European Journal of Pharmacok~3.... Molecular Pharmacoh#,,y Section, 226 (1992/273-277

273

cO 1992 Elsevier Science Publishers B.V. All rights reserved 0922-41(16/92/$05.0(1

EJPMOL 90322

Ipriflavone inhibits phosphoinositide hydrolysis and in the osteoblast-like UMR-106 cells

C a 2+

uptake

M a r i a A n g e l a S o r t i n o a, G r a z i a A l e p p o ", U m b e r t o S c a p a g n i n i ~ a n d P i e r L u i g i C a n o n i c o h " Institute ~f Pharmacology, University of Catania. School r~fMedicine, Catania. Italy, and j' Chair of Pharmacology, School of Dentistry, Unicersity o]"Pacia, Pacia. Italy

Received 14 February 1992, accepted 24 March 1992

The mechanism of action of ipriflavone, an isoflavone derivative, was studied in the osteoblastic-likc UMR-106 cell line. Ipriflavonc affected both phosphoinositidc hydrolysis and 45Ca2 + uptake. A repeated trcatn,.cnt of UMR-106 cells (once a day, for 3 days) with ipriflavonc decreased, in a concentration-dependent manner, [3H]innsitol monophosphate accumulation. This effect was also achieved after single addition of high concentrations of ipriflavone or 100 nM [Asul'7]eel-calcitonin, a semi-synthetic analog of ce! calcitonin. When rcpcated!y added to UMR-106 cells, 17/3-cstradiol produced a marked inhibition of [3H]inositol monophosphate accumulation, an effect which appeared significant only at a concentration of 1 /zM and which was accompanied by a reduced incorporation of [3H]inositol into membrane phospholipids. A repeated treatment with ipriflavone ": .......... reduced '~5Ca2" uptake as ~ell. Thib effect was obscrved also after a .... ,~,. . . . . ."~:'~"~ . . . . . . "~" [A~'_,L7]eel-calcitonin but not following single or repeated treatment with 17/3-estradiol. The present data indicate the osteoblast as a direct and specific target for ipriflavone and suggest that this compound may share intracelhtlar transducing mechanisms with other antiosteoporotic hormones such as estrogen and calcitonin. Ipriflavone; Phosphoinositidcs; UMR-106 cells; 4SCa2 ~ uptake

I. Introduction Ipriflavone is an isoflavone derivative which is emerging asapromisingantiosteoporoticnon-hormonal drug. In vitro studies have shown that ipriflavone is able to inhibit bone resorption as indicated by a redueed 4SCa2' release from rat fetal radii and ulnae (Tsuda et al., 1986). In human bone cell cultures, ipriflavone has been shown to stimulate collagen synthesis (Sziklai and Ribari, 1985), suggesling a stimulatory role for thia c o m p o u n d in protein matrix formation. T h e antir~.,sorptive action of iprillavone is also supported by in vivo studies, which have indicated ipriflavone as a potent inhibitor of bone loss obtained in rats using different experimental models of osteoporosis, such as those induced by ovariectomy (Yamazaki et al., 1986b), streptozotocin (Shino et al., 19861, or prednisolone t r e a t m e n t (Yamazaki et al., 1986a). T h e mechanism of action of ipriflavone in

Correspondence to: Dr. Maria Angela Sortino, MD., Institute of Pharmacology, University of Catania, School of Medicine. Viale A. Doria 6, 95125, Catania, Italy. Tel. (95) 338647; Fax to5) 333219.

inhibiting bone resorpti,~n is still poorly understood. lpriflavone may ac~ oJ:~ bone cells either dZ~ctly or indirectly. A potentiating action of iprifiavone on both estrogen effect on bone metabolism and calcitonin release induced by subliminal estrogen concentrations has been suggested (Yamazaki and Kimoshita, 1986). The action of ipriflavone has been studied in an osteosarcoma-derived tumora! cell line, the osteoblasticlike U M R - 1 0 6 cells. In this cell line ipriflavone, at micromolar concentrations, counteracts the stimutatory effect of parathyroid hormone on cyclic A M P accumulation and significantly increases alkaline phosphatase activity (Benvenuti et al., 1991). In o r d e r to analyze further the mechanisms involved in the action of ipriflavone at the osteoblast, we studled the effect of the drug in vitro, utilizing the U M R 106 cells, a cell line widely used to assess the role of different hormones and drugs which affect bone metabolism and cell growth. In this cell line we have ,;tudied the effect of ipriflavone on two intracellular transducing mechanisms, phosphoinositide hydrolysis and 45Ca2+ influx, which play a crucial role in the modulation of cell division and differentiation (reviewed in Bcrridge, 1987).

274

2. Materials and methods UMR-I06 cells were grown under sterile condition~ at 37°C, in a humidified almosphere of 95q~. air/5%: C'O:, in l)ulbecco's modified Eagle's m e d i u m / H a m ' s FI2 ( D M E M / F 1 2 ) ( 1 : t ; (Jib,co Grand Ishmd. NY, USA) supl)lemcntcd with I00 U / m l penicillin, l ( 1 0 # g / r o t streptomycin (Sigma, St, t.ouis, MO, USA) and 5Q fet;tl calf serum ((3ibco). Ccils were passagcd once a week and only cells of passages 2--13 have been utilized for the present stud},. UMR-t06 ceils wcrc grown in 75 cmz flasks (Falcon, Milan, Italy) at a density of 2.5-5 × 10" ce!ls/fla,& and detached with uypsin (0.5 mg/ml). Cells to be used in experinaents were plated inlo 24-wcll multiwell plates (Falcon) at a density of 5 × 104 cells/ml, lpriflavone (Chiesi Farinaccutici, Parma, Italy) and 17/3-cstradiol (Sigma) (both initially dissolved in 1(111c'~ ethanol) were added to chronically treated cells every day starting on day 1. All controls received an equal voh, mc of an ettmno~ somtion that never exceeded 0.01c4 of total volume.

2.1. Measurement of [ ~tt]ino.sitol motlopho.whate 24 tt before the experiments, UMR-106 cells were incubated with ! u C i / m l 2-[~H]myo-inosito[ (New England Nuclear, Florence, Italy: specific activity 15.6 Ci/mmol). Tiffs labelling time was chosen because it reprcscnts the steady state for [3H]inositol incorporation into membranal phosphoinositides. After the 24-h labelling period, cells were washcd twicc with a KrebsHenseleit st~lution containing I0 mM LiCI (Sigma) 19 rain later, drugs wcrc added for 45 min and the incubation was carried out in the continuous presence of an O, flow. Reaction was stopped by adding m e t h a n o l / chhr,,J:' , water ( i : 1 : t ) and after centrifugation, the aqueous . . . . . . h;>~es were separated. [3H]lnositol ,mmop!,~., ',~ ,,,?~ was separated by ion cxchange ~:m~ using a Dowcx |ormate form resin (i~ .... ~': Bio-Rad gaboratoric:,, Richmond. CA. ~_ : . ~; +:> elutcd with a solution of 0.2 M amm,',~q,ma ,..,~ ~, ~ ,u,- acid, according :e tl'~e tllCtht ' .! ' -, " '." . . . . ~.'~ a!. (!982). Ra-

....

/.tCi/ml. Drugs were then added for an additional I0 rain. Radioactive calcium influx was blocked by three rapid washings at 4°C with a chelating buffer containing Tris, EGTA and choline chloride, lntrace!lu!ar 4Sea2' was extracted with NaOH and after neutralization with hydrochloric acid, radioactivity was determined by scintillation spectrometry. ) *,

..o. Statistical analy:,is Data were analyzed by Student's t-test and one-way analysis of variance followed by a multiple range test for significance.

3. Results

3.1. l:;tfectofil>riJlat'oneon the accumulation ofinositol pho.qgmtes in I/MR-f06 cells A repeated treatment with ipriflavone (once a day, for 3 days) iuduced an inhibition of [3!l]lnsP accumuhtt ion. This effect was concentration-dependent, maxima! at a concentration of i0 # M ipriflavone (fig. 1) and independent of an action of ipriflavone on phospholipid synthesis. A repeated treatment with ipriflavone, in fact, did not modify, the incorporation of [3H]inositot into the phospholipid fraction (179.8 +_ 5.7 and 183.6_+ 7 d p m / m g protein x 103 for basal and ipriflavone-treated cultures respectively). An acute treatment with 1 / , M ipriflavone inhibited phosphoinositide hydrolysis as well. However, this ef-

a ?

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~ .t

~r\

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~--..I

~ 4 A

i,

\K, k

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[ JMR "',: ':I s, grov,q'~ : .... ~ t~ v,,,.' plates at a ,.k.z~;uy of 5 Y ~{~a celb~/m~ . . . . . . . . . ~shed ~wice ",~i~h :, ' .... i.... >~olution (pH 7.4) containing 154 mM NaC1. 5.~ . , ' J KC]. 3.6 mM NaHCO 3. 1 mM MgCI:. 1,3 mM CaCI.. 5.6 v.aM glucose and 1(1 mM HEPES (at! from Sigma). Cells were incubated for 10 .Is(, " +

rain in tile presence of " ._a- (New England Nuclear; s p e c i f i c a c t i v i t y 23 ('i/~figI at a c o r l c e n t r a t i o n of 1

T

0 -

-r--qc k-il0

~ ........8~

/'/

a'

,5' -n

- log N [Iprlflavone] Fig. 1. Conccmration-responsecurve of i|~riflavoneeffect o'~ phosphoinositide hydrolysis in U M R - 1 0 6 cells, lorifiavone at different to g r o w i n g UMR-106 cells. Values are m e a n ± S , E M of lwo i n d c p e n d e n t experiments.

concentrations, was added eve~. day for 3 days

275 8"~

10 I

O Control **



1 7 ~ - E z ( 1 0 pM)

0

6

8

E

%

2

t

%

II o

Control

Acute CT

I_

Repeo~ed Acute -{prlflovone-

Fig. 2. Effect of single or repeated t r e a t m e n t with 1 # M ipriflavone and 100 nM [Asul'7]eel-calcitonin (CT) on [31t]inositol monophosphatc a~.cumuk~tkm h~ U M R - 1 0 6 ~t.q!.-...,',dues arc - - c r ~ ..r a~Cah_, ~.~.M . . . . 3 - 5 i n d e p e n d e n t studies. * P < 0.01 versus control values. ** P < 0.1)5 versus control values.

TABLE I

0

[3H]lnsP (dpm/mgprotein)×103

I

I

I

I

I

I0

9

8

?

fl

- -

log id [ t p r l f t c l v ~ n a ]

i1 i.iUl- -t of Fig. 4. Effect of 17/J-estradiol (,7/3.r:2, 10 pM) on the nlllJ " ' '~':" [31t]inositol m o n n p h o s p h a t e productiol: induced by different eoneentr',tions of ipriflavone. 173-Estradiol and ipriflavone were added simultaneously for 3 days since day 1 in cul:ure. Values are mean+_

Effect of a single addition of ipriflavone ar, d parathyroid hormone (PTH) on [3H]InsP accumulation in U M R - ; 0 6 cells. Values are mean ± S . E . M . N" three i n d e p e n d e n t experiments,

I

C

S.E.M. of three independent experiments.

fect was not as marked as that produced by a chronic treatment with ipriflavone (fig. 2). A n acute treatment with 100 n M [ A s u l ' 7 ] e e l c a l c i t o n i n c a u s e d an inhibition of I n s P f o r m a t i o n simi-

7.7±0.3 7.6 ± 0 2 5.4±9.4" 5.3+0.4"

Control

P T H (100 nM) lpriflavone (1 ~ M )

ipriflavone+ PTH

lar to that induced by a repeated treatment with ipriflavone (fig. 2). The inhibitory effect of ipriflavone on phosphcino~itide hydrolysi'~ was mimicked by a re-

~' P < 0.01 versus c~mtrol values

[Asul'7]eol-calcltonln 8

7

7

I

x ,-,

4.

( 1 0 0 hid)

,,

6

fi ,>.

Q.

Q-

~.

'~.

3

2

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f.I.

2

~-

Control

Repeated

AcuCe

17/~-E2

17B-Ez

--

t.,p • Fig, 3. Effect of a repea~ed (diagonal bar) '~r single tu, ,~h,.d bar) exposure of U M R - 1 0 6 cells to 1 /xM 17/3-estradiol (17/3-E2). Values are mean +_S.E.M. of three independent experiments. * P < (HII versus cm~trol values,

t

0 ~--~-~

C

I

, 8

-

~ 7

,

G

5

log M [[?rlfla~on~]

Fig. 5. Concentration-response cun, e , ' the inhibimry effect of ipriflavone on 45Ca2' uptake in UMR-~ "~ ceils. The effect of ipriflavone is compared to that produced b. l!JO r!M [Asul'Tleelcatcitonin. Values arc mean -~ S . E . M of two ir~d,.n..erldel~t studies.

27{~

-IABH 2 Eftcot of)' chronic treatment ~i|h 17/5-cstradiolon 'a~('-:t:' influxin UMP,-lt~,~cclb;, valuc~, rcplcscm mean,- SEM ~,f a rcprcscnlati~'.:cxr~¢timcnI rult in qmtdrup!icaTc,

................................. ~m,,-g.a.V:~x~l~i,i[~ ................... ( d p n / m g 1t[{11¢111f1II111}'* Ill 2

C;,,n,,~ 17/3-cshadio!

... 3.1 t~.2

pc)ted (but uot a single) treatment with 17/3-eslradiol which, at a concentration of 1 #M, produced a marked redt, clion of[3H]lnsP accumt_,lation (fig. 3). This effect wits accompaedcd by a n-duced incorporation of [3H]inositoi int~ the phospholipid fraction (179.8 -+_:5.7 and 15f.6 -± 5•3 tlpn~/rng p r o t c i n × I/) 3, for control and 17/3-eslradiol-treatcd group, ~espeetivcly: P < ().Ill by ~:{itidclti'.-,t-ics~), A concomitant repeated treatment with a low concentration of 17[3-estradiol and increasing conccntrations of ipriflavonc produced a shift It) the left of the inhibitory curve mdu:co b.y iFriflawmc alone (fig. 4). Parathyroid hormone (P fl-l: Peninsula, Merscysidc, UK). at it concentration o5 lilt) nM, did not affect [q-illnsP formation nor did it prcvent the inhibitory effect of ipriflavonc (table 1). 3.2. Effect ¢~j"ipriflat'one on 45Ca-'* influx in UMR-106

cell.~ A chronic treatmcnt with ipriflavo:~e dccre:,.sed 45Ca-" influx in UMR-10(~ cells. This effect was significadt only aT a concentration of ill ~M (fig. 5J. A single addition of [Asu~'V]eel-caleitonin (ISF, Milan, Italy) reduced ) : C a : ' inqux to a similar extent (fig. 5). Convcrs~:ly, a singlc or repeated trcalmcnt ",:'it!,. !7~estradiol did not modify the uptake oi external Ca 2 ' in this cell line (table 2).

4. Discussion lprifiavonc has been sho~n to affect bone metabolism and to act directly on the osteoblast as :d,,own by an incrcase of alkaline phosphatase activity and an inhibition o[ PTH-stinmiated cAMP .:wcun ~' ~tiox~ in LiMR-:0(~ cells (genvcnuti ct al., 1091). The. forrncr etfcc~ i,- potentiated in thc presence of t.25(OHtzI).~ (P;,:.~i ct al.. 1991)), suggesting a possible modulation by ipriflavone of the stimulz,,tory action of other hormones on ostecblasts The present data confirm a direct action of iprif!avone on the ostcob~ast and demonshatc that this compound can afiect different in'~racettutar second messengers, The inhibitory effect o1 ipriflavone on phosphoinositide tr:drots:

Ipriflavone inhibits phosphoinositide hydrolysis and Ca2+ uptake in the osteoblast-like UMR-106 cells.

The mechanism of action of ipriflavone, an isoflavone derivative, was studied in the osteoblastic-like UMR-106 cell line. Ipriflavone affected both ph...
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