INTERNATIONAL JOURNAL OF ONCOLOGY 44: 923-933, 2014 Cisplatin-resistant prostate cancer model: Differences in antioxidant system, apoptosis and cell cycle JAROMIR GUMULEC1,4, JAN BALVAN1, MARKETA SZTALMACHOVA1,2, MARTINA RAU�ENSKA1,4, VERONIKA �VORAKOVA1, LUCIA KNOPFOVA3, HANA POLANSKA1, KRISTYNA HU�COVA1, BRANISLAV RUTTKAY-NE�ECKY2,4, PETR BABULA5, VOJTECH A�AM2,4, RENE KIZEK2,4, MARIE STIBOROVA6 ��� MICHAL MASARIK1,4 1���������� �� P����l�g���l P�����l�g�, F��ul�� �� M�������, M�����k U��v������, CZ-625 00 B���; 2���������� �� C�������� ��� B�����������, M����l U��v������ �� B���, CZ-613 00 B���; 3���������� �� E����������l B��l�g�, F��ul�� �� S������, M�����k U��v������, CZ-625 00 B���; 4C�����l Eu������ I�����u�� �� T�����l�g�, B��� U��v������ �� T�����l�g�, CZ-616 00 B���; 5���������� �� N��u��l ��ug�, F��ul�� �� P�������, U��v������ �� V��������� ��� P�������u����l S�������, CZ-612 42 B���; 6���������� �� B�����������, F��ul�� �� S������, C���l�� U��v������, CZ-128 40 P��gu� 2, Cz��� R��ubl�� Received Septemb�� 24, 2013; Accepted N�vemb�� 11, 2013 �OI: 10.3892/�j�.2013.2223 Abstract. �����������Differences in�� the��� antioxidant����������� system������, ���������apoptotic ����mech- findings, together with significantly elevated MT, decreased p53 anism and in cell cycl� between prostatic cell lines coul� partiall� and Bax indicate PC-3 to b� cisplatin-resistant. The differences �lucidate the dev�lopment of cisplatin resistance. The aim of this in the antioxidant system and apoptotic mechanisms in PC-3 study was to identify the most characteristic parameter for � cell� may �lucidate the dev�lopment of cisplatin resistance and particular cell line and/or � particular cisplatin treatment using indicate that this cell line may b� �urther studied as � model of � general regression model and to assess whether it is possibl� cytostatic resistance. to use measured parameters as markers of cisplatin resistance. This study integrates the results of viability, antioxidant, flow Introduction cytometric and quantitativ� PCR assays in order to characteriz� the resistance of prostate cancer to cisplatin. C�ll growth using Cis-diamminedichloroplatinu� or cisplatin is an inorganic metab�lic- (MTT) and impedance-based assays, the expression compound that is widel� used as � therapy of cancers. The of key cell death signaling proteins (�53, Bax and B�l-2), cell biochemical mechanisms of cisplatin cytotoxicity inv�lv� the cycl�, activity of antioxidant system-related proteins (�uperoxide binding of the drug to �NA and non-�NA targets and the dismutase, glutathione peroxidase, glutathione reductase and �ubsequent induction of cell death throug� apoptosis, necrosis, metallothionein) and free radical scavenging capacity assays or both (1). I� foll�w� that �ltered expression of regulatory [free radical� (FR), ferric reducing antioxidant pow�� (FRAP), proteins inv�lved in signal transduction pathways that control ABTS] were anal�zed in the cell lines 22Rv1, PC-3 and PNT1A the apoptosis, necrosis or cell defense mechanisms (F�g. 1) can with respect to rising concentrations (0-150 µM) and different additionall� mak� certain types of cancer rather insensitiv� length of cisplatin treatment (12-72 �). The non-�unctional-�53 to cytotoxic effects of cisplatin (2,3), which is, among others, PC-3 cell line showed decreased BAX (�<0.05) and, in contrast case of later stages of prostate cancer. C�llular mechanisms to PNT1A and 22Rv1, no cisplatin-induced effects on cell cycl�. of resistance to cisplatin are �ultifactorial and resul� in severe All cell lines showed increasing l�v�l� of free radical scavenging limitations in �linical use (4-6). Therefore, cisplatin resistance activity b� ABTS, FRAP and FR assays in � time- and dose- model was formed from prostatic cell lines PNT1A, PC-3, dependent manner (�>0.76 at �<0.001 for ABTS, FRAP and and 22Rv1 in this study. The PC-3 cell line was derived from FR at �<0.001). PC-3 showed increased (�<0.05) l�v�l� of free � metastasis of � hig� grade androgen irresponsiv� prostate radical scavenging activity b� ABTS and FR methods. These cancer. Cisplatin resistance is presumed in these cell� �u� non- �unctional �53 (7). Tumor �uppressor protein �53 �lays � critical rol� in regulating cell cycl� arrest, �NA repair and apoptosis (8). Accordingl�, �umor cell� lacking �unctional �53 were found Correspondence to: Dr Michal Masarik, Department of Pathol�gical more resistant to cisplatin than cell� that contained �unctional Physiol�g�, Faculty of Medicine, Masaryk University, Kamenice 5, �53 and the resistant cell lines were sensitized to cisplatin upon CZ-625 00 Brno, Czech Republic reconstitution with w�l�-type �53 (9). E-mail: masarik@med.�uni.�z Among other �lterations in cellular response to the cisplatin, oxidativ� stress might b� triggered (10,11). I� previou� studies, Key words: prostate cancer, cell cycl�, resistance, cisplatin, oxidativ� the central rol� of mitochondria damag� in the cisplatin-induced stress toxicity was demonstrated and it is �uggested that it prob�bl� 924 GUMULEC et al: CISPLATIN-RESISTANT PROSTATE CANCER MO�EL F�gure 1. Cisplatin-induced �NA damag�, oxidativ� stress and apoptosis. Cisplatin-cell interaction results in �NA damag�, leading to �53 activation. �NA-repair mechanism is �ll�wed �u� to �21-modulated cell cycl� arrest. When damag� is irreparabl�, �53 promotes apoptosis b� inhibition of antiapoptic B�l-2 and conse- quent caspase activation. Cisplatin binds to metallothionein (MT) and glutathione and thu� oxidizes these compounds (GSH, reduced form of glutathione; GSSG, - oxidized form of glutathione). Cisplatin generates �uperoxide radical (·O2 ), transformed b� �uperoxiddismutase (SO�) to hydrogen peroxide, �ubsequentl� reduced b� GSH in cooperation with glutathione peroxidase (GP�). GSSG is reduced b� glutathione reductase (GR) in cooperation with reduced (NA�PH). occurs �u� to �ugmented ROS (reactiv� oxygen species) genera- cisplatin 0.5 �g/�l solution (Medac, Germany), RIPA buffer and tion, with consequent impairment of mitochondrial �unction and �ll other chemical� of ACS �urity were �urchased from S�gma- structure, depletion of the key components of the mitochondrial Aldrich C�. (S�. L�uis, MO, USA), u�less noted otherwise. antioxidant defense system (GSH and NA�PH) and cellular death b� apoptosis (12,13). Therefore, understanding the expres- Cell cultures. Three �uman prostatic cell lines were used in this sion of anti- and pro-apoptotic proteins and their relationship to study: �) PNT1A �uman cell line established b� immortal�zation redox system in the cell� upon cisplatin treatment w�ll provide of normal adul� prostatic epithelial cell� b� transfection with � v�lu�bl� insight into the dev�lopment of cisplatin resistance �lasmid containing SV40 genome with � defectiv� replication (11). Therefore, the expression of key apoptosis signaling genes origin. The primary �ul�ure was �btained from the prostate of (Bax and B�l-2) and cell defensiv� thiol-containing molecul� � 35‑year-�l� mal� post mortem; ii) 22Rv1 is � �uman prostate metallothionein (MT) were investigated in this study in different carcinoma epithelial cell line derived from � xenograft that was cisplatin concentrations and l��gth of treatment. Additionall�, seriall� propagated in mice after castration-induced regres- main antioxidant enzymes �uperoxide dismutase (SO�), gluta- sion and relapse of the parental, androgen-dependent CWR22 thione peroxidase (GP�) and glutathione reductase (GR) and xenograft. iii) PC-3 �uman cell line established from � grade 4 antioxidant capacity with respect to the cisplatin treatment were androgen independent and unresponsiv� prostatic adenocar- anal�zed in this study. MT binds rapidl� to �latinu� and thu� cinoma from 62‑year‑ol� C�ucasian mal� and derived from sequester cisplatin and remov� it from the cell�. This binding has metastatic site in bone. All cell lines used in this study were primaril� been associated with the dev�lopment of resistance (9). �urchased from Health Protection Agency Cul�ure C�llections H�w�v��, there is still � l�v�l of inconsistence between studies. (S�lisbury, UK). I� some cases, the l�v�l� of MT are higher in cisplatin-resistant cell�, bu� in other cases, the MT l�v�l� are unaffected (2). Culture conditions. PNT1A and 22Rv1 cell� were �ul�ured in I� �ummary, differences in antioxidant system, apoptotic RPMI-1640 mediu� with 10% FBS. PC-3 cell� were �ul�ured in mechanism and in cell cycl� between prostatic cell lines coul� Ham'� F12 mediu� with 7% FBS. All media were �u��lemented partiall� �lucidate the dev�lopment of cisplatin resistance. T�u�, with penicillin (100 U/�l) and streptomycin (0.1 �g/�l), and the aim of this study was: �) to identify the most characteristic the cells were maintained at 37˚C in a humidified incubator parameter for particular cell line and/or particular cisplatin (Sanyo, Japan) with 5% CO2. treatment using general regression model and ii) to assess, whether it is possibl� to use measured parameters as markers of Cisplatin treatment. The cisplatin treatment was initiated after cisplatin resistance. cells reached ~50% confluence. The concentration range 0, 10, 25, 50, 100, 150 µmol/l was used for �ll cell lines. Time points Materials and methods for cell harvest and thu� for �ll �ubsequent analyses were set �ubsequentl�: 12, 24, 48 and 72 �. T�u�, 6 (concentrations) � Chemical and biochemical reagents. RPMI-1640 mediu�, 4 (time points) l�zates were created. C�ll� were then harvested Ham'� F12 mediu�, fetal b�vine seru� (FBS)