Cellular and biomolecular responses of human ovarian cancer cells to cytostatic dinuclear platinum(II) complexes

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Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly
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  ORIGINAL PAPER Cellular and biomolecular responses of human ovarian cancercells to cytostatic dinuclear platinum(II) complexes Miaoxin Lin  • Xiaoyong Wang  • Jianhui Zhu  • Damin Fan  • Yangmiao Zhang  • Junfeng Zhang  • Zijian Guo Published online: 25 November 2010   Springer Science+Business Media, LLC 2010 Abstract  Polynuclear platinum(II) complexes represent aclass of potential anticancer agents that have shown prom-ising pharmacological properties in preclinical studies. Thenature of cellular responses induced by these complexes,however, is poorly understood. In this research, the cellularresponses of human ovarian cancer COC1 cells to dinuclearplatinum(II) complexes {[ cis -Pt(NH 3 ) 2 Cl] 2 L 1 }(NO 3 ) 2  ( 1 )and {[ cis -Pt(NH 3 ) 2 Cl] 2 L 2 }(NO 3 ) 2  ( 2 ) (L 1 =  a , a 0 -diamino-  p -xylene, L 2 =  4,4 0 -methylenedianiline) has been studiedusing cisplatin as a reference. The effect of platinumcomplexes on the proliferation, death mode, mitochondrialmembrane potential, and cell cycle progression has beenexamined by MTT assay and flow cytometry. The activa-tion of cell cycle checkpoint kinases (CHK1/2), extracel-lular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) of the cells by thecomplexes has also been analyzed using phospho-specificflow cytometry. Complex  1  is more cytotoxic than complex 2  and cisplatin at most concentrations; complex  2  and cis-platin are comparably cytotoxic. These complexes kill thecells through an apoptotic or apoptosis-like pathway char-acterized by exposure of phosphatidylserine and dissipationof mitochondrial membrane potential. Complex  1  shows thestrongest inductive effect on the morphological changes of the cells, followed by cisplatin and complex  2 . Complexes  1 and  2  arrest the cell cycle in G2 or M phase, while cisplatinarrests the cell cycle in S phase. The influence of thesecomplexes on CHK1/2, ERK1/2, and p38 MAPK varieswith the dose of the drugs or reaction time. Activation of phospho-ERK1/2 and phospho-p38 MAPK by these com-plexes is closely related to the cytostatic activity. Theresults demonstrate that dinuclear platinum(II) complexescan induce some cellular responses different from thosecaused by cisplatin. Keywords  Apoptosis    Cytotoxicity    Dinuclear platinumcomplex    Flow cytometry    Signaling pathway Introduction Platinum complexes have been extensively studied aspotential anticancer agents since the successful use of cisplatin in chemotherapy [1–3]. Polynuclear platinum complexes constitute a novel class of prospective antican-cer agents that have shown some peculiar activities ascompared with mononuclear platinum compounds [4, 5]. Unlike cisplatin and its analogues that predominantly form1,2-d(GpG) and 1,2-d(ApG) intrastrand crosslinks withDNA [6], polynuclear platinum complexes mainly formlong range intra- and interstrand crosslinks [4, 7]. The Pt-DNA crosslinks can simulate a series of intracellularresponses and lead to the death of cancer cells [6]. So far,much attention has been focused on the molecular inter-actions between polynuclear platinum complexes and DNA[8, 9], whereas studies on cellular responses to these complexes are relatively rare [10]. Lack of such knowledgehas hindered further rational design and development of polynuclear platinum complexes as anticancer agents. M. Lin    J. Zhu    D. Fan    Y. Zhang    Z. Guo ( & )State Key Laboratory of Coordination Chemistry,School of Chemistry and Chemical Engineering, NanjingUniversity, Nanjing 210093, People’s Republic of Chinae-mail: zguo@nju.edu.cnX. Wang ( & )    J. ZhangState Key Laboratory of Pharmaceutical Biotechnology,School of Life Sciences, Nanjing University,Nanjing 210093, People’s Republic of Chinae-mail: boxwxy@nju.edu.cn  1 3 Apoptosis (2011) 16:288–300DOI 10.1007/s10495-010-0562-0  Recently, we discovered that dinuclear platinum(II) com-plexes  1  and  2  (Fig. 1) demonstrate unique DNA bindingmode in comparison with cisplatin [11], which may suggestthat unusual DNA damages could induce distinct deathpathways in cancer cells.It is known that cisplatin induces two different modes of cell death: apoptosis and necrosis [12]. Apoptosis is char-acterized by exposure of phosphatidylserine (PS) on the cellsurface, membrane blebbing, cytoplasmic shrinkage, chro-matin condensation, and the formation of apoptotic bodies.Necrosis, however, is characterized by cytoplasmic swell-ing, loss of membrane integrity, swelling of cytoplasmicorganelles, and moderate chromatin condensation [13].Since necrosis occurs equally in cells with or without func-tional apoptosis, cell death with both apoptotic and necroticfeatures may exist in the cisplatin-treated cells [14].Apoptotic cell death is usually determined by bindingAnnexin V to the exposed PS or by detecting caspase-cleaved proteins or fragmented DNA [15]. When DNA isdamaged by cisplatin, the cell cycle is arrested to providetime for repair. The G1 or S checkpoint allows DNA res-toration before replication, and the G2 or M checkpointfacilitates the reparation of DNA damaged during S or G2phases to prevent cells from initiating mitosis [16]. Cis-platin induces G2 or S phase arrest but does not cause G1arrest in all cases [14]. The cell cycle arrest after DNAdamage is regulated by ataxia telangiectasia mutated(ATM) and Rad3-related (ATR) kinases, which activatecheckpoint kinases CHK1 and CHK2 through phosphory-lation [17, 18]. Cells incapable of repairing DNA damages caused by cisplatin would get into apoptosis [19]. On theother hand, cisplatin-DNA damage also modulates severalsignal transduction pathways, such as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated proteinkinase (p38 MAPK) pathways [12]. These pathwayscoordinately regulate diverse cellular activities includingcell cycle arrest, mitosis, proliferation, and apoptosis [20].Referring to the above information on cisplatin, in thiswork we focus on the cellular responses and death mode of the human ovarian cancer COC1 cell line in the presence of complexes  1  and  2 . Various factors of concern, such ascytotoxicity, apoptotic character, mitochondrial membranepotential ( DW m ), cell cycle progression, and activation of kinases CHK1, CHK2, ERK1/2 and p38 MAPK, areinvestigated and discussed. Experimental ChemicalsComplexes  1  and  2  were prepared as described previously[11, 21]. Cisplatin was purchased from Aldrich. Solutions of complexes  1 ,  2 , and cisplatin (2.5 mM) were preparedwith deionized water and stored at  - 20  C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) was purchased from Sigma. Common reagents usedin the experiments were of analytical grade.Cell cultureHuman ovarian cancer cells COC1 were purchased fromChina Center for Type Culture Collection (CCTCC) andwere maintained in RPMI medium 1640 (Gibco, Carlsbad,CA) with heat-inactivated fetal bovine serum (10%). Cellswere cultured at 37  C in an atmosphere of 5% CO 2  and100% relative humidity.CytotoxicityThe cytotoxicity of the platinum complexes in COC1 cellswas measured by MTT assay in 96-well microtiter plates ata predetermined cell density [22]. Specifically, cells werecultured over night after inoculation and complex  1 ,  2 , orcisplatin with different concentrations (0.1, 1, 10, 100  l M)was added to the culture medium. The culture was incu-bated for additional 24, 36, or 48 h, and MTT (20  l l,5 mg ml - 1 ) in PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 gNa 2 HPO 4 , and 0.24 g KH 2 PO 4  per liter) was added to eachculture and incubated for 4 h. The culture medium wasremoved after centrifugation. Formazan was solubilized bydimethyl sulfoxide (DMSO; 150  l l) and the absorbance of each sample was measured at 570 nm on an ELISA platereader. IC 50  values of the platinum complexes wereobtained from the fitted inhibition curves at 48 h.Apoptotic assayThe apoptosis-inducing property of complexes  1 ,  2  andcisplatin in COC1 cells was evaluated by flow cytometryusing Annexin V conjugated with fluorescein isothiocya-nate (Annexin V-FITC) (Bipec Biopharma Corporation, H 2 NNH 2 PtPtNH 3 ClNH 3 NH 3 H 3 NClNH 2 PtPtNH 2 NH 3 ClNH 3 H 3 NH 3 NClPtH 3 NClH 3 NCl cisplatin complex  1 complex  2 2+2+ Fig. 1  Chemical structures of cisplatin, complexes  1  and  2 Apoptosis (2011) 16:288–300 289  1 3  Cambridge, MA) and propidium iodide (PI) counterstain-ing. Exponentially growing COC1 cells were inoculated in12-well plates and cultured overnight. Platinum complex  1 , 2 , or cisplatin was added to the culture to reach a finalconcentration of 1.0, 2.5 or 10  l M, respectively. Afterincubation for 48 h, cells were harvested, washed twicewith PBS, and re-suspended in binding buffer (100  l l) at aconcentration of 10 6 cells ml - 1 . The cells were incubatedwith Annexin V-FITC solution (5  l l) at 4 - 8  C for 15 minin the dark and for another 5 min after addition of PI(10  l l, 20  l g ml - 1 ) under the same condition. Cells wereanalyzed on the FACSCalibur flow fluorocytometer(Becton–Dickinson).Detection of   DW m Effect of complexes  1 ,  2  and cisplatin on the mitochondrialmembrane potential  DW m  was tested using a lipophiliccationic fluorochrome 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1) (DePsipherKit, R&D Systems, Minneapolis, MN) as a voltage sensi-tive probe. COC1 cells were inoculated in 12-well platesand cultured overnight before each platinum complex wasadded. After incubation for 48 h, cells were harvested,washed with PBS, and incubated with JC-1 (5  l g ml - 1 ) at37  C in the dark in an incubator with 5% CO 2  for 20 min.Cells were then washed with PBS twice, re-suspended inPBS (500  l l) and analyzed by flow cytometry.Cell cycle analysisCOC1 cells were inoculated in 12-well plates and culturedovernight. Complex  1 ,  2 , or cisplatin was added to theculture medium respectively to reach the specific concen-trations. After incubation for 24 h, the cells were harvested,washed with icy PBS, fixed in ethanol (70%) at 4  C for12 h, and centrifuged to get rid of ethanol. The cells werefurther washed twice with PBS, re-suspended in PBS(100  l l) and incubated with RNase A solution (4  l l,100 mg ml - 1 , Sigma) at 37  C for 10 min. PI (50  l g ml - 1 ,0.1% Triton X-100 in 200  l l PBS) was added to thesolution to stain the cells in the dark at room temperaturefor 15 min. The solution was then diluted with PBS to500  l l and the content of DNA was analyzed by flowcytometry. Cell cycle distribution curves were producedwith OriginPro 7.0 and the percentage of the cells in eachphase was calculated with FCS Express V3 (De NovoSoftware, Los Angeles, CA).Detection of phospho-CHK1/CHK2COC1 cells were inoculated in 12-well plates and culturedovernight before addition of complex  1 ,  2 , or cisplatin (2 or10  l M). After incubation for 12 h, the cells were collectedby centrifugation, re-suspended in PBS (0.5 ml), fixed withformaldehyde (2% final concentration) at 37  C for 10 minand centrifuged. The cells were then permeabilized in icymethanol (90%) for 30 min, rinsed twice with incubationbuffer (5 mg ml - 1 bovine serum albumin in PBS), centri-fuged, re-suspended in incubation buffer (100  l l), andblocked for 10 min at room temperature. Intracellularphospho-protein staining was performed as described in theprotocol of Cell Signaling Technology (CST, Danvers,MA) and the literature [23, 24]. Specifically, phospho- specific primary antibody (CST, Danvers, MA) was addedto the cells and incubated at 4  C overnight. The cells wererinsed in incubation buffer and incubated with AlexaFluor  488 F(ab 0 ) 2  fragment of goat anti-rabbit IgG(H  ?  L) (Molecular Probes, Carlsbad, CA) for 30 min atroom temperature. After rinse, cells were re-suspended inPBS and analyzed on flow cytometry. Cells cultured in adrug-free medium were used as controls. The data wereprocessed with WinMDI 2.9 and the curves were generatedand smoothed with OriginPro 7.0 software.Phosphorylation of ERK1/2 and p38 MAPK The time-dependent effect of complex  1 ,  2 , or cisplatin onthe phosphorylation of ERK1/2 and p38 MAPK wasevaluated by intracellular staining following the procedureas described in the immediate above section. Cells culturedin a medium without drug were used as controls. Rabbitmonoclonal antibodies against phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204, D13.14.4E) and phospho-p38MAPK (Thr180/Tyr182, 3D7) were purchased from CST(Danvers, MA), and the secondary antibody is the same asdescribed above. In the negative and positive controlexperiments, cells were incubated with inhibitors 1,4-dia-mino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene(U0126, CST, Danvers, MA; 25  l M), 4-[5-(4-fluoro-phenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]-pyridine (SB203580, Beyotime Institute of Biotechnology;25  l M) and stimulator phorbol 12-myristate 13-acetate(PMA; 50 ng ml - 1 ), respectively, for 2 h before harvest. InMAPK inhibition experiments, cells were pretreated withinhibitor U0126 (25  l M) or SB203580 (25  l M) for 30 minand kept coexisting with each complex throughout theexperiment. Results and discussion CytotoxicityThe cytotoxicity of complexes  1  and  2  against humanovarian cancer COC1 cells was tested by MTT assay at 290 Apoptosis (2011) 16:288–300  1 3  four representative concentrations after 24, 36 and 48 h,respectively, using cisplatin as a reference. More detailedcytotoxic profiles were tested at 48 h and the IC 50  values of the complexes were obtained from the fitted inhibitioncurves. As Fig. 2 shows, complex  1  appears more cytotoxicthan complex  2  and cisplatin at most concentrations; whilecomplex  2  is almost as cytotoxic as cisplatin at all testedconcentrations. The IC 50  values of complexes  1 ,  2  andcisplatin at 48 h are 1.08  ±  0.05, 4.97  ±  0.87 and7.02  ±  1.64  l M, respectively. Complex  1  is more effec-tive than cisplatin, particularly at low concentrations( \ 10  l M). In our previous studies, complexes  1  and  2  alsoshowed high cytotoxicity against the human non-small-celllung cancer A-549 cells [11], and so did complex  2  againstthe murine leukemia P-388 cells [21].Mode of cell deathApoptosis and necrosis are common cellular responses toanticancer drugs, especially the former. Cells in earlyapoptosis display the characteristic phosphatidylserine (PS)exposure on the outer surface of the plasma membrane[15], which can be quantitatively detected by flow cyto-metric assay using Annexin V-FITC combined withpropidium iodide (PI) [25]. The apoptotic cells can bedistinguished from the intact cells according to theAnnexin V variations (Annexin V ?  vs. Annexin V - ). PIstaining allows further distinction between early stageapoptotic (Annexin V ?  /PI - ) and late stage apoptotic ornecrotic (Annexin V ?  /PI ? ) cells, in that PS exposureusually precedes the loss of plasma membrane integrityduring apoptosis, while both events tend to synchronize innecrosis [26]. As the density plots in Fig. 3 show, in comparison with the untreated control, complexes  1 ,  2  andcisplatin markedly increase the proportion of COC1 cellswith PS exposure (Annexin V ? ) and/or loss of plasmamembrane integrity (PI ? ). The percentage of cells at dif-ferent stages is presented in Fig. 4. Complex  1  shows thestrongest impact on COC1 cells among the three com-plexes at all tested concentrations. For example, at 2.5  l M,only 32.9% of the cells remain intact (Annexin V -  /PI - )after treatment with complex  1 ; while 82.6 and 71.1% of the cells remain intact in the case of complex  2  and cis-platin, respectively. Complex  2  is comparable to cisplatinat 1.0  l M, but is less effective at higher concentrations.Although the influence of these complexes on the PSexposure and loss of plasma membrane integrity varies inextent, the cell death induced by them follows a similarpathway, i.e. from the lower left quadrant (Annexin V -  / PI - ) to the lower right quadrant (Annexin V ?  /PI - ) andthen to the upper right quadrant (Annexin V ?  /PI ? ). Owingto complexes  1 ,  2  and cisplatin all induce COC1 cells toundergo the intermediate Annexin V ?  /PI -  stage, thepredominant death mode in this case is apoptosis.The morphological distinction among the dead cellsinduced by complexes  1 ,  2  and cisplatin was detected byanalyzing their light-scattering properties with flowcytometry. The amount of forward scatter (FSC) of laser Fig. 2  The cytotoxicity of complexes  1 ,  2  and cisplatinagainst human ovarian cancerCOC1 cells at differentconcentrations after incubationfor 24, 36 and 48 h, and thefitted inhibition profiles at 48 h.Data are expressed as mean(%)  ±  standard deviations(S. D.) of at least threeindependent experimentsApoptosis (2011) 16:288–300 291  1 3  light is generally used to evaluate the cellular size, whilethat of side scatter (SSC) is commonly adopted to revealthe cellular granularity [26]. These parameters can beobtained on the FACSCalibur flow fluorocytometer. AsFig. 5 presents, COC1 cells in drug-free control are largelyin normal size and granularity (green dots), which includemainly PI -  cells. After incubation with each complex,most cells undergo similar changes in size and granularity.FSC-positive spread of the green dots representing thecytoplasmic swelling is not evident, suggesting that thesecomplexes do not induce typical necrosis in COC1 cellsunder the test conditions. SSC-positive spread of the dotsmay result from the plasma membrane blebs of apoptoticcells, which cause increased diffraction of the laser beam[26]. In the lower left corner of the plots, the population of PI ?  red dots (ellipse I) may represent the shrunken cellsand subsequent apoptotic bodies containing DNA, and thepopulation of PI -  blue dots (ellipse II) may manifest thereleased apoptotic particles without DNA. If apoptotic cellsare not taken up by phagocytosis, they become secondarynecrotic cells that share many features of primary necrosis[26]. The dots in ellipse III might represent the overlap of such secondary necrotic cells and early-apoptotic cells.Among these complexes,  1  is the most effective inducer forthe morphological changes of COC1 cells, followed bycisplatin and  2 . The results indicate that complexes  1 ,  2  and Fig. 3  Flow cytometricanalysis of COC1 cells afterincubation with complex  1 ,  2  orcisplatin for 48 h andsubsequent staining withAnnexin V and PI. Dots withAnnexin V -  /PI - , AnnexinV ?  /PI - , and Annexin V ?  /PI ? features represent intact, early-apoptotic, and dead cells,respectively292 Apoptosis (2011) 16:288–300  1 3
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