 200

140 f 10

IC5o

HI

histone

A

> 200

> 200

> 200

’ 200

Results are the means (It range) of 2-4 independent experiments.

IC50 values (>200 PM; Table 2).

Similar

results

were

obtained

for

all

PKC

subspecies tested. An earlier

(25) has shown that,

report

with protamine sulfate as a substrate,

neither phospholipid nor DC is needed for PKC activation. DPEs exhibited inhibitory actions with either Hl histone (in the presence of PS and 1,2-diolein)

100 .?? . .?I 2

100

80

80

60

60

q-

40

40

z L

20

20

0

0

10

20

30

40

0

10

20

30

Phosphatidylserine

40

0

10

20

concentration

30

40

0

10

20

30

40

50

0

(pg/ml)

Figure 3 : Effect of diphenylethylene derivatives on type III (a) protein kinase C activity as a function of phosphatidylserine concentration (udml) measured as described under experimental procedures. (m) vehicle alone, (0) 100 uM DPE derivatives [DPE 1 (panel A), DPE2 (panel B), DPE 3 (panel C), DPE 4 (panel D)], @) 0.2 uB/ml 1,2-diolein. Results are expressed as a percentage of the maximum activity obtained in the presence of PS. The error ranges of the control values in this typical experiment are less than 5% and the maximum activity (100%) corresponded to about 90,000 cpm. 1475

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BIOCHEMICAL

166, No. 3, 1990

0.4

0.6

t

0.2

AND BIOPHYSICAL

0.4

0.6

t

0.2

Caz+concentration

0.4

0.6

RESEARCH COMMUNICATIONS

t

0.2

0.4

0.6

-

(m&l)

Finure 4 : Effect of diphenylethylene derivativeson type III (a) proteinkinaseC activity asa functionof addedCa2+concentration (n&i) measured asdescribed underexperimental procedures. The arrows represent0.5 mM EGTA. (a) 2 &ml PS, (0) 2 &ml PS plus 100pM DPE derivatives [DPE 1 (pane1A), DPE 2. (panelB), DPE 2 (panel C), DPE & (panel D)], @) 2 &ml PS plus 0.2 pg/ml 1,2-diolein. Resultsare expressedas a percentageof the maximumactivity obtainedin thepresence of PS, 1,2-dioleinandcalcium.Theerrorrangesof the control valuesin this typical experimentare lessthan 5% andthe maximumactivity (100%) correspondedto about100,000cpm.

or protamine fragment

sulfate as the substrate (Table 2).

of PKC (PKM)

was inhibited

The activity

of the proteolytic

by most of the tested DPEs (Table 2).

These results seem to indicate that the inhibitory action of DPEs is exerted on the catalytic domain of the enzyme rather than on the regulatory domain. None of these DPEs influenced the activity

of the catalytic

subunit of PKA (Table 2).

DISCUSSION Unlike

other

polyaromatic

hydrophobic

compounds

charged amino side chain, such as phenothiazines

substituted

with

(26) and triphenylethylene

-60 -40 -20

0

0.1

0.2

0

0.1

1,2-diolein

0.2

0

concentration

0.1

0.2

0

0.1

0.2

110

0.3

(pglml)

Figure 5 : Effect of diphenylethylene derivativeson typeIII (a) proteinkinaseC activity in the presence of 2 rig/mlPSasa functionof 1,2-dioleinconcentration(pg/ml) measured asdescribed under experimentalprocedures. (B) vehicle alone, (0) 100 pM DPE derivatives [DPE 1 (panelA), DPE 2 (panelB), DPE 3 (panelC), DPE & (pane1D)]. Resultsare expressed asa percentageof the maximumactivity obtainedin the presence of PSand 1,2-diolein.The error rangesof the controlvaluesin thistypical experimentarelessthan5% andthe maximumactivity (100%)corresponded to about60,000cpm. 1476

a

Vol.

166, No. 3, 1990

derivatives,

BIOCHEMICAL

which inhibit

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

the enzyme (15-18), DPEs show a dual effect on PKC.

DPEs appear to act as mixed-type inhibitors, increasing the affinity of the enzyme for PS and Caz+ and reducing its maximum catalytic activity. This dual effect of DPEs is probably due to their interaction with the catalytic domain as described above. The interaction of DPEs may occur at the same site as for a previously studied triphenylethylene hydroxyphenyl)ethylene

derivatives structure.

Whereas polar or aromatic substitutions

CN and phenol of triphenylethylene double bond reinforce

(16) as a result of a common l,l-di(pderivative)

this inhibition,

(e.g.

at carbon C2 of the ethylene

saturated or hydrophobic

substitutions

(DPEs) tend to derepress it. Some of the compounds thus become stimulatory under certain conditions. The activation of PKC by DPEs, having no fatty acyl moiety and no DG-like structure, appears unrelated to that of unsaturated fatty acids or DG (21), and also to that of other synthetic activates

PKC

derivatives

show a biphasic

concentrations Hydrophobic

activators.

The Ca2+ channel blocker,

even at high PS and DG concentrations (0.1

uM)

effect

on PKC,

and inhibiting

(27).

Flavonoid

activating

the enzyme

at low

it at higher

concentrations

(28).

side-chain substituted naphthalene sulfonamide derivatives

PKC in a manner similar to PS (29). It is plausible, therefore, derivatives are unique in the way that they modify PKC activity. activity

felopidine

of di- and tri-phenylethylene

derivatives

activate that DPE How the

on PKC could possibly

related to their other actions on human breast cancer cells is presently investigation

by multiparametric

be

under

analysis.

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15. O’Brian, C. A., Liskamp, R. M., Solomon, D. H., and Weinstein, I. B. (1986) J.N.C.I. 76, 1243-1246. 16. Bignon, E., Ogita, K., Kishimoto, A., Gilbert, J., Abecassis, J., Miquel, J.-F., and Nishizuka, Y. (1989) Biochem. Biophys. Res.Commun. 163, 1377-1389. 17. Su, H.-D, Mazzei, G. J., Vogler, W. R., and Kuo, 3. F. (1985) Biochem. Pharmacol. 34, 3649-3653. 18. O’Brian, C. A., Liskamp, R. M., Solomon, D. H., and Weinstein, I. B. (1985) Cancer Res. 45, 2462-2465. 19. Miquel, J.-F., Wahlstam, H., Olsson, K., and Junbeck, J. (1963) J. Med. Chem. 6,774-780. 20. Kissane,J. M., and Robins, E. (1958) 3. Biol. Chem. 233, 184-193. 21. Sekiguchi, K., Tsukuda, M., Ase, K., Kikkawa, U., and Nishizuka, Y. (1988) 3. B&hem. 103, 759-765. 22. Kishimoto, A., Kajikawa, N., Shiota, M., and Nishizuka, Y. (1983) J. Biol. Chem. 258, 1156-l 164. 23. Bechtel, P. J., Beavo, J. A., and Krebs E. G. (1977) J. Biol. Chem. 252, 2691-2697. 24. Kosaka, Y., Ogita, K., Ase, K., Nomura, H., Kikkawa, U., and Nishizuka, Y. (1988) Biochem. Biophys. Res. Commun. 151, 973-981. 25. Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mot?, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. 26. Mori, T., Takai, Y., Minakuchi, R., Yu, B., and Nishizuka, Y. (1980) J. Biol. Chem. 255, 8378-8380. 27. Sutherland, C., and Walsh, M. P. (1989) Biochem. Pharmacol. 38, 1263-1270. 28. Picq, M., Dubois, M., Murani-Silem, Y., Prigent, A.-F., and Pacheco, H. (1989) Life Sciences44, 1563-1571. 29. Ito, M., Tanaka, T., Inagaki, M., Nakanishi, K., and Hidaka, H. (1986) Biochemistry 25, 4179-4184.

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