YU ISSN 0354-7310

UDC 616-006

OFINSIruTEOF ONCOLOGY

SREMSKA KAMENICA, NOVI SADYL]GOSLAVIA

7t4Archive of Oncology 1999

rVisit the Journal's World Wide Web site at

http: //WWW.ONK.NS.AC.YU.htto: //WWW.ONCO-AID.ORG.\U.

trln aitro analyses of antiproliferativeactivity of novel platinum(ll)complexes with Sulfur-containingligands 0n MCFT human breast cancercell lineVesna KO]ICGordana BOGDANOVICTatjana SRDICDimitar TAKIMOVZivadinD, nUCnnOCMilob l. DURANDr agana C ETOE VIC -SIMINMirjana BALTiCVukadin LEOVAC

Original articleLrDC: 618.19-006:512.055.1:it6.i5i. i

Vesna KOJIC'Gordana BOGDANOVIC,Tatjana SRDIC'Dimitar JAKIMOV'Zivadin D. BUGARaIC,Milo5 I. DURAN'Dragana CETOJEVIC-SIMIN,Mirjana BALTIC'Vukadin LEOVAC,

1NSTITUTE OF ONCOLOGY SREMSKA KAMENICA, NOVI SAD,SREMSK{ KAMENICA, YUGOSLAVIA,FACULTY

OF SCIENCE, DEPARTMENT OF CHEMISIRY, UNIVER-SITY OF KMCUJEVAC, YUGOSLAVIA,FACULIY

OF SCIENCE, DEPARTMENI OF CHEMISTRY, UNIVER.SITY OF NOVI SAD, YUGOSLAVIA

C)

v

--r

remska Kamenica, Noai Sad, yugoslauia

ln uitro analyses of antiproliferativeactivily of novel platinum(Il) complexeswith Sulfur-containing ligands onMCFT human breast cancer cell line

Cbsfnacr,:,, I,::B^a7kground: The effects of two noael cisplatin analogues: cislpt(ClSCH2CH1SCHIrC;-

lPL:yd cis-[Pt(DMSo)rCl2I @t) on growth, induction if apoptosis and cell-cycle ltarnirt:::; .-MCFT humanbreost cancer cell line analysed alone or simultaneousty with Taxol are presoi;,;. :,..

this paper

M at eri al and tneth o d s : The plntinum(il) comprexes, cis-dichroro-1,2-bis(methylthio)ethaneplatinum(Il), (Ptl) and cis-dichlorobis(dimethylsulfoxide)platiturur1171, rp,.)cisplatin(cisPt) and Taxol (T) hatte been tested at different equimolai concentrations" Cells irer;exposed to complexes t'or 2h and let't to recooer in fresh medium t'or 24h, 48h or 72h, Groiuth irtltii-:-tion was messuredby tetrazoliumWsTl assay. Analyses of the cell cycle and apoptosis u)ere pL1-

form_ed by flow cytometry, at the same expostffe times, Thi lCuoaatui of each p:t conrylexas i( r.r, ,; jcombination index (Cl; Pt complex + Taxol) t'or uarious cytotoxicity leaels were detennined bu rr:;,i:-an elfecls analysis.

Results: MCFT cells uere t'ound to be sensitiae to both Pt complexes. The noael analogues irfi:r-enced the cell growth more effectioely as compared to Cisplatin. Cytotoxic effect was cotrcatrrttiot:ancl time-dependent, Profound growth inhibitory effect was obseroed t'or Pti complex, across its ttiiconcentrotions at all recottery peiods. A plateau effect wqs achieoed three days at'ter the treatnmi.;.:at PtL concentrstions S lpM. Pt2 , howeoer, decreased MCFT cells suntioal oity fo, the t'irst 2lhrangingbetween 50-55%' Pt2 cytotoxicity sharply decrensed thereafter, approaiiing zh - treatlrcntcytotoxicity level. The ffiedian lC50 values for pt1 and pt2 were similar (O.\SZ anA 0,30511114, ,rrprr_tiaely) but only t'ot the first 24h. The lC56 aalues for PtL stuongty depend on the recooery peiort, Onsimu,Itaneous exposure of cells to Taxol nnd Pt complexes no consisi;tent et'fect was t'ound. Tlrc CIs f orcowbinations of Taxol with-Ptl or Pt2 reuealed cytotoxic elfects that wiie in most cases synergistic(Ptl) or less than sdditioe (PtZ). Ftow cytometry analysis has shown that each pt complex incltrcelnpoptosis in MCFT cells' The lettel of apoptosis correlated zuith cytotoxicity leoel t'or the range con-centrqtions. Both Pt complexes, at lCq6 concentrations, increased the n *iq of MCFT cells in Gt)G1phase of ceII cycle' Pt2-treated cells remqined arrested in GsGl phase up to 72 h after the treatntent.Combination of Pt2 and Taxol caused further arrest of cetls in GsGl phase (2afi parallel with strongdecrement of G2M phase cells"

Conclusion: This study showed that hao noael Pt complexes containing sulfur thio ligandsinfluenu the MCFT cells growth more effectioely as compared to the parent irug. They diffir howe,-er, in their cytotoxicity prot'iles and in their interaction with Taxol as well, Thi ull cycti'ctrangesand induction of apoptosis in MCFT celts hnplicate a programmed cell death pathuay in celt-iiling,

Key zoords: Platinum complexesl CerI growth; fitoptosis; Ceil cycle; Breast cancer ceg line

Addr ess carr e spo nrience to :

Vesna Koirc. lnstilute al Oncology Srentsh Knmenica,Noui iad., Instrtulski put q,21[al Sremskn Knnrcnrca,Yrgoslaaui

Tlrc ruaruLscript uas receiaed: 25.11.1999.

Proaisionally accEted: 01.12.1999.

Acceated t'or ptLbltcation: 13.12.19gg.

(0 1999, institute of oncologv rn Sremska Kamenica, yugoslavia

*rix,

Clsplatin, cis-[PtC12(NH3)2], is a widely usedanticancer drug. The drug's greatest therapeuti-cal impact was found to be on testicular andovarian cancers (1). It has also proved to be ofbenefit in the treatment of wide variety of othersolid tumors (head and neck, lung, bladder, col-orectal and breast cancer) in combinationchemoiherapy regimens. Because of its severe

toxicty profile and the spontaneous develop-

ment of drug resistance in tumors, numerous pt

(ll) and Pt (lV) complexes have been sr.nthe-sized and tested for antitumor actrvrtr. tir Th.principle goal of these investigations rs obtain-ing an antitumor drug with higher solubilrtr,,,better antitumor activity, anci iower torrcitr.However, at present, only severai platinumtlilcompiexes (Carboplatin and Oxaliplatin) haveshown substantial anticancer activity in clinicajsettings. Despite this progress, the search forother platinum chemotherapeuiic agents is con-tinuing, since the carboplatin and other second-

153

Koji6 V.

generation platinum(ll) complexes, although

less toxic than cisplatin, appear to be highly

uoss-resistant with cisplatin.

The great majority of the second-generation

antitumor active platinum complexes are sttuc-

tural analogues of cisplatin with two amine or

amine groups in cis position. The presence ofthe N-H groups on the platinum antitumor

active complex is 1ikely required for a hydrogen

bond donor function, although steric effects can-

not be excluded a priori (3). But, most importantis that new compounds should lack cross-resis-

tance io cisplatin and carboplatin (4). It is

already known that this requirement can be

reached by using non-ammonia ligands.

Crsplatin and Taxol are highly suited for

combination chemoiherapy since they have dis-

tinct mechanisms of action (5). Taxol is used incombination with cisplatin in treatment ofmetastatic breast cancer patients who have

experienced resistance or refractory to anthracy-

clines (1,6). Taxol, unlike other common antimi-

crotubule agents, promotes microiubule assem-

bly (enhances the polymerization of tubule) and

thus disrupts the dynamic equilibrium withinthe microtubule system . Due to that, cells are

blocked in the late G2lM phase of cell cycle fol-

lowed by inhibition of ce11 proliferation.

The aim of the study was to examine the

effects of iwo cisplatin analogues with sulfurligands on the growth, induction of apoptosis

and cell-cvc1e parameters of MCFT human

breast cancer cell line. The platinum complexes:

cis-[Pt(cH3scH2cH2scH3)cl2] (Pt1) and crs-

[Pt(DMSO)2C12]) ft2) were analysed as single

agents or simultaneously with Taxol at equimo-

lar concentrations.

:,,,.,:..i I m.Ei il i[:filrffil-$ffiiiiiiiiiiiii:.ii.:..r:

lVIETHO.DS

CELL LINEMCF7, human breast adenocarcinoma,

estrogen receptor positive (ER+) cells grow as

monolayer in Dulbecco's modified Eagle's

medium (DMEM ) with 4.5% of glucose, sup-

plemeted with 10% of fetal caif serum (FCS,

NNNS) and antibiotics: 100 IU/m1 of penicillin

and 100 pglmg of streptomycin (lCN Gale-

nika). Cells were cultivated in flasks (Costar,

25cm') at 37"C in the atmosphere of 100%

humidity and 5% of CO2 (Heraeus). Expone-

ntially growing viable cells were used through

the assays. The viable cells were determined by

dye exclusion test (DET) with trypan blue (7).

TESTED SUBSTANCES

Synthesis and characterization of platinum

complexes

Distilled water was demineralized and puri-

fied to a resistance greater than 10 MO.cm. The

0 1999, Institute of oncology in Sremska Kamenica, Yugoslavia

compounds K2[PtCla], dimethyl sulfoxide(DMSO) were obtained from Aldrich Chemical

Co. and used without further purification. The

ligand CH3SCH2CH2SCH3 was prepared by

adding of hvo equivalents of CH3J to the corre-

sponding sodium salt of dithiol ligand in water

solution.

Al1 common chemicals were of reagent

grade.

The complexes crs-[Pt(CH3SCH2CH2SCH3)Cl2]

and cls-[Pt(DMSO)2C12] were prepared from

K2PtC14 according to literature procedures (8, 9).

The purity of these complexes was checked by

elemental microanalyses and 1H NMR measure-

ments.

cls-[Pt(CH3SCH2CH2SCH3)C12]

This complex was prepared by adding of

0.0785 g (6.42 . 10'mol) of CH3SCH2CH2SCH3

to a solution containing 0.266 g(6.42. 10*- mol)

of K2PtCia in 15 ml of water. The brown mixture

was stirred at room temperature for 3h. The

resulting light-yellow solution was left in refrig-

erator and stood overnight. The crystalline pow-der was filtered off, washed with ethanol, and

ether and then dried in vacuo for 10h. Yield

0.186 g or 74.6 l'kl. Anal. Calcd for cis-

[PI(CH3SCH2CH2SCHrC12l = CaHroSzClzPt

(FW = 388.15): C, 12,38; 11, 2.60, Found: C, 1 1.47;

H,2,54[',/,).

cls-[Pt(DMSO)2CI2]

This complex was prepared by adding of

0.009 mol of the dimethyl sulfoxide to a solution

containing 0.003 mol of K2PtCla in 10 ml ofwaier. The mixture was allowed to stand at

room temperature until ye11ow crysials precipi-

tated. The complex was filtered, washed withwater, ethanol, and ether, and dried in vacuo for

4 h. Anal" Calcd for cls-[Pt(DMSO)rC121 =C4H1 2S2O2C1rP t iFW = 422.24) : C, i 1.38; H, 2.86.

Found: C, 71.44; H, 2.77 ["/,).

Elementai microanalyses were perforrned

by the Microanalytical Laboratory, Faculty of

Chemistry, Universiiy of Belgrade and

Department of Chemistry, Iowa State University(USA). Al1 necessary pH measurements were

made at 22"C. The pH meter (lskra MA 5704)

H"C

-- s. ,clI \/zlPtl-- ,/ \sctH"c/

H.C.

s' ctl"c' \ ,'/-PrH"C ,/ \' .s, .clH.c"'

P12

Figyr.q 1, Platinum([):complqxeq,usd,iin, lhis,studi

was calibrated with Fischer certified buffer solu-

tions of pH 4.00 and 7.00. Proton NMR spectra

of D2O solutions containing TSP (3trimethylsi-lylpropane-1-sulfonate) as internal reference

were recorded with Varian Gemini 200 spec-

trometer (8,9).

The complexes llere tested alone t-rr sinui:a-neouslv with Taxol ai equimolar concentrations

ranging from 10{ to 10'M.

Drugs

Commercially a',,ailable solution for i. r'

administration of Cisplaiin, and Taxol (Ebeive,

Austria), served as stock solution. The range of

drugs concentrations, 104-10o M, were used, in

order to define IC56 concentration for particular

time point, The substances of adequate concen-

trations were added in volume of 10 ttl/we1l.

WST1 ASSAY

Cytotoxicity was evaluated by tetrazolium

colorimetric WST1 assay (Boehringer Mann-

heim). Exponentially growing cells were har-

vested and plated into 96-wel1 microtitar plates

(Costar) at seeding density of 5 x 103 ce1ls in a

volume of 90pl per well, and preincubated incomplete medium at 37oC for 24 hours (h).

Tested substances, at twice the required finai

concentration, in growth medium (10 pi/we1l)

were added to all wells except control.

Microplates were incubated for 2 h. After the

exposure period medium was changed anci cells

were left to recover for 24, 48 and 72 h respec-

tively. The wells containing cells wrthoui tested

substances served as control. Two hours before

the end of incubation 10 pl of WST1 solution

was added to all wells. Optical density was mea-

sured on a spectrophotometer plate reader

(Multiscan MCC340, Labsysiems) at

4921690nm. The wells without cells containinq

complete medium and WST1 onlv acted as

blanks. lnhibition of growth was expressed as a

percent of control and cytotoxicity was calculat-

ed according to the formuia:(1-ODrestloDControt) x 100. The substance

potency was expressed as the ICE(, (509i,

inhibitory concentration).

FLOW CYTOMETRY

Cell cycle analysis

Cell suspension (1x106/ml) was treated with1ml 0. 1% TRITON-X-1O0 for 5 min at 4oC, fol-

lowed by 20 pl RNA-ase (1mg/ml) in PBS,

stained with propidium iodide (PI) and ana-

lyzedby standard procedure. Flow cytometry

was performed on FACS Calibur (Becton

Dickinson) flow cytometcr,

Measurement of apoptosis

Cell suspension (1x10'/ml) was incubated

in dark, in staining buffer containing 20 pL of PI

and 20 pL of Annexin-V in HEPES buffer(Annexin-V-FLUOS kit, Boehringer). After the

incubation period stained cells were resuspend-

ed in HEPES buffer and analyzed by standard

procedure. Flow cytometry was performed on

FACS Calibur (Becton Dickinson) flow cytometer,

t

I

154

C) .ltttiproliintttite actit'ity and platinunt(ll) complexes uith sult'ru ligands

D.{TA A\ALYSISThe IC56 of Pt complexes and Tarol as rr'e1i

a: the rnteraction betu'een them tr'ere deter-

mined bi' median effect analysis (10, 11). The

anallsrs compares the effects of drug combina-

ii..ns to the effects of individual drug across the

enirre dose-effect range, indicatlng if the interac-

rrrn is svnergistic, additive or antagonistic. Data

Ln tables and figures represent the mean of the

quadruoie wel1s.

Ali calculations were performed with the

PC siatistical computing package.

REST.ILTS

Ihis study evaluates iwo novel cispiatin

anaiogues for their potential to inhibit growth of

\lCF human breast cancer cell line. MCFT cells

rvere exposed to platinum complexes alone or

srmultaneously with Taxol for 2 hours. The sur-

vivai of the cells was evaluated by the end of the

n'eatment or after recovery period of 24,48 and

72 hours respectively. The effects of drug com-

brnaiions at ihe IC25, iC56 and IC75 level were

cietermined by median effect analysis. The abili-

n'to induce apoptosis and cell cycle changes

lr'ere anaiyzed as well.

1ml

li:.. :aiiing behleen 50-559, The cviotoxiciiv

sharplv decreased thereafter, approaching 2h -

heatment cvtotoricity level. lncreasing the con-

centration above 0.1pM resulted in no addition-

al cytotoxicity resembling Pt1 cytotoxicity pat-

tern. The IC5g values for Pt1 and Pt2 are similar,

but only for the first 24h (0.337 and 0.3051pM,

respectively). The IC56 values for Pt1 decreased

by recovery period for the factor 10.

Figure 3. Compiiiion ot growth inhibitory effeii oiPi1 ind Pt2 complex to cisplatin at 72h and 2ah post

;;.,;:,;.,,;;;;; of MCFT ce,,s were

noticed for both complexes above particular

concentrations (>1pM for Pt1 and >0.1pM

respectively). In order to check out whether the

number of survived ce11s was really increased or

there was only change in the cell metabolic

activity, DET assay was performed under the

same experimenial conditions. The results

obtained highlv corresponded to those of tetra-

zolium assay. The linear correlation coefficients

were rptl=0.9361 and rptz=0.9086 respectively

(data not shown).

Combination of Pt-complexes and Taxol

were analyzed by median effect method, pri-

marily under the assumption that drug mecha-

nisms of action were mutually nonexclusive i.e.

were completely independent. A drug combina-

tion index (CI) was caiculated for three different

levels of cytotoxicity (25;50 and 75%). On simul-

taneous exposure of cells to Taxol and Pt com-

plexes no consistent effeci was found. The CIs

for drug combinations revealed cytotoxic effects

that were in most cases synergistic (Pt1) and less

than addrtive (Pt2). Median effect analysis

showed that interaction of Pt-complexes and

Taxol vary depending not only on the type of

complex, but also on the cytotoxicity level

(Figure 4).

It is well known that cisplatin can lnduce

apoptosis in various cells, in any cell cycle

phase, as a function of drug concentration and

exposure duration, but a period of cell cycle

"stasis" precedes the onset of apoptosis (5, 12,

13). We examined whether Pt complexes can

-.-rc -r-Tdd 1-PE+Tdol

"-"":T:.X:*,,":" :;H; ";,"."".;;;;;* ---

Figure 4. Effects of Pt complexes,alone and in combi-

naflon with Taxol on MCFT cell line survjval, deter-

mined,24h, 48h and TZh pgqt tieatment 2{h after

piating oeiis {5i1ff) were exposed to com:plexes ior

2h: Medium'wis,, ctr;n*.d and, Cetls were left to

recover. :Suivival was evaluated by tetrazolium

WSII assay atindiiated trme poinis Bars rePresents

mean of quadruple welts,,:::induce apoptotic cell death in MCFT cel1s. Flou

cytometry analysis has shown that each Pt com-

piex induced apoptosis in MCFi cells.

Prolonging the exposure iime of cells io Plcom-

plexes, at IC56 concentration, from 2 to 24h,

increased the totai number of apoptotic cells.

Both Pt1 and Pt2 induce higher apoptosrs ievei

at lower concentrations during the first 24h-po,"t

treatment that correlates with cytotoxicitv pro-

Table 1, Time-related effect of Pi complexes ort

induction of apoptosis rn MCFT cells Pt complexes

wue applied at ICig ioncenkations. The apoptosis

was analyzed Uy nnnexin-V ruUOS assay. Data are

given as a percent,of ap6rplobc iell number

;.;o*,*,r,o not shown)

Taxol alone induced strong apoptotic

response (38.22"1,), while in combination with

Pt2 under the same experimental conditions,

total number of apoptotic cells deueased

(30.25%). Pt1 induced higher total number of

apoptotic cells than Pt2, (24.15 and 13.88 respec-

tively ) at 24h post treatment but in all other

time points the values were similar (Table 1.)

8e'E;{

Pt1

\,

:!eoE:

-.-r.z* |

!

0., ,

Figure 2. Effect of Pt complexes on MCF/ ceil line

survival. 24h afterplating, celli (5x10) were exposed

io Pt complexes,,{or 2h' Medium whs changd and

cells were left to reiover fot 241;"48 and V''lh rcspec-

nvely. Survival wai,evaluated by tetrazolium WSTI

assay at indicated hme pointsr'Bars repres€nts mean

of ouadruple weils. ::

MCFT ceils were found to be sensitive to

both Pt1 and Pt2 complexes (Figure 2). The cyto-

toxicity profiles of these two novel Pt complexes

are different but each of them influences the ce1l

growth more effectively as compared to cis-

platin at equimolar concentrationsn (Figure 3).

Surr,ival rate depends on concentration and

recovery time.

A profound growth inhibitory effect was

observed for Pi1 complex, across its al1 concen-

trations at each recovery period. Prolonging the

recovery time from 24-to72h increased cytotox-

icity 0f Pt1, reaching plateau 72h post treatment,

at concentrations <1pM. Howevet, PtZ

decreased MCFT cells surv'ival only for the first

O 1999, Institute of oncology in Sremska Kamenica, Yugoslavia

155

i. -:r. 1-.

-..:: -.::. ,-.si:1atrr, rs ci cle-phase nonspecrf-

- -.. -. ::.::ed 'rr-rth ct'toiovc cisplatin concen-

-r::-,-.: ::.a.. remain arrested ai one or more

,:.:. : : ::.e cell cvcle for up to severai dar.s prior. :. :eath t1l) Both Pt complexes also

: - -:.i :ell ci'cle perturbations. Cells treated at

--: ,--:.entraiions oi Pi1 and Pt2 accumuiaied

, . :ra,.e of ceil o'cle, Pt1 transientlv (24h)

- - ::::i the number of cells in G2M phase of

-: -.,.: Ptl-treated cells remaineci arrested in. . -: 1.. l2h posi exposure. Taxol inhibits cell

. - : irr', €rS€ at the G2M phase junction (15). It,. :. -::.i that Pt2 in combination wiih Taxol

-,,-.,: ::aher arrest of ce11s in GgGl phase (24h)

:,-, .. ,.:th strong decrease of cells in G2M

:-,:.,:,-: fable2).

D I S S Cu Sl.S.I ,N,t:......;.,..,..1iti,i,i 1..t.l.

T::,e I iieits of Pt compleres on DNA content of:, -:- - '-

. : :--,::,:,:re-. were appti,*!,,{ffiCI,,tn.n!ennaiio*st:,

-:. l\-i contrnt, ll,inaiy :i:b,y:ii$ig$iBiii :

...::i.. ]:ta are,pvm.,a5. e1peiCerr1..oi:,4*i.nunitbep;14i.

:::.r.,; iell cvcle phase. ,

.- .,:,.: ::':i1*?3f jlI,lliX',..i}'?Y "

,n this paper we report on in aitro results of

.:.:^:umor activity of some non-classical plat-

:.:nill) complexes againsi human breast can-

-=: cell line. This study showed that iwo Pt

.-:rpiexes containing sulfur ligands strongly

,:::bited growth of MCFT cells in a dose and

::'e-ciependent manner. They also induced

,;;ptosis and cell cycle changes in treated cells.

-:.e rnteraction of Pt-complexes and Taxol var-

..: depending on the type of complex and the

:',':otoxicity level.

The iested Pt complexes are clsplatin ana-

-':ues. Ii is recognized that manipulation of the

::ructure of the leaving groups appears to influ-

=:.;e tissue and intracelular distribution of the

:..r,rp1exes, but upon interacting with DNA, the

.:able, carrier groups presumably determine the

s:ruclure of the adduct. The ultimate aim of the

:rodifications of the parent drug is to make ana-

,.rgues that produce different spectrum of DNA.esions and so circumvent the problem of resis-

iance to cisplatinum (1). It seems that differ-

ences related to carrier ligands influence both

;i-pes and frequencies of DNA lesions formed,

and consequently, various growths inhibiting

acti',,ity could be expected.

O 1999, Institute of oncology in Sremska Kamenica, Yugoslavra

Cis configuration of the tested Pi complexes

u'as identified as potentially critical for antineo-

plastic activity. They are platinum(ll) structures

assuming planar shape. Both ofthem have chlo-

rine atoms for so-called leaving groups. The

"carrier" hgands are cyciic moieties in Pt1, butnot in Pt2 compiex. It is already known that

structural difference of the carrier ligand may

greatly alter the spectrum of antitumor activity

of platinum complexes. The carrier ligand of Pticomplex consists of five-member dng, bound to

Pt atom by two bonds that make complex achelate, and more stable than monodentate ana-

logue Pt2, The carrier ligand of Pt2 complex con-

sists of two molecules of dimethylsulfoxide. The

lower toxicity of Pt2 might be attributed io itsfaster detoxification. As data on pharmacokinet-

ics and pharmacodinamics of the novel Pt com-

plexes have not been ar,ailable so far we can

only speculate on their structure-related activityassuming that, as clsplatin analogues, cytotoxic

mechanism(s) similar to that of the parent drugcould be expected.

The differences among the complexes were

also found r.vhen the effects of combination withTaxol were studied. Various kinetics of apopto-

sis - induction and cell cycle changes induced by

individual Pt complex, can explain, in part,

resulting differences of drug combinations. Pt

complexes, especially Pt2, induce apoptosis ear-

lier and arrest cells in GgCl phase of cell cycle.

Taxol inhibiis cell cycle traverse at the G2M

phase junction (15). By arresting cells in G0G1

phase of cell cycle Pt2 inhibited both Taxol-

induced mitotic arrest and apoptotic death. So,

less than additive cvtotoxic effect of Pt2 and

Taxol combination corresponds to early and

strong arrest of cells in G0G1 phase by Pt2.

We want to point out that the interaction of

Taxol and Pt-complexes was evaluated only on

simultaneous exposure of MCFT cells to the

drugs. It is known that interaction of Taxol and

clsplatin is highly schedule - and cell-dependent

(16,17). Kano et all. found that on sequential

exposure to Paclitaxel first, followed by cis-

platin, additive effects were observed in differ-ent cell lines including MCFT cells. On simulta-

neous exposure to the drugs additive and sub-

additive effects were obtained in 4549, MCFT

and PA1 cells. Our results with novel Pt com-

plexes at least in paft correspond with theirresults. Different mechanisms by which clsplatin

may exert dominance over Taxol suggested insome studies (5) must be kept in mind as wellas when analysis of Pt2 and Taxol interaction is

concerned.

CONCLUSIONThis study showed that trvo novel Pt com-

plexes containing sulfur ligands influence the

MCFT cells growth more effectively compared

to the parent drug. However, they differ in therr

cytotoxicity profiles and in their interaction rvith

Taxol as well. The cell circle changes and induc-

tion of apoptosis in MCFT cells implicate a pro-

grammed cell death pathway in cell-killing

Acknowledgment: The authors wish tothank Prof. Assist, Aleksandar Kerenji for crit-

ical reading of the manuscript and useful sug-

gestions and Mrs Ljiljana Krmpot for excelient

technical assistance^

.....i..i' G .R.ENG:E$.' ,,',,, '..:ri ,.,

1. O'Dwver JP, johnson W, Hamillton C. Cispiaiin and itsanalogues. In: De Vita TV, Hellman JR.S, Rosenrerg A5, eds.

Canccr principies and practice of oncologv. Philadeiphia:

Lipprncott-Raven, 7997 :37 5-572.

2. Bleomink MJ, Reedijk J. In: Sigel A, Sigel H, eds. lletalsions in biological systems. Basel: Marcel Dekker,lnc,

1.996;32:647 85.

3. Lempers ELM, Reedijk j, Interaction of Platinum AminrCompounds with Sulfur-Containing Biomoiecuies and

DNA Fragmenis. Adv Inorg Chem 7992;37.1t'5'217

4. Reedijk |. Improved Understanding in PlatinumAntitumor Chemistry . Chem Comm i996:801-6.

5. Judson LP, Watson MJ, Geluig AP, Forvler CW, iiaskill tS.

Cisplatin hhibits paclitaxel-induced apoptosis rn cisplatur

resistant ovarian cancer cell lines: Possible explanation for

falure of combinahon therapy. Cancer Res 1999j9:2425 32.

6. Cerald K, Evoy MC, Paclitaxel. In: Cerald K, Ei,oy N{C,

eds, Drug information. Amencan Hosprtai FormuiarvService, 1997:841-5.

7. Bogdanovii G, Raletri-Savii J, Markovii N. In vrtro assays

for antitumor-drug screening on human tumor cell lines:

Dve exclusion test and colorimetric cvtotoxicitv assav.

Arclrive of Oncolo gy 199 4;2:181-1,

8. Price J H, Wrlliamson AN, Schramm RF, trVayiand BB.

Palladium(li) and Platinum(ll) Alkyl Suifoxide Complexes.

Examples of. Sulfur-Bonded, Mixed Sulfur- and Oxvgen,

Bonded, and Totally Oxigen-Bonded Complexes. inorgChem i972;11:1280-4.

9. Appleton TG, Bailey AJ, Bedgood DR ft, Hall JR. AminoAcid Complexes of Palladium(ll). 1.NMR Studv of the

Reactions of the Diaqua (ethylendiamine) palladium(ll)

Cation with Ammonia, Betaine and the Amino Acids+NHu(CHf nCOOl (n=1-3) 1 inorg Chem 199 4;33'.217 -26.

10, Perz PR, Codwin KA, Handel ML, Hamilton CT A com-

parison of clonogenrc, microtetrazolium and sulforho-

damine B asseys for determhation of cisplatin cytotoxicitv

in human ovarian carcinoma cell iines. Eur T Canccr

1993;29A(3):395-99.

11. Chou TC, Talalay P. Quantitative analysis of dose -effect

relahonships: the combines effects of muitiple drugs or

eruvme inhibitors. Adv Enzyme Reg 1984;22:27-55.

12. Huddart RA, Titley J, Robertson D, Williams GT,

Honvich A, Cooper CS. Programmed cell death in response

156

'ir danotho-apeutic agmE in human gtrm cell tumouriines, Ew ICarrer 1995,31A(5):E9.16.

13. Hong \'1, Lai I{D. Lin YS, tai MZ. Antagonism of p53-

dependent apoptosis by mitogen signals. Cancer Res

199:592W-57.

1{. Steien iV, Schimke T5, Sc}rimke TR. Induction of apop-

tosis bv cell<vde phase specific drugs. Apoptosis 1994;223-

15. Siur SY, Yue P, Wu GS, El-Deiry WS, Stroot B, Hong WK.'rcun R Implication of p53 in growth arrest and apoptosis

urduced by the synthetic rethoid CD437 in human lung can-

;er cells. Cancer Res 1999;59:282933.

16, Kano Y, Akutsu M, Tsunoda S, Suzuki K, Yazawa Y. In:'itro rhedule -dependent interaction behveen paclitaxel

-.J cisplatin in human carcinoma cell lines. Cancer

flemother Pharm acol 1996 ;37 :525-30.

li. Cordes N, Plasswilm L. Cell line and schedule-depen-

Jert *totoxicity of paclitaxel (Taxol): role of the solvent

Cremophor ELlethanol. Anticancer Res 1998;18:1851-7.

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