Cellular Uptake, DNA Binding and Apoptosis Induction of Cytotoxic Trans-[PtCl2(N,N-dimethylamine)(Isopropylamine)] in A2780cisR Ovarian Tumor Cells

Trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] is a novel trans-platinum compound that shows cytotoxic activity in several cisplatin resistant cell lines. The aim of this paper was to analyse, by means of molecular cell biology techniques and total reflection X-ray fluorescence (TXRF), the cytotoxic activity, the induction of apoptosis, the cellular uptake and the DNA binding of trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] in the cisplatin resistant cell line A2780cisR. The results show that this drug is more cytotoxic and induces a higher amount of apoptotic cells than cisplatin in A2780cisR cells. However, the intracellular accumulation and extent of binding to DNA of trans-[PtCl2(N,N-dimethylamine)( isopropylamine)] is lower than that of cis-DDP. Moreover, trans-[PtCl2(N,N-dimethylamine)(isopropylaminae)] is partially inactivated by intracellular levels of glulathione. The result suggest that circumvention of ciplatin resistance by trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] in A2780cisR cells might be related with the ability of this drug to induce apoptosis.


INTRODUCTION
Cisplatin [cis-diammmedichloroplatinum (II), cis-DDP] is one of the most widely used drugs in the treatment of cancer. It shows remarkable activity alone or in combination with other drugs in the treatment of several tumors, including those of the lung, ovary, testes and bladder [1,2]. The success of cis-DDP as an antiturnor drug has been attributed to different factors, including penetration of the cellular membrane, accumulation in the tumor cell. and efficiency in coordinating with chromosomal DNA [3,4]. The extent of DNA lesions induced by cis-DDP in the cell nuclei and the cell killing potential of the drug is believed to depend on the cellular level of reactive platinum species and the persistence of platinum within the cells [5,6].
Despite the success of cis-DDP against certain types of cancer, the patients treated with the drug suffer from severe side effects including nephrotoxicity, nausea and vomiting, myelosuppression and ototoxicity [2,3]. Moreover, quite often tumors become resistant to cisplatin [7]. The cis-DDP resistance may take place at various levels, including cellular accumulation, interaction with glutathione (GSH) and/or metallothioneins, DNA repair and defective apoptotic program [8].
Although transplatin or trans-DDP (the stereoisomer of cisplatin) is clinically inactive, several classes of trans-platinum complexes show antitumor activity and some of them are able to circumvent cisplatin resistance [9]. Trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] ( Figure 1) is a novel trans-platinum(II) complex with mixed aliphatic amine ligands which show cytotoxic activity in tumor cell lines sensitive and resistant to cisplatin [ 10]. Moreover, trans.
[PtC12(N,N-dimethylamine)(isopropylamine)] induces tumor cell death through apoptosis [ 11 ]. The A2780cisR human ovarian tumor cell line may be considered a model to study cisplatinresistance because it exhibits acquired resistance to cis-DDP from a combination of decreased uptake, enhanced DNA repair/tolerance and elevated GSH levels [ 1.2, 13 ]. The results reported in this paper indicate that trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] is able to overcome cisplatin-resistance in A2780cisR cells through apoptoss induction. Trans-[PtClz(N,Ndimethylamine)(isopropylamine)] induces a higher percentage of apoptotic cells than cisoDDP both in A2780 and A2780cisR cell lines. However, the intracellular accurnulation and extent of binding to DNA of trans-[PtClz(N,N-dimethylamine)(isopropylanine)] ir A2780cisR cells is lower than that of cis-DDP. Interestingly, the cytotoxic activity and the binding to DNA of trans-[PtC12(N, N-dirnethylamine)0sopropylarnine)] in A2780eP eell increases by previous cel!  10]. Stock solutions of the compounds (1 mg/nfl) in DMEM medium (Dulbecco's modified Eagles Medium) were freshly prepared before use. Cell Lines and Culture Conditions. The pair of human ovarian tumor cell lines (A2780/A2780cisR) were cultured in DMEM medium (Dulbecco's modified Eagles Medium) supplemented with 10% FCS (foetal calf serum) together with 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37C in an atmosphere of 95% of air and 5% CO. Drugs Cytotoxicity. Cell death was evaluated by using a system based on the tetrazolium compound MTT which is reduced by living cells to yield a soluble formazan product that can be assayed colorimetrically 14]. E.xnentially growing A2780 and A2780cisR cells [ 15] were plated in 96-well sterile plates, at a densi.ty of 10 cells/well in 100 Ixl of medium, and were incubated for 3-4 hours. Stock solutions of the compounds dissolved in DMEM were added to the wells at final concentrations from 0 to 300 lxM, in a volume of 100d/well. After twenty-four hours of incubation, 50 lxl of a freshly diluted MTT solution (1/5 in culture medium) was added to a final concentration of 1 mg/ml into each well and the plate was further incubated for 5 hours. Cell survival was evaluated by measuring the absorbance at 520 nrn, using a Whittaker Microplate reader 2001. IC50 values (drug concentrations that induces 50% of cell death) were calculated from curves consmacted by plotting cell survival (%) versus compound concentration (tM). All experimems were made in quadruplicate.
Quantification of apoptosis by annexin V binding and flow cytometry. A2780 and A2780cisR cells were exposed to 2xlC50 of the platinum drugs for 24 hours. Attached and detached cells were recovered,, mixed and resuspended in annexin V binding buffer (PharMingen). 2.5 tl of propidium iodide (PI, Sigma) and I tg/ml of annexin V-fluorescein isothiocyanate (PharMingen) were added, and the cells were left at room temperature before flow cytometric analysis in a FACScalibur Beckton-Dickinson apparatus. The percentage of apoptotic cells induced by each platinum drug (percentage of annexin V-positive/Pl negative cells) was calculated from the annexinV/PI scattergrams. ,,fetal Based Drugs Vol. 8, Nr. 1,2001 Measurements ofplatinum accumulation. Cultures plates containing exponentially growing A2780cisR cells in 10 ml of DMEM medium (cell density 2 x 10 cells/ml) were exposed to 50 lxM of the platinum drugs dissolved in DMEM medium for 1, 3 or 24 hours. Cells were washed with ice-cold PBS, scraped and resuspended in 700 [xl of lysis buffer containing 20 mM Tris.HC1, pH 7.5, 2 mM EDTA and 0.4% Triton X-100. incubated at 4C for 15 min and centrifuged at 12.000 rpm for 15 min in a centrifuge. Afterwards, supernatants were treated for 3 hours at 37C with 20 txg/ml ofproteinase K (Boehringer). The platinum content in the samples was determined by TXRF (total. reflection X-ray fluorescence). Experiments were camed out in triplicate.
Determination of platinum binding to DNA in vivo. Culture plates containing exponentially growing A2780cisR cells in 10 ml of DMEM medium (cell density 2 x 10 cells/ml) were exposed to 50 laM of the platinum drugs dissolved in DMEM. The plates were incubated for 1, 3 or 24 hours under the conditions described above. Following drug incubation, culture medium was removed from the plates and the cell plates were washed with PBS. Subsequently, the cells were lysed with 700 lxl of a buffer solution containing 150 mM Tris.HCl pH 8.0, 100 mM EDTA and 100 mM NaCI, incubated for 15 minutes at 4C and centrifuged at 12.000 rpm for 15 min in a microfuge. Supernatants were treated for 3 hours at 37C with 20 lxg/ml of proteinase K (Boehringer). Afterwards, supematants were incubated for 16 hours at 37C with 100tg/ml of RNase A (Boehringer). Finally, DNA was extracted with a volume of phenol-chloroform-isoamyl alcohol (50 + 49 + 1), precipitated with 2.5 volumes of cold ethanol and 0.1 volumes of 3 M sodium acetate, washed with 75% of ethanol, dried and resuspended in 1 ml of water. The DNA content in each sample was measured by UV spectrophotometry at 260 nm in a Shimadzu UV-240 spectrophotometer and platinum bound to DNA was determined by TXRF. Experiments were carried out in triplicate.
Total reflection X-ray fluorescence measurements. The analysis by TXRF was performed using a Seifert Extra-II spectrometer (Seifert, Ahrensburg, Germany). TXRF determinations were carried out according to a procedure previously reported 16]. Briefly, a 100 lal sample of either cell supernatants or cellular DNAs from the cell cultures was introduced in a test tube of 2 ml. This solution was standardised with 100 ng/ml of Vanadium [Merck (Darmstadt,Germany) ICP Vanadium standard solution]. Afterwards, the sample was introduced into a high-purity nitrogen flow concentrator at a temperature of 70C until the vohtme was reduced five times. An aliquot of 5 tl was then taken, deposited on a previously clean quartz-made reflector and dried on a ceramic plate at a temperature of 50C. The entire process was done in a laminate flow chamber (Model A-100). The samples were analysed following the X-ray Molybdenum line under working conditions of 50 kV and 20 mA with a live-time of 1000 s and a dead time of 35%. Spectra were recorded between 0 and 20 keV. The following 15 elements were simultaneously analysed: P, S, K, Ca, V, Fe, Cu, Zn, As, Br, Rb, Sr, Ni. Mn and Pt, in order to obtain a correct deconvolution of profiles associated with the general spectrum. The Pt line was used for Pt quantification. The analytical sensitivity of the TXRF measurements was 0.3 to 22.4 ng Pt in a solution volume of 100 tl, with repeatability between 2 and 8% (n 3). lntracellular GSH content, lntracellular GSH levels were determined, in A2780 and A2780cisR cells growing as specified for the Cytotoxicity tests. Approximately 5 xl05 cells/ml were seeded into P100 plates, and, after overnight incubation, cells were washed twice with ice-cold PBS. Cellular GSH was then extracted using 2 ml of ice-cold 0.6% sulfosalicilic acid followed by a 10 min incubation at 4C. Total GSH content in the extract was then determined according to the method of Griffiths [ 18]. Protein quantification was carded out after solubilization in 2 ml of sodium hydroxide 1M using the Lowry assay [19]. The GSH levels were expressed as nmol/mg protein.
Depletion of GSH levels in A2780cisR cells. A2780cisR cells were pre-exposed for 24 hours to 50 pM of Lbuthionine sulfoximine (L-BSO). This resulted in an approximately 80% reduction in GSH levels 17]. The growth inhibitory effect of the platimm compounds after 24 hours of drug exposure was then determined using the MTT method.  sulfoximine) to decrease the levels of GSH in A2780cisR cells prior to drug-treatment. Our determinations of GSH intracellular content indicated that the A2780cisR cell line possesses about 6-times higher glutathione levels than its parental A2780 cell line (GSH levels: nmol/mg protein, A2780 8.5 _ _ . 025; A2780cisR 50 .+_ 0.8; p<0.01). These data are in agreement with previous data reported in the literature and indicate that the A2780cis R cell line has high intrinsic levels of GSH [ 13]. Interestingly, Table 1 shows that while potentiation of cvtotoxicity in A2780cis R cells was only slight for cis-DDP it was significantly laigh for the two {rans complexes trans-[PtCl(N,N-dimethylamine)(lsopropylamine)] and trans-DDP (p<0.01).
Altogether these results iiadicate that trans-[PtClz(N,N-dirnethylamine)(isopropylamine)] is able to circumvent cisplatin resistance in A2780cisR cells. In addition, the cytotoxcity data obtained in the presence or absence of preoexposure to L-BSO also suggest that as previously found for other trans-platinum conplexes [22, 23], trans-[PtC12(N,N-dimethvlamine ) (isopopvlamine)] may be also more susceptle to inactivation by reaction with GSH {han cIs-DDP.  phase contrast microscopy (data not showaa). Cell detachment has been previously reported as an indication of apoptosis induction [ 11,12]. Both detached and attached cells were mixed and assayed by a flow cytometric annexin V binding assay [24]. Annexin V binds phosphatidyl serine residues that are asymmetrically distributed to the inner plasma membrane but move to the outer plasma membrane early in apoptosis. Figure 2 shows that treatment with trans-[PtCl(N,Ndimethylamine)(isopropylamine)] induced a higher increase in the annexin V-positive/PI negative cell population (right bottom quadrant) than. treatment with cis-DDP or trans-DDP both in A2780 ( Fig. 2: panels B, C and D, respectively) and A2780cis cells (Fig 2. panels F, G and H, respectively) The annexin V-positive/PI negative cell population constitutes the fraction of apoptotic cells [24]. Table II shows the percentage of apoptotic cells induced by the platinum compounds in A2780 and A2780cisR cells as calculated from the scattergrams of Figure 2. As can be observed in Table II, all the platinum drugs induced a higher percentage of apoptotic cells in the A2780 line than in the A2780cisR line. Thus, trans-[PtCl(N,N-dimethylamine)(isopropylamine)] killed most of A2780 cells through apoptosis (91.97 %). Moreover, the percentage of apoptotic A2780 cells induced by trans-[PtC12(N,N-dimethylamine)(isopropylamine)] was two-times and three-times higher than those induced by cs-DDP (45.20%) and trans-DDP (29.26%), respectively. In addition, trans-[PtClz(NiN-dimethylamine)(isopropylamine)]also induced a significant percentage of apoptotic cells in the A2780eisR line (58.38%). This percentage was 1.7-times and 5.0 times higher than those induced by cis-DDP (34.24 %) and trans-DDP (10.59 %), respectively. Altogether, the results indicate that the lower the dose of drug needed to kill the cells the higher the percentage of apoptosis induction [25]. Thus, trans-[PtCl:(N,Ndimethylamine)(isopropylamine)] has a higher ability to induce apoptosis than both cis-DDP and trans-DDP in the pair of cell lines A2780/A2780cisR. Inversely, the data also suggest that the higher the doe of drug needed to kill the cells the higher the percentage of induction of necrosis [25]. Thu, it is interesting to note that trans-DDP induced high percentages of necrosis both n Me.tal Based Drugs VoL 8, Nr. 1,2001 .A2780 (46,08%) grad A27g0ciR cells (60,62%)..   the percentage of intracellular platinum in A2780cisR cells relative to the platinum input was 82%, 74% and 60%, respectively. These data indicate that intracellular accumulation of trans-[PtCl(N,N-dimethylamine) (isopropylamine)] in A2780cisR cells is lower than that of both cis-DDP and trans-DDP. We think that the lower cellular uptake of trans-[PtCl2(N.Ndimethylamine) (isopropylamine)] relative to cis-DDP and trans-DDP might be related to its larger rnolecular size.  and trans-DDP to DNA was respectively 180, 40 and 20 nmol/g DNA after 1 hour of incubation and progressively increased to reach respectively 810, 590 and 480 nmol/g DNA after 24 hours of incubation.
We also quantified the levels of platinum binding to DNA when the A2780cisR cells were pre-exposed to L-BSO before treatment with the platinum drugs. Figure 5 shows that under these conditions the level of DNA binding shown by cis-DDP, trans-[PtCl2(N,Ndimethylamine)(isopropylamine)] and trans-DDP increased relative to that found in A2780cisR cells not pre-exposed to L-BSO. Interestingly, the higher increase in DNA binding was shown by trans-[PtC12(N,N-dimethylamine)(isopropylamine)]. Thus, the kinetics of DNA binding of trans-[PtCl(N,N-dimethylamine)(isopropylamine)] was very similar to that of cis-DDP in A2780cisR cells pre-exposed to L-BSO. These results support the cytotoxicity data reported above and suggests that trans-[PtCl2(N,N-dimethylamine)(isopropylamine)] is significantly more affected in its reaction with DNA by cellular levels of GSH than cs-DDP. and trans-DDP (more than 13.6-times as potent). Moreover, trans-[PtCl_(N.Ndimethylamine)(isopropylamine)] is a better inductor of apoptosis in A2780cis R cells than both cis-DDP (1.7-times as better) and trans-DDP (5.5-times as better). These results are in agreement with previously reported data, which showed that trans-[PtCl2(N,N-dimethylamine) (isopropylamine)] is also able to circumvent cisplatin resistance and induces apoptosis in Pam 212-ras murine keratinocytes [ 10,11 ]. It has been reported that the A2780cisR human ovarian tumor cell line e.xhibits acquired resistance to cis-DDP from a combination of decreased uptake, enhanced DNA repair/tolerance and elevated GSH levels [12,13]. The aim of the present study was to investigate whether the cellular uptake, the reaction with GSH and the level ot platinum dimethylamine)(isopropylamine)] seems to be inactivated by reaction with GSH at a higher extent than cisDDP. In fact, either the cytotoxic activity or the amount of binding to DNA of trans-[PtCl.(N, N-dimethylamine)(isopropylamine)] in A2780cisR cells significantly increased when intracellular levels of GSH were depleted with L-BSO before treatment with the platirum drug. In contrast, both the cytotoxic activity and the amount of cis-DDP binding to .OqA are not significtly altered when GSH levels in A2780cisR cells are depleted with L-BSO prior to cis-l)DP treatment. Therefore, the intracellular accumulation, the reaction with GSH md fle level of platinum binding to DNA are factors, which cannot explain the circumvention of clsplatin re,sistance shown by trans-[PtCl2(N,N-dimethylamine) (isopropylamine)] in A2780cisR cells.
Emerging evidence suggest that an important number of cases of cisplatin resistance might be the result of the inability of cis-DDP to induce cell death through apoptosis in particular cell lines [8,25]. We think that circumvention of ciplatin resistance by trans-[]?tCl(N,N-dimethylamine) (isopropylamine)] might be directly related with the ability of this drug to induce apoptosis in A2780cisR cells. It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of platinum drugs [2 6]. [PtC12(N,N-dimethylamine)(isopropylamine)] might induce apoptosis in A2780cisR cells through interaction with other targets (i.e., proteins, phospholipids, cytoskeleton etc.) and ubscquent cellular damage. Further research is warranted to test these two hypotheses.