Growth Effects of Some Platinum(II) Complexes with Sulfur-Containing Carrier Ligands on MCF7 Human Breast Cancer Cell Line upon Simultaneous Administration with Taxol

The platinum (II)complexes, cis-[PtCl2(CH3SCH2CH2SCH3)] (Pt1), cis-[PtCl2(dmso)2] (dmso is dimethylsulfoxide; Pt2) and cis-[PtCl2(NH3)2] (cisplatin), and taxol (T) have been tested at different equimolar concentrations. Cells were exposed to complexes for 2 h and left to recover in fresh medium for 24, 48 or 72 h. Growth inhibition was measured by tetrazolium WST1 assay Analyses of the cell cycle, and apoptosis were performed by flow cytometry, at the same exposure times. The IC50 value of each platinum(II) complex as well as combination index (CI; platinum(II) complex + taxol) for various cytotoxicity levels were determined by median effects analysis. MCF7 cells were found to be sensitive to both Pt1 and Pt2 complexe These cisplatin analogues influenced the cell growth more effectively as compared to cisplatin. Cytotoxic effect was concentration and time-dependent. Profound growth inhibitory effect was observed for Pt1 complex, across all its concentrations at all recovery periods. A plateau effect was achieved three days after treatment at Pt1 concentrations ≤ 1 μM. Pt2, however, decreased MCF7 cells survival only for the first 24 h ranging between 50-55%. Pt2 cytotoxicity sharply decreased thereafter, approaching 2 h - treatment cytotoxicity level. The median IC50 values for Pt1 and Pt2 were similar (0.337 and 0.3051 μM, respectively) but only for the first 24 h. The IC50 values for Pt1 strongly depend on the recovery period. On simultaneos exposure of cells to taxol and platinum(II) complexes no consistent effect was found. The Cls for combinations of taxol with Pt1 or Pt2 revealed cytotoxic effects that were in most Cases synergistic (Pt1) or less than addtiive (Pt2). Flow cytometry analysis has shown that each platinum(II) complex induced apoptosis in MCF7 cells. The level of apoptosis correlated with cytotoxicity level for the range concentrations. Both cisplatin analogues, at IC50 concentrations, increased the number of MCF7 cells in G0G1 phase of cell cycle. Pt2-treated cells remained arrested in G0G1 phase up to 72 h after treatment. Combination of Pt2 and taxol caused further arrest of cells in G0G1 phase (24 h) in parallel with strong decrement of G2M phase cells.


INTRODUCTION
Cisplatin, cis-[PtCl2(NHs)2], is a widely used antieaneer drug. The drug's greatest therapeutical impact was found to be on testieular and ovarian cancers/1/. It has also proved to be of benefit in the treatment of wide variety of other solid tumors (head and neck, lung, bladder, eoloreetal and breast cancer) in combination chemotherapy regimens. Because of its severe toxicity profile and the spontaneous development of drug resistance in tumors, numerous Pt(II) and Pt(IV) complexes have been synthesized and tested for antitumor activity/3/. The principal goal of these investigations is obtaining an antitumor drug with higher solubility, better antitumor activity, and lower toxicity. However, at present, only several platinum(II) complexes (earboplatin and oxaliplatin) have shown substantial antieaneer activity in clinical settings. Despite this progress, the search for other platinum chemotherapeutic agents is continuing, since the carboplatin and other second-generation platinum(ll) complexes, although less toxic than cisplatin, appear to be highly crossresistant with cisplatin.
The great majority of the second-generation antitumor active platinum complexes are structural analogues of cisplatin with two ammine or amine groups in cis-position. The presence of the N-H groups on the platinum antitumor active complex is likely required for a hydrogen bond donor function, although steric effects cannot be excluded a priori/3/. But, most important is that new compounds should lack crossresistance to cisplatin and carboplatin/4/, it is already known that this requirement can be reached by using non-ammonia ligands. Cisplatin and taxol are highly suited for combination chemotherapy since they have distinct mechanisms of action/5/. Taxol is used in combination with cisplatin in treatment of metastatic breast cancer patients who have experienced resistance or refractory effects to anthracyclines /1,6/. Taxol, unlike other common antimicrotubule agents, promotes microtubule assembly (enhances the polymerization of tubule) and thus disrupts the dynamic equilibrium within the microtubule system. Due to that, cells are blocked in the late G2/M phase of cell cycle followed by inhibition of cell proliferation.
The aim of the study was to examine the effects of two cisplatin analogues with sulfur-containing carrier ligands on the growth, induction of apoptosis and cell-cycle parameters of MCF7 human breast cancer cell line. The platinum(ll) complexes, cis-[PtCI2(CHaSCHCH2SCH3)] (Ptl) and cis-[PtCl(dmso)z]) (Pt2), were analyzed as single agents or simultaneously with taxol at equimolar concentrations.

EXPERIMENTAL Chemicals and Materials
Distilled water was demineralized and purified to a resistance greater than 10 Mr) cm. The compounds K2[PtCI4], dimethylsulfoxide (dmso) were obtained from Aldrich Chemical Co. and used without further purification. The ligand CH3SCH2CH2SCH3 was prepared by adding two equivalents of CH3I to the corresponding sodium salt of dithiol ligand in water solution/7/.
All common chemicals were of reagent grade.
Cell line MCF7, human breast adenocarcinoma, estrogen receptor positive (ER+) cells grow as monolayer in Dulbecco's modified Eagle's medium (DMEM). with 4.5% of glucose, supplemented with 10% of fetal calf serum (FCS, NIVNS) and antibiotics: 100 IU/ml of penicillin and 100 lag/mg of streptomycin (ICN Galenika). Cells were cultivated in flasks (Costar, 25cm2) at 310 K in the atmosphere of 100% humidity and 5% of COz (Heraeus). Exponentially growing viable cells were used through the assays. The viable cells were determined by dye exclusion test (DET) with trypan blue/8/.

Platinum Complexes
The complexes cis-[PtC12(CHsSCH2CH2SCH3)] and cis-[PtCl2(dmso)2] were prepared from K2PtCI4 according to literature procedures /9,10/. The purity of these complexes was checked by elemental microanalyses and 1H NMR measurements. The complexes were tested alone or simultaneously with taxol at equimolar concentrations ranging fi'om 10 -4 to 10 -8 M.

Drugs
Commercially available solution for i.v. administration of cisplatin, and taxo! (Ebewe, Austria), served as stock solution. The range of drugs concentrations, 104-10-9 M, were used, in order to define ICso concentration for particular time point. The substances of adequate concentrations were added in volume of 10 tL/well.

WST1 assay
Cytotoxicity was evaluated by tetrazolium colorimetric WST1 assay (Boehringer Mannheim). Exponentially growing cells were harvested and plated into 96-well microtitar plates (Costar) at seeding density of 5 x 103 cells in a volume of 90 lal per well, and preincubated in complete medium at 310 K for 24 hours (h). Tested substances, at twice the required final concentration, in growth medium (10 tl/well) were added to all wells except control. Microplates were incubated for 2 h. After the exposure period medium was I'oi. 9. Nos. [1][2]2002 Growth fl'ects of Some Platimtm(li)Complexes" with Sulfitr-Containing Carrier Ligands changed and cells were left to recover for 24, 48 and 72 h respectively. The wells containing cells without tested substances served as control. Two hours before the end of incubation 10 lal of WST1 solution was added to all wells. Optical density was measured on a spectrophotometer plate reader (Multiscan MCC340, Labsystems) at 492/690 rim. The wells without cells containing complete medium and WST1 only acted as blanks. Inhibition of growth was expressed as a percent of control and cytotoxicity was calculated according to the formula: (1-OD/ODco,t,.o) x 100. The substance potency was expressed as the ICs0 (50% inhibitory concentration).

Flow Cytometry
Cell cycle analysis. Cell suspension (lx106/ml) was treated with ml 0.1% TRITON-X-100 for 5 rain at 277 K, followed by 20 ILtl RNA-ase (1 mg/ml) in PBS, stained with propidium iodide (PI) and analyzed by standard procedure. Flow cytometry was performed on FACS Calibur (Becton Dickinson) flow cytometer. Measurement of apoptosis. Cell suspension (lxl06/ml) was incubated in the dark, in staining buffer containing 20 gL of PI and 20 laL of Annexin-V in HEPES buffer (Annexin-V-FLUOS kit, Boehringer).
After the incubation period stained cells were resuspended in HEPES buffer and analyzed by standard procedure. Flow cytometry was performed on a FACS Calibur (Becton Dickinson) flow cytometer.

Data analysis
The IC50 of platinum(II) complexes and taxol as well as the interaction between them were determined by median effect analysis/11, 12/. The analysis compares the effects of drug combinations to the effects of individual drugs across the entire dose-effect range, indicating if the interaction is synergistic, additive or antagonistic. Data in tables and figures represent the mean of the quadruplicate wells.

RESULTS
This study evaluates two cisplatin analogues, cis-[PtCI2(CH3SCH2CH2SCH3)] (Ptl) and cis-[PtCIE(dmso)2] (Pt2; dmso is dimethylsulfoxide), for their potential to inhibit growth of MCF human Ptl Pt2 breast cancer cell line. MCF7 cells were exposed to platinum(II) complexes alone or simultaneously with taxol for 2 hours.The survival of the cells was evaluated by the end of the treatment or after recovery period of 24, 48 and 72 hours respectively. The effects of drug combinations at the IC25, IC50 and IC75 level were determined by median effect analysis. The ability to induce apoptosis and cell cycle changes were analyzed as well.
MCF7 cells were found to be sensitive to both Ptl and Pt2 complexes ( Figure 2). The cytotoxicity profiles of these two platinum(II) complexes are different but each of them influences the cell growth more effectively as compared to eisplatin at equimolar concentrations ( Figure 3). Survival rate depends on concentration and recovery time. were exposed to complexes for 2 h, 24 h after plating. Medium was changed and cells were left to recover for 24, 48 and 72 h respectively. Cytotoxicity was evaluated by tetrazolium WST1 assay at indicated time points. Bars represents mean of quadriplicate wells.
A profound growth inhibitory effect was observed for Ptl complex, across all its concentrations at each recovery period. Prolonging the recovery time from 24-72 h increased cytotoxicity of Ptl, reaching plateau The increased survival of MCF7 cells was noticed for both complexes above particular concentrations (> laM for Ptl and >0.1 tM respectively). In order to check out whether the number of surviving cells was really increased or there was only change in the cell metabolic activity, DET assay was performed under the same experimental conditions. The results obtained highly corresponded to those of tetrazolium assay. The linear correlation coefficients were rptl 0.9361 and rpt2 0.9086 respectively (data not shown).
Combinations of platinum(II) complexes and taxol were analyzed by median effect method, primarily under the assumption that drug mechanisms of action were mutually, nonexclusive i.e. were completely independent. A drug combination index (CI) was calculated for three different levels of cytotoxicity (25; 50 and 75%). On simultaneous exposure of cells to taxol and platinum(II) complexes no consistent effect was found. The CIs for drug combinations revealed cytotoxic effects that were in most cases synergistic (Pt l) and less than additive (Pt2). Median effect analysis showed that interaction of platinum(If) complexes and taxol varies, depending not only on the type of complex, but also on the cytotoxicity level (Figures 4 and 5).
It is well known that cisplatin can induce 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 [5,13,14]. We examined whether platinum(If) complexes can induce apoptotic cell death in MCF7 cells. Flow cytometry analysis has shown that each platinum(II) complex induced apoptosis in MCF7 cells. Prolonging the exposure time of cells to platinum(II) complexes, at IC50 concentration, from 2 to 24 h, increased the total number of apoptotic cells. Both Ptl and Pt2 induce higher apoptosis level at lower concentrations during the first 24 h post treatment that correlates with cytotoxicity profile of the complexes (data not shown). Medium was changed and cells were left to recover. Survival was evaluated by tetrazolium WST1 assay at indicated time points. Bars represents mean of quadruplicate wells.   (Table 1). Although cisplatin is cycle-phase nonspecific, cells treated with cytotoxic cisplatin concentrations may remain arrested at one or more steps of the cell cycle for up to several days prior to cell death/15/. Both platinum(II) complexes also induced cell cycle perturbations. Cells treated at IC50 concentrations of Ptl and Pt2 accumulated in G0G phase of cell cycle. Ptl transiently (24 h) increased the number of cells in S phase of cell cycle. Pt2-treated cells remained arrested in G0G up to 72 h post exposure. Taxol inhibits cell cycle traverse at the G2M phase junction/16/. It was found that Pt2 in combination with taxol caused further arrest of cells in G0G phase (24 h) in parallelwith strong decrease of cells in G2M phase cells ( Table 2). ND ND Platinum(lI) complexes were applied at IC50 concentrations. The DNA content was analyzed by propidium iodide. Data are given as a percent of cell number at particular cell cycle phase.

DISCUSSION
In this paper we report on in vitro results of antitumor activity, of some non-classical platinum(II) complexes against human breast cancer cell line. This study showed that two platinum(ll) complexes, Ptl and Pt2, with sulfur-containing carrier ligands, strongly inhibited growth of MCF7 cells in a dose and timedependent manner. They also induced apoptosis and cell cycle changes in treated cells. The interaction of platinum(ll) complexes and taxol varied depending on the type of complex and the cytotoxicity level.
The tested platinum(ll) complexes are cisplatin analogues. It is recognized that manipulation of the structure of the leaving groups appears to influence tissue and intracellular distribution of the complexes, but upon interacting with DNA, the stable, carrier groups presumably determine the structure of the adduct. The ultimate aim of the modifications of the parent drug is to make analogues that produce a different spectrum of DNA lesions and so circumvent the problem of resistance to cisplatin/1/. It seems that differences related to carrier ligands influence both types and frequencies of DNA lesions formed, and consequently, various growths inhibiting activity could be expected.
Cis-configuration of the tested platinum(ll) complexes was identified as potentially critical for antineoplastic activity. They are platinum(ll) structures assuming planar shape. Both of them have chlorine atoms for so-called leaving groups. The "carrier" ligands are cyclic moieties in Ptl, but not in Pt2 complex. It is already known that structural difference of the carrier ligand may greatly alter the spectrum of antitumor activity of platinum(ll) complexes. The carrier ligand of Ptl comp,lex consists of two thioether (-SCH3) groups and its chelation to the Pt center forms a very stable five-member ring. This ring contributes to higher stability of Ptl than the corresponding Pt2 complex. Difference in the stability between these two complexes can also be correlated with difference in their toxicity. The carrier ligand of Pt2 complex consists of two molecules of dimethylsulfoxide. The lower toxicity of Pt2 might be attributed to its faster detoxification. As data on pharmaeokinetics and pharmacodinamics of the Ptl and Pt2 complexes have not been available so far, we can only speculate on their structure-related activity assuming that, as cisplatin analogues, cytotoxic mechanism(s) similar to that of the parent drug could be expected.
The differences among the complexes were also found when the effects of combination with taxol were studied. Various kinetic of apoptosis-induction and cell cycle changes, induced by individual platinum(II) complex, can explain, in part, resulting differences of drug combinations. Platinum(II) complexes, especially Pt2, induce apoptosis earlier and arrest cells in G0G phase of cell cycle. Taxol inhibits cell cycle traverse at the G2M phase junction/16/. By arresting cells in G0G phase of cell cycle Pt2 inhibited both taxol-induced mitotic arrest and apoptotic death. So, less than additive cytotoxic effect of Pt2 and taxol combination corresponds to early and strong arrest of cells in G0G phase by Pt2.
We want to point out that the interaction of taxol and Pt-complexes was evaluated only on simultaneous exposure of MCF7 cells to the drugs. It is known that interaction of taxol and cisplatin is highly scheduleand cell-dependent/17,18/. Kano et al. found that on sequential exposure to paclitaxel first, followed by eisplatin, additive effects were observed in different cell lines including MCF7 cells/17/. On simultaneous exposure to the drugs additive and subadditive effects were obtained in A549, MCF7 and PAl cells. Our results with Ptl and Pt2 complexes at least in part correspond with their results. Different mechanisms by which eisplatin may exert dominance over taxol, suggested in some studies/5/, must be kept in mind as well as when analysis of Pt2 and taxol interaction is concerned.

CONCLUSION
This study showed that two Ptl and Pt2 complexes containing thio ligands influence the MCF7 cells growth more effectively as compared to the parent drug. However, they differ in their cytotoxicity profiles and in their interaction with taxol as well. The cell cycle changes and induction of apoptosis in MCF7 cells implicate a programmed cell death pathway in cell-killing.