research article antioxidant enzyme inhibitor role...

Research ArticleAntioxidant Enzyme Inhibitor Role of Phosphine MetalComplexes in Lung and Leukemia Cell Lines

Burcu SaygJdeLer Demir1 TuLba KeleG2 and Osman SerindaL3

1Department of Chemistry Science and Letters Faculty Osmaniye Korkut Ata University Fakıusagı 80000 Osmaniye Turkey2Department of Chemistry and Chemical Technology University of Bayburt 69000 Bayburt Turkey3Science Institute Kanuni University 01170 Adana Turkey

Correspondence should be addressed to Burcu Saygıdeger Demir burcudemirosmaniyeedutr

Received 2 May 2014 Accepted 1 December 2014 Published 28 December 2014

Academic Editor Claudio Pettinari

Copyright copy 2014 Burcu Saygıdeger Demir et alThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Phosphine metal complexes have been recently evaluated in the field of cancer therapy In this research the cyto-toxic effects of some metal phosphines [PdCl

2((CH2OH)2PCH2)2NCH

3] (C1) [RuCl

2(((CH

2OH)2PCH2)2NCH

3)2] (C2)

[PtCl2((Ph2PCH2)2NCH

3)(timin)

2] (C3) on K562 (human myelogenous leukemia cell line) and A549 (adenocarcinomic human

alveolar basal epithelial cells) cells were investigated using the MTT test C1 and C2 are water-soluble metal complexes which mayhave some advantages in in vitro and in vivo studiesThe effects of the above-mentioned metal complexes on thioredoxin reductase(TrxR) (EC 1819) glutathione peroxidase (GPx) (EC 11119) and catalase (Cat) (EC 11116) enzymeswere also testedThe resultsof this research showed that all three metal complexes indicated dose-dependent cytotoxicity on A549 and K562 cell lines and thatthe complexes inhibited different percentages of the TrxR GPx and Cat enzymes of these tumor cells

1 Introduction

Some anticancer agents act through production of ROS(reactive oxygen species) to kill tumor cells Reported studieshave shown that cells with high levels of antioxidant enzymesare resistant to some anticancer agents [1 2] The inhibitionof these enzymes is an indicator of apoptotic pathways andorganometallic compounds have been most recently used asthe first step of cancer drug discovery [3ndash5]

In the last three decades metal complexes have been ofinterest to cancer therapy researchersThe international com-munity has widely recognized that while some rutheniumcomplexes exhibit low toxicity to normal cells they are easilyabsorbed by tumor tissue and rapidly excreted from the body[3ndash7] and ruthenium complexes bearing promising anti-cancer activity have successfully entered into clinical trials [38ndash10] In addition to ruthenium complexes the investigationof platinum and palladium complexes is also important forthe treatment of some human cancers [11] Many of the prop-erties that make metal-phosphine complexes attractive foruse in catalysis are also relevant for medicinal applications

such as anti-inflammatory antibacterial and antitumoralstudies The earliest example of an antitumoral application ofa metal-phosphine complex is the gold(I) complex auranofin[12] Several phosphine metal complexes have been synthe-sized to treat cancer cells after the discovery of auranofin [13ndash16]

Antioxidant enzymes in cancer cells such as GPx GRand especially TrxR are major targets for recent antitumordrug studies Several different clinical antitumor agents havebeen reported to inactivate TrxR However the relationshipbetween TrxR inactivation and apoptosis has been less fullyreported [17] In normal cells TrxR1 is necessary for redoxhomeostasis and protection against oxidative damage andmutation Once transformation into a malignant cell hastaken place TrxR1 supports tumor growth and progressionIn recent years strong connections have been identifiedbetween the thioredoxin system and the apoptosis regulatorprotein p53 [5] One of the reasons for preferring TrxR as amolecular target is the fact that it is a selenoprotein con-taining a selenocysteine on the flexible C-terminal arm of itsactive site (-Gly-Cys-SeCys-Gly-) which is very accessible

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2014 Article ID 717421 10 pageshttpdxdoiorg1011552014717421

2 Bioinorganic Chemistry and Applications

during catalysis Many electrophilic compounds selectivelyand irreversibly modify the active site amino acids of TrxR[18ndash22] Since GPx has cysteine and selenocysteine residuein its active site the inactivation mechanism of GPx withmetal complexes resembles that of TrxR [23] Inactivation ofCat having histidine aspartic acid and phenyl alanine aminoacids in its active site might be carried out via coordinationof these amino acids to metal complexes or coordination ofother amino acids outside the catalytic site [24]

The cytotoxic activities of Ru(II) Pd(II) and Pt(II)phosphine complexes on A549 and K562 cell lines and theinactivation of the GPx Cat and TrxR enzymes of these cellsvia the metal phosphine complexes have been investigated inthis study

2 Material and Method

21 Synthesis All reactions were carried out under puri-fied nitrogen using standard Schlenk techniques Solventswere purified by standard methods and distilled undernitrogen prior to use [PdCl

2((CH2OH)2PCH2)2NCH3] (C1)

[PtCl2((Ph2PCH2)2NCH3) (timin)

2] (C3) was prepared

according to the procedure described in the literature [2526] [RuCl

2(((CH

2OH)2PCH2)2NCH3)2] was synthesized

according to the new method for this study NMR spectrawere recorded on a BrukerARX-300 spectrometer usingD

2O

and CDCl3as solvents In the NMR spectra the 1H and 13C

chemical shifts are reported in ppm downfield from the inter-nal standard SiMe

4 The 31P NMR (1215MHz) spectra were

recorded with 85 H3PO4as an external standard and pos-

itive chemical shifts lie downfield of the standard Elementalanalyses were performed by the Inonu University ResearchLaboratory in Malatya Turkey All reagents were purchasedfrom Aldrich Chemical Co and were used without furtherpurification

211 Synthesis of [RuCl2(((CH

2OH)2PCH2)2NCH3)2] (C2)

An aqueous solution (10mL) of [((CH2OH)2PCH2)2NCH3]

(2mmol) was added dropwise to the ruthenium precursor[Ru(COD)Cl

2] (095mmol) in toluene (10mL) at 40∘C with

constant stirring The mixture was further stirred for 48 hand the aqueous layer was separated from the organic layerThe aqueous solution was concentrated to 5mL in vacuumand evaporated slowly at room temperature to afford thegreen colored complex C2 at 78 yield

Anal Calcd for [RuCl2(((CH

2OH)2PCH2)2NCH3)2]

(C2) C 256H 58 N 43 Found C 271H 65 N409 1HNMR (D

2O 25∘C) 120575 36 (s 16H PCH

2OH) 120575 32

(s 8H PCH2N) 120575 25 (s 6H NCH

3) 31P-[1H]NMR (D

2O

25∘C) 120575 65 ppm (s Ru-P) 13C NMR (d-DMSO 25∘C) 12057559 ppm (s ndashPCH

2OH) 120575 47 ppm (m ndashNCH

3) 120575 245 (s

PCH2N) FT-IR (KBr cmminus1) 1250 (CndashOH) 1050 (NndashC) 1460

(CndashH) 1150 (PndashCndashN(R)ndashCndashP) 3200ndash3400 (OndashH)

22 Cell Culture In order to examine the anticancer activitiesof metal phosphine complexes two different human cancercell lines were used a K562 cell line provided by CukurovaUniversityrsquosHematologyClinic and anA549 cell line supplied

byGaziantepUniversityrsquos Cell Culture LaboratoryThe cancercells weremaintained in the logarithmic phase at 37∘C in a 5carbon dioxide atmosphere using a culture medium con-taining 10 fetal bovine serum 1 penicillin and 1 strep-tomycin RPMI-1640 (Sigma) (developed by Roswell ParkMemorial Institute)

23 MTT Test (Cytotoxicity Test) The growth inhibitoryeffect towards cancer cell lines was evaluated by means ofMTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide a yellow tetrazole) assay [27] Briefly 3 times 103 cellswell dependent upon the growth characteristics of the cellline were seeded in 96-well microplates in the growthmedium (100120583L) and then incubated at 37∘C in a 5 carbondioxide atmosphere After 24 h the medium was removedand replaced with a freshmedium containing the appropriateconcentrations (4762 3801 2857 1905 952 and 476iusdotmLminus1) of the phosphine metal complexes being studiedTriplicate cultures were established for each treatment After48 h each well was treated with 10 120583L of a 5mgsdotmLminus1 MTTsaline solution and following 5 h of incubation 100 120583L ofdimethyl sulfoxide (DMSO) was addedThe inhibition of cellgrowth induced by the phosphine metal complexes wasdetected after incubation (30 minutes) by measuring theabsorbance of eachwell at 570 nmusing aBio-Rad 680micro-plate reader The IC

50values represent the concentrations of

phosphinemetal complexes that reduce themean absorbanceat 570 nm to 50 of those in the untreated control wellscontaining only culture medium Thus cytotoxicities of thecompounds were determined All data were from at leastthree independent experiments and are expressed as mean plusmnstandard deviation

24 Catalase Activity Assay Catalase activity was measuredas described by Claiborne [28] 3 times 103 cellswell were platedin 96-well microplates in a growthmedium (100 120583L) and thenincubated at 37∘C in a 5 carbon dioxide atmosphere After24 h the medium was removed and replaced with a fresh onecontaining the IC

50values of the metal complexes for each

cell for 48 hThe cells werewashed twice in a phosphate buffersolution (PBS) and then collected a protease inhibitor cock-tail purchased from Sigma was added into the cell suspensionand sonicated in a 50mM potassium phosphate buffer (PBpH 70) on ice for 25 s (using Bandelin SONOPULS HD 2200ultrasonic homogenizer) at a 10 output and 80 duty cycleAfter the sonication procedure the protein concentrationwas determined by Lowry at alrsquos method for Cat and otherenzyme activity assays [29]

Cell extracts (200ndash400mg) were added to 3mL of a10mM H

2O2in 50mM potassium phosphate buffer (pH 78)

and the disappearance of H2O2(extinction coefficient (120576) of

00396 cm2sdot120583molminus1)was immediatelymeasured at 240 nm for60 s at 15 s intervals Catalase activity was expressed in unitsper grams of protein

25 Glutathione Peroxidase Activity Assay After applying thesame sonicating procedure to the cells each 5 120583L sample ofthe cell content was incubated for 10min at 37∘C in a 495 120583L

Bioinorganic Chemistry and Applications 3

Table 1 Specific activities of TrxR GPx and Cat enzymes in the given period after incubation of A549 and K562 cells with IC50 values ofmetal complexes

A549 K562Umg protein Umg protein

TrxRControl

C1C2C3

05080ax plusmn (000458)y04740a plusmn (000529)02600b plusmn (001114)02960b plusmn (000557)

TrxRControl

C1C2C3

10640a plusmn (000529)05867d plusmn (001155)07200b plusmn (005568)06070c plusmn (002000)

GPxControl

C1C2C3

01520a plusmn (000265)00680c plusmn (000854)01067b plusmn (000351)00460d plusmn (000361)

GPxControl

C1C2C3

02367a plusmn (000577)00200b plusmn (000000)00000b plusmn (000000)00000b plusmn (000000)

CatControl

C1C2C3


CatControl

C1C2C3

3549300a plusmn (327432)1318900b plusmn (147401)1219100c plusmn (180602)1088163d plusmn (278970)

Among groups xmean value (119899 = 3) ystandard deviation andashdSignificant differences (1 119875 le 005 2 119875 le 001 3 119875 le 0001) in a row

incubation mixture containing 50120583L of a 100mM potassiumphosphate buffer (pH 70) 5 120583L of 100mM GSH 10 120583L of200mMEDTA 5 120583L of 400mMsodiumazide 50 120583L of 2mMNADPH 320 120583L distilled water and 50120583L GR (10 usdotmLminus1)After the 10min incubation period the reaction was initiatedby the addition of 5 120583L of 10mmolLminus1 of H

2O2The decrease

in the absorbance of the system was measured for 30 s at340 nmA similarmixture excludingGSHwas used as a blanksample [30] A unit of activity (U) was defined as the amountof enzymes that catalyzed the oxidation of one micromole ofNADPH (120576 = 622mMminus1) to NADP+ in one minute underthese conditions

26 Thioredoxin Reductase Activity Assay Enzyme activitywas determined spectrophotometrically by monitoring theNADPH dependent production of 2-nitro-5-thiobenzoate(120576 = 13600Mminus1sdotcmminus1) at 412 nmand at 37∘C [31]The sonica-tion step was achieved using the same sonicating procedureas in the other two enzyme assays Forty microliters of thesample was added to an assay mixture of 100mM sodiumphosphate pH 74 2mM EDTA and 3mM DTNB The reac-tion was initiated by adding 02mM NADPH The activitiesof the enzymes were monitored for 60 s The reaction waslinear throughout the entire the experimental period Aunit of thioredoxin reductase activity was expressed as onemicromole of NADPH oxidized to NADP+ in 1min underassay conditions

27 Statistical Analyses To determine whether the differ-ences between the activities of the enzymes of two differentcancer cells were significant depending on the IC

50values of

the metal complexes analysis of variance and then Tukeyrsquostest were used [32]Three replicates were used as comparisonsfor each experiment Results were also given asmean valuesplusmnstandard errors of the three replicates in Table 1 Differencesbetween data were assumed significant at (1 119875 le 005 2119875 le 001 3 119875 le 0001) All statistical analyses were carriedout using SPSS 115

3 Results and Discussion

31 Results

311 Synthesis and Characterization of Metal ComplexesNN-bis (hydroxymethyl phosphinomethyl) aminomethyl[((CH

2OH)2PCH2)2NCH3] (dppam) was synthesized by

using the reported procedure [25] The water-soluble charac-teristics of these (hydroxymethyl) phosphines presented theprospect of investigating their coordination chemistry inwater or under biphasic conditions Upon interaction with[Ru(COD)Cl

2] in toluene dppam in water produced the

complex [RuCl2(((CH

2OH)2PCH2)2NCH3)2] at 78 yield

(Figure 2) The chemical constitution of C2 was confirmedby elemental analysis and 1H 13C 31P NMR and FABmass spectroscopies FAB mass spectrometry was used toidentify the molecular ions for the ligand parent ionsat [M+ H+] and mz 2453 [25] Due to very highmolecular weight of C2 molecular mass spectrum for[RuCl

2(((CH

2OH)2PCH2)2NCH3)2] could not be clarified

Analysis of the FT-IR spectrum of the metal complex C2proves that the OndashH stretch peaks of phosphine exhibit aband between 3300 and 3400 cmminus1 The peak at 1050 cmminus1 isassigned to the NndashC stretch whereas 1050 cmminus1 is assigned tothe asymmetric bending plane of CH

3 Consequently when

the IR spectra of free ligands and the metal complex werecompared and they were found to be similar [25] The31PNMR spectrum of C2 consisted of a single resonance at65 ppm indicating a significant downfield shift compared tothe free ligand (minus173 ppm) upon coordination of the phos-phine units to the ruthenium (II) center which is consistentwith the literature [33] The 1H NMR spectrum of C2 showsmultiplets centered at 39 ppm for PndashCH

2ndashOH protons sug-

gesting that there is a slight downfield shift compared to thechemical shift of the free ligand (36 ppm) with singlet peaksat 32 ppm and 25 ppm respectively for NndashCH

2ndashP protons

and NndashCH3protons The 13C NMR spectrum of C2 shows

the similar peaks ligand at 245 ppm for the PndashCH2ndashN carbon

peak 475 ppm for the NndashCH3carbon peak and 59 ppm for


C1

PdCl

Cl

P

P

N

HOH2C

HOH2C

H3C

CH2OH

CH2OH

(a)

P

N

P

Ru

P

P

N

Cl

Cl

C2

HOH2CHOH2C

HOH2C

HOH2C

H3C

CH2OHCH2OH

CH3

CH2OH

CH2OH

(b)

N

PPt

HN

O

N

O

N

NH

O O

C3

H3C

CH3

(H3C)3

PPh2

Ph2

(c)

Figure 1 Molecular structures of the phosphine metal complexes used

PndashCH2OH In addition the literature dates and elemental

analysis results show that the complex ratio is 1 2 [34]

312 Cytotoxicity of Metal Complexes Platin palladium andruthenium complexes of phosphines were used to investigatetheir cytotoxic activity towards two different cell lines A549and K562 Cytotoxicity was evaluated by means of the MTTtest after 24 48 72 and 120 hours of treatmentwith increasingconcentrations of the aforementioned compounds The IC

50

values which were calculated from dose dependent curvescan be seen in Figures 3 and 4 The results showed that A549cells were resistant to death whereas K562 cells have lowresistance in the presence of metal complexes Consideringthe resistances of the cell lines the 120-hour treatment periodforK562 cells and the 24-hour treatment period forA549 cellswere not studied for cytotoxicity of metal complexes Amongthe 24 h treatment group only C1 for K562 had cytotoxicactivity whereas the cell morphology of the others remainedrelatively unchanged due to its slow penetration of the cells(Figure 5) In the 24-hour treatment of the tested compoundC3 showed better cytotoxic activity for the A549 and K562cell lines at 0158mM and 005mM respectively The othertested compounds C1 and C2 showed different cytotoxicitieson each cell (C1 onA549 7981mM 4625mM and 4575mMfor 48 h 72 h and 120 h resp C1 on K562 2625mM3352mM and 2396mM for 24 h 48 h and 72 h resp and

C2 on A549 7436mM and 5302mM for 72 h and 120 hresp) (Figures 3 and 4) The assignment of microscopicimages proved that after treating with C2 most of the A549and K562 cells were still alive after 24 h likewise after 48 hK562 cells treated with C2 were still alive based on themicroscopic view So these incubation times were ignored forC1 and C2

313 Enzyme Studies Inhibition of the enzymes CAT TrxRand GPx by organometallic and other metal compounds inthe treatment of cancer has been widely studied The metalphosphine complexes C1 C2 and C3 have been studied toexamine the inhibition of the above-mentioned enzymes(Figure 1) The general results of the inhibition ratios havebeen found to be consistent with those of the values in theliterature [15 35]

The enzyme activities of the cells incubated with the IC50

values of metal complexes tended to decrease at 72 h whencompared with untreated A549 and K562 cell lines Table 1shows a statistically significant decrease in CAT GPx andTrxR activities inA549 andK562 cells in the presence ofmetalcomplexes

The TrxR activities of A549 and K562 control cells(untreated with any complex) were found to be 0508Umgprotein and 1064Umg protein respectively and these activ-ities were accepted as 100 activity The TrxR activity of


P

N

P

Ru

P

PP

N

P

RuCl

ClCl

+ N

Cl

HOH2C

HOH2C

CH2OH HOH2C HOH2C

HOH2CHOH2C

CH2OH CH2OH

CH2OHCH2OHCH2OH

H3C H3C CH3

48h40

∘C

Figure 2 Synthesis of [RuCl2(((CH

2OH)2PCH2)2NCH

3)2] (C2) complex

C1 C2 C3

120 saat

48 saat72 saat

Metal complexes

0

4

2

6

8

10

IC50

valu

es (m

M)

7981 7436

46254575

5302

0158

Figure 3 IC50

values of metal complexes at 48 72 and 120 h forA549 cells

the K562 cells was higher than that of the A549 cells TheTrxR activity of the A549 cells treated with C1 (4625mM72 h) decreased by 669 The TrxR activity of the A549cells treated with C3 (0158mM 48 h) decreased by 4173(Figure 7) In a reported study five different rutheniumcomplexes which had values of 1ndash100120583M IC

50inhibited the

TrxR enzyme of A549 cells by 50 to 100 [36] In ourstudy the inhibition of TrxR inA549 cells with C2 (7436mM72 h) was found to be 4882 (Figure 7) which is close tothe literature values As is well known the complexes of thesame ligand with various metals exhibit distinct inhibitionsdepending on the metal For instance the gold complexof hydrophilic alkyl phosphine ligands showed 100 TrxRinhibition in A549 cells whereas the silver complex of thesame ligand showed 70 inhibition [13] The C1 and C2 usedin this study demonstrated similar metal-originated resultsAfter incubation of the K562 cells C1 (2625mM 24 h) andC2 (4918mM 72 h) inhibited TrxR by 4455 and 3233respectively compared with the control (Figure 6)ThemetalcomplexC3 (0050mM 48 h) inhibited theTrxR ofK562 cellsby 4296 (Figure 8) and the TrxR of A549 cells by 4173(Figure 7) According to the results of this study the TrxRenzyme was inhibited via metal complexes which indicatedthat cell deathmay have occurred by an apoptotic pathway In2006 Zhao et al revealed that there was a linear correlationbetweenTrxR activity and cell life growth and apoptosis andthey demonstrated the inhibition of TrxRrsquos relationship with

0

1

2

3

4

5

C1 C2 C3

2625

3352

005

2396

4918

24 h48 h72 h

Metal complexesIC

50

valu

es (m

M)

Figure 4 IC50values ofmetal complexes at 24 48 and 72 h for K562

cells

apoptosis [17] AlthoughC2 inhibited the TrxR of K562 a littlebit more than it did that of A549 it actually showed goodinhibition for both types of cells (sim50) Witte et al in 2005defended the theory that some well-known anticancer agentssuch as Platinol Oxaliplatin and MHC have good inhibitoreffects against TrxR (50ndash60) [37]

The GPx activity of A549 cells as the control group wasfound to be 0152Umg protein After incubation of theA549 cells with C1 (4625mM 72 h) C2 (7436mM 72 h)and C3 (0158mM 48 h) their specific activities were calcu-lated as 0068Umg 0107Umg and 0046Umg proteinrespectively C3 had the most effective inhibition (6974)C1 reduced the TrxR enzyme activity in A549 cells by 5526and C2 reduced it by 2962 (Figure 7)

A metal complex has different effects on the sameenzymes in two different cells which indicates that metalcomplexes are cell selective In a study with gold phosphinecomplexes concerning cell selectivity most of the complexesshowed good inhibitory effects (more than 80) on the TrxRof A549 cells but percentages were different in the othertested cell lines In the same study it was observed thatenzymes were inhibited disparately by the metal complexesWhile a gold complex inhibited TrxR by more than 80 itinhibited both GPx and GR (glutathione reductase) enzymesof the same cell (A549) by under 50 [4] C1 caused a9167 decrease in the GPx activity of K562 while C2 and C3inhibited it completely (Figure 8)


H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)

Figure 5 Microscope images (20x) of K562 cells with controls (H2O and DMSO) and IC

50values of metal complexes for 48 and 72 hours

(a) Image of K562 cells at 72 h with distilled water as a control (b) and (c) images of the cells at 72 h with IC50values of C1 and C2 complexes

respectively (d) image of the cells at 48 h incubated with DMSO as a control of C3 complex (e) image of the cells at 48 h incubated withIC50value of C3

H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)

Figure 6 Microscope images (20x) of A549 cells with controls (H2O and DMSO) and IC


(a) image of A549 cells at 72 h with distilled water as a control (b) and (c) images of the cells at 72 h with IC50values of C1 and C2 complexes



0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()

Figure 7 Percentages of inhibition of TrxR GPx and Cat enzymesof A549 cells in the given period after incubation with IC

50values

of C1 C2 and C3 complexes

The catalase activities of both types of cells were found tobe at higher levels than those of the other two enzymesrsquo activ-ities The catalase activity of healthy human cells is alreadyhigher than GPx and TrxR activity The catalase-specificactivities of the A549 and K562 control cells (untreated withany complex) were found to be 85473Umg protein and35493Umg protein respectively and these activities wereaccepted as 100 activity Some of the tested compoundsshowed good catalase inhibitor properties After incubationof the A549 cells with C1 (4625mM 72 h) C2 (7436mM72 h) and C3 (0158mM 48 h) Cat was inhibited at 60393794 and 7047 respectively (Figure 7) ConsequentlyC3 was more effective at Cat inhibition in A549 cells thanwere C1 and C2 The same inhibition effect of C3 (6934)was observed in K562 cells (Figure 8) While C1 inhibited theCat ofK562 cells (6284) at almost the same level as it did theCat of A549 cells C2 inhibited the Cat of K562 cells (6565)more than it did the Cat of A549 cells

314 Statistical Comparison The statistical findings of thisstudy showed that while there was no significant differencebetween C2 and C3 in terms of TrxR inhibition both differedsignificantly from the control in the A549 cell line Therewas a remarkable difference in TrxR inhibition in the K562cell line between all three complexes and the control groupStatistically C1 inhibited the TrxR enzyme of K562 cellsfar more than C2 did but only slightly more than C3 did(Table 1)

There were significant differences between all three testedcompounds and the control group in terms of GPx inhibitionin the A549 cell line (Table 1) While the least difference wasseen between C2 and the control the greatest difference wasbetween C3 and the control The results showed no respect-able differences among the three complexes when applied toK562 cells but there was considerable difference between allthree of them and the control group

0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes

Figure 8 Percentages of inhibition of TrxR GPx and Cat enzymesof K562 cells in the given period after incubation with IC

50values


Themost significant differences of Cat inhibition in A549cells were between C3 and the control group The differencesbetween C1 and the control were greater than those foundbetween C3 and the control while those between C2 and thecontrol were the smallest There was significant disparitybetween all three of the compounds tested and the controlgroup with regard to Cat inhibition with the greatest differ-ence being between C3 and the control and the least betweenC1 and the control (Table 1)

32 Discussion In this study three original metal phosphinecomplexes were found to have variable cytotoxic activitiesat the mM level in A549 and K562 cells In addition tothe cytotoxic activities measurement of the activities of theenzymes TrxR GPx and Cat was attempted by use of metalcomplexes as inhibitors in both cells Phosphines are widelyused as a ligand group in the treatment of cancer via theinhibition of antioxidant enzymes [4 13 35 38] Platinumcomplexes belong to a group of metal complexes with goodTrxR-inhibiting properties [5 19 36] but ruthenium com-plexes are new in this area and they exhibit properties of goodinhibitors of TrxR and of some other related enzymes [3 38ndash40] Palladium complexes are one of the least commonmetalsto be used at the inhibition of these enzymes

Since high levels of the enzyme TrxR in many humancancer cell lines prevent anticancer agents from inducingapoptosis [39] inactivation of the TrxR enzyme was investi-gated in this studyThe inactivation of TrxR and of the closelyrelated enzyme glutathione peroxidase was also examinedAlthough both of the enzymes belong to the TrxR systemmetal complexes exhibit enzyme-selective or cell-selectiveinhibition according to the structure of the metal complex orligand It is already known that anticancer activity is closelyconnected to the chemical structure of the metal complexes[3] as the results of this study show In some reported studiesphosphine-Au derivatives inhibited TrxR more than GR [4142] For instance the results of our study demonstrated


that the TrxR inhibition effects of all three tested metalphosphine compounds were higher in A549 cells than theirGPx inhibition effects However the same complexes showedless inhibition in K562 cells

Most of the electrophilic compounds (like metal com-plexes) interact selectively and irreversibly with the SHSe-group at the active site of the enzyme thus becominginhibitors of TrxR [43ndash45] For example in one reportedstudy the crystal structure of phosphole-gold(I) complexesshowed a coordination bond between one phosphole-goldunit and the host enzyme And the second gold atom formeda linear SndashAundashS bond by losing its chloride atoms duringinteraction with the active site [39] Selenolates are softerdonor ligands compared to thiolates So they behave better assubstrates for some metal ions [5 46] Moreover otherproperties of ligands such as size charge and lipophilicityare important for biological activity

A sequential thiol-exchange mechanism in which thio-lates act as soft ligands forming covalent bonds with the softmetal ions is suggested to explain the reactivity and cellu-lar distribution of the tested phosphine compounds whichwere used for inactivation of the TrxR and GPx enzymesin A549 and K562 cells For instance Becker at al suggestthat selenocysteine residue is a suitable site for platination inhTrxR and that the mechanism involves a selenolate-thiolateexchange with the ligand of the Pt(II) compounds Thisinactivation viametal complexesmay also cause an inhibitionof DNA synthesis [18] On the other hand we estimate thatbecause they act as inhibitors of TrxR and related enzymes(ie Cat and GPx) Pd(II) Pt(II) and Ru(II) phosphinecomplexes may also make a modification of the redox stateof the cells Thus they cause an increased production ofH2O2and oxidation of the components of the enzyme system

therefore creating the conditions for cell death as reportedin some other studies [4 47ndash49] And it is known thata significant increase in the intracellular H

2O2production

causes apoptotic cell death in tumor cells [50] There is muchexperimental evidence that cancer cells are more susceptibleto H2O2-induced cell death than normal cells [51 52]

Increasing the cellular levels of H2O2by using H

2O2generat-

ing systems instead of direct application of H2O2may be one

of the most efficient ways to kill cancer cells [53]

4 Conclusion

Our studies have contributed to understanding of metalphosphinesrsquo new role in cancer cells The results have indi-cated that tested metal phosphine compounds were effec-tivein terms of cell death on K562 and A549 cell lines in vitroConsequently the Pd Ru and Pt complexes of phosphinesare potentially novel therapeutic agents for K562 and A549carcinoma cells It should be noted that the molecularstructures of the compounds subscribe to their cytotoxic andmentioned enzyme inhibitor effects

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to pediatric immunology ProfessorDr Mustafa Yılmaz for sharing his academic experiences Inaddition the authors are thankful to Cukurova UniversityrsquosHematology Laboratory for allowing them to use their facili-ties and Gaziantep Universityrsquos Cell Laboratory for providingcell lines

References

[1] W Zhong T Yan R Lim and L W Oberley ldquoExpression ofsuperoxide dismutases catalase and glutathione peroxidase inglioma cellsrdquo Free Radical Biology and Medicine vol 27 no 11-12 pp 1334ndash1345 1999

[2] D Deeb X Gao H Jiang et al ldquoOleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancercells through a ROS-dependent mechanismrdquo Biochemical Phar-macology vol 79 no 3 pp 350ndash360 2010

[3] X Yang L Chen Y Liu et al ldquoRuthenium methylimidazolecomplexes induced apoptosis in lung cancer A549 cells throughintrinsic mitochondrial pathwayrdquo Biochimie vol 94 no 2 pp345ndash353 2012

[4] V Gandin A P Fernandes M P Rigobello et al ldquoCancer celldeath induced by phosphine gold(I) compounds targetingthioredoxin reductaserdquo Biochemical Pharmacology vol 79 no2 pp 90ndash101 2010

[5] S Urig and K Becker ldquoOn the potential of thioredoxin reduc-tase inhibitors for cancer therapyrdquo Seminars in Cancer Biologyvol 16 no 6 pp 452ndash465 2006

[6] O Zava S M Zakeeruddin C Danelon H Vogel M Gratzeland P J Dyson ldquoA cytotoxic ruthenium tris(Bipyridyl) complexthat accumulates at plasmamembranesrdquoChemBioChem vol 10no 11 pp 1796ndash1800 2009

[7] F Linares M A Galindo S Galli M A Romero J A RNavarro and E Barea ldquoTetranuclear coordination assembliesbased on half-sandwich ruthenium(II) complexes noncovalentbinding to DNA and cytotoxicityrdquo Inorganic Chemistry vol 48no 15 pp 7413ndash7420 2009

[8] S H van Rijt A J Hebden T Amaresekera et al ldquoAmide link-age isomerism as an activity switch for organometallic osmiumand ruthenium anticancer complexesrdquo Journal of MedicinalChemistry vol 52 no 23 pp 7753ndash7764 2009

[9] X Meng M L Leyva M Jenny et al ldquoA ruthenium-containingorganometallic compound reduces tumor growth throughinduction of the endoplasmic reticulum stress gene CHOPrdquoCancer Research vol 69 no 13 pp 5458ndash5466 2009

[10] E Meggers G E Atilla-Gokcumen K Grundler C Frias andA Prokop ldquoInert ruthenium half-sandwich complexes withanticancer activityrdquo Dalton Transactions no 48 pp 10882ndash10888 2009

[11] N Farrell Y Qu U Bierbach M Valsecchi and E MentaCis-Platin Chemistry and Biochemistry of a Leading AnticancerDrug Wiley Weinheim Germany 1999

[12] C K Mirabelli R K Johnson C M Sung L Faucette KMuirhead and S T Crooke ldquoEvaluation of the in vivo anti-tumor activity and in vitro cytotoxic properties of auranofin acoordinated gold compound in murine tumor modelsrdquo CancerResearch vol 45 no 1 pp 32ndash39 1985

[13] C Santini M Pellei G Papini et al ldquoIn vitro antitumouractivity of water soluble Cu(I) Ag(I) and Au(I) complexes


supported by hydrophilic alkyl phosphine ligandsrdquo Journal ofInorganic Biochemistry vol 105 no 2 pp 232ndash240 2011

[14] N Manav A K Mishra and N K Kaushik ldquoTriphenyl phos-phine adducts of platinum(IV) and palladium(II) dithiocarba-mates complexes a spectral and in vitro studyrdquo SpectrochimicaActamdashPart AMolecular and Biomolecular Spectroscopy vol 60no 13 pp 3087ndash3092 2004

[15] CWetzel P CKunzMUKassack et al ldquoGold(I) complexes ofwater-soluble diphos-type ligands synthesis anticancer activ-ity apoptosis and thioredoxin reductase inhibitionrdquo DaltonTransactions vol 40 no 36 pp 9212ndash9220 2011

[16] O Rackham S J Nichols P J Leedman S J Berners-Price andA Filipovska ldquoA gold(I) phosphine complex selectively inducesapoptosis in breast cancer cells implications for anticancer ther-apeutics targeted to mitochondriardquo Biochemical Pharmacologyvol 74 no 7 pp 992ndash1002 2007

[17] F Zhao J Yan S Deng et al ldquoA thioredoxin reductase inhibitorinduces growth inhibition and apoptosis in five cultured humancarcinoma cell linesrdquo Cancer Letters vol 236 no 1 pp 46ndash532006

[18] D Mustacich and G Powis ldquoThioredoxin reductaserdquo Biochem-ical Journal vol 346 no 1 pp 1ndash8 2000

[19] K Becker C Herold-Mende J J Park G Lowe and R HeinerSchirmer ldquoHuman thioredoxin reductase is efficiently inhib-ited by (2210158406101584021015840-terpyridine)platinum(II) complexes Possibleimplications for a novel antitumor strategyrdquo Journal of Medici-nal Chemistry vol 44 no 17 pp 2784ndash2792 2001

[20] L Zhong and A Holmgren ldquoEssential role of selenium inthe catalytic activities of mammalian thioredoxin reductaserevealed by characterization of recombinant enzymes withselenocysteine mutationsrdquo Journal of Biological Chemistry vol275 no 24 pp 18121ndash18128 2000

[21] S Gromer J Wissing D Behne et al ldquoA hypothesis on the cat-alytic mechanism of the selenoenzyme thioredoxin reductaserdquoBiochemical Journal vol 332 part 2 pp 591ndash592 1998

[22] C H Williams Jr L David Arscott S Muller et al ldquoThiore-doxin reductase twomodes of catalysis have evolvedrdquoEuropeanJournal of Biochemistry vol 267 no 20 pp 6110ndash6117 2000

[23] P M Scarbrough K A Mapuskar D M Mattson D GiusW H Watson and D R Spitz ldquoSimultaneous inhibition ofglutathione- and thioredoxin-dependent metabolism is nec-essary to potentiate 17AAG-induced cancer cell killing viaoxidative stressrdquo Free Radical Biology and Medicine vol 52 no2 pp 436ndash443 2012

[24] C D Putnam A S Arvai Y Bourne and J A Tainer ldquoActiveand inhibited human catalase structures ligand and NADPHbinding and catalytic mechanismrdquo Journal of Molecular Biologyvol 296 no 1 pp 295ndash309 2000

[25] T Keles ldquoSynthesis of new water soluble aminomethylphos-phine and their metal complexes and investigation of their bio-logical activityrdquo Department of Chemistry Institue of Naturaland Applied Sciences University of Cukurova ADANA 2013

[26] B Akkus Synthesis of Transitıon Metal Complexes withMixed Ligands and Characterization Department of Chem-istry Institue of Natural and Applied Sciences University ofCukurova Adana Turkey 2009

[27] S Tardito C Isella E Medico et al ldquoThe thioxotriazole copper(II) complex A0 induces endoplasmic reticulum stress andparaptotic death in human cancer cellsrdquo Journal of BiologicalChemistry vol 284 no 36 pp 24306ndash24319 2009

[28] A Claiborne ldquoCatalase activityrdquo in Handbook of Methods forOxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985

[29] O H Lowry N J Rosebrough A L Farr and R J RondallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[30] E Beutler and E Beutler ldquoRed cell metabolismrdquo inAManual ofBiochemical Methods pp 66ndash68 Grone amp Stratton New YorkNY USA 1971

[31] S Gromer L D Arscott CHWilliams Jr R H Schirmeri andK Becker ldquoHuman placenta thioredoxin reductase Isolation ofthe selenoenzyme steady state kinetics and inhibition by ther-apeutic gold compoundsrdquo The Journal of Biological Chemistryvol 273 no 32 pp 20096ndash20101 1998

[32] D G Kleinbaum L L Kupper K E Muller and A NizanApplied Regression Analysis and Other Multivariate MethodsDuxbury Press Boston Mass USA 1998

[33] P E Garrou ldquoΔ119877ring contributions to 31P NMR parameters

of transition-metal-phosphorus chelate complexesrdquo ChemicalReviews vol 81 no 3 pp 229ndash266 1981

[34] F R Pavan G V Poelhsitz M I F Barbosa et al ldquoRuthe-nium(II) phosphinediiminepicolinate complexes inorganiccompounds as agents against tuberculosisrdquo European Journal ofMedicinal Chemistry vol 46 no 10 pp 5099ndash5107 2011

[35] G Lupidi L Avenali M Bramucci et al ldquoSynthesis propertiesand antitumor effects of a new mixed phosphine gold(I)compound in human colon cancer cellsrdquo Journal of InorganicBiochemistry vol 124 pp 78ndash87 2013

[36] P Mura M Camalli A Bindoli et al ldquoActivity of rat cytosolicthioredoxin reductase is strongly decreased by trans-[bis(2-amino-5-methylthiazole)tetrachlororuthenate(III)] first reportof relevant thioredoxin reductase inhibition for a rutheniumcompoundrdquo Journal of Medicinal Chemistry vol 50 no 24 pp5871ndash5874 2007

[37] A-B Witte K Anestal E Jerremalm H Ehrsson and E S JArner ldquoInhibition of thioredoxin reductase but not of glu-tathione reductase by the major classes of alkylating andplatinum-containing anticancer compoundsrdquo Free Radical Biol-ogy amp Medicine vol 39 no 5 pp 696ndash703 2005

[38] A K Renfrew A E Egger R Scopelliti C G Hartınger and PJ Dyson ldquoSynthesis and characterisation of the water solublebis -phosphine complex [Ru(1205786-cymene)(PPh

2(o-C6H4O)-

1198962minus 119875119874)(pta)]+ and an investigation of its cytotoxic effectsrdquo

Comptes Rendus Chimie vol 13 no 8-9 pp 1144ndash1150 2010[39] P C A Bruijnincx and P J Sadler ldquoNew trends for metal com-

plexes with anticancer activityrdquo Current Opinion in ChemicalBiology vol 12 no 2 pp 197ndash206 2008

[40] S M Guichard R Else E Reid et al ldquoAnti-tumour activity innon-small cell lung cancermodels and toxicity profiles for novelruthenium(II) based organo-metallic compoundsrdquo BiochemicalPharmacology vol 71 no 4 pp 408ndash415 2006

[41] S Gromer L D Arscott CHWilliams Jr R H Schirmeri andK Becker ldquoHumanplacenta thioredoxin reductaserdquoThe Journalof Biological Chemistry vol 273 no 32 pp 20096ndash20101 1998

[42] E Vergara A Casini F Sorrentino et al ldquoAnticancer thera-peutics that target selenoenzymes synthesis characterizationin vitro cytotoxicity and thioredoxin reductase inhibition of aseries of gold(I) complexes containing hydrophilic phosphineligandsrdquo ChemMedChem vol 5 no 1 pp 96ndash102 2010

[43] K Becker S Gromer R H Schirmer and S Muller ldquoThiore-doxin reductase as a pathophysiological factor and drug targetrdquo


European Journal of Biochemistry vol 267 no 20 pp 6118ndash61252000

[44] Y Nishinaka H Nakamura H Masutani and J Yodoi ldquoRedoxcontrol of cellular function by thioredoxin a new therapeuticdirection in host defencerdquoArchivum Immunologiae etTherapiaeExperimentalis vol 49 no 4 pp 285ndash292 2001

[45] J Nordberg and E S J Arner ldquoReactive oxygen speciesantioxidants and the mammalian thioredoxin systemrdquo FreeRadical Biology amp Medicine vol 31 no 11 pp 1287ndash1312 2001

[46] R G Pearson ldquoAcids and basesrdquo Science vol 151 no 3707 pp172ndash177 1966

[47] A Cingolani J V Hanna M Pellei et al ldquoCrystal structuresand vibrational and solution and solid-state (CPMAS) NMRspectroscopic studies in triphenyl phosphine arsine and stibinesilver(I) bromate systems (R

3E)xAgBrO

3(E = P As Sb 119909 =

1minus4)rdquo Inorganic Chemistry vol 42 no 16 pp 4938ndash4948 2003[48] K G Daniel P Gupta R H Harbach W C Guida and Q P

Dou ldquoOrganic copper complexes as a new class of proteasomeinhibitors and apoptosis inducers in human cancer cellsrdquoBiochemical Pharmacology vol 67 no 6 pp 1139ndash1151 2004

[49] C Marzano V Gandin A Folda G Scutari A Bindoli andMP Rigobello ldquoInhibition of thioredoxin reductase by auranofininduces apoptosis in cisplatin-resistant human ovarian cancercellsrdquo Free Radical Biology amp Medicine vol 42 no 6 pp 872ndash881 2007

[50] J L HirparaM-V Clement and S Pervaiz ldquoIntracellular acidi-fication triggered by mitochondrial-derived hydrogen peroxideis an effector mechanism for drug-induced apoptosis in tumorcellsrdquo Journal of Biological Chemistry vol 276 no 1 pp 514ndash5212001

[51] Q Chen M G Espey M C Krishna et al ldquoPharamacologicascorbic acid concentrations selectively kill cancer cells actionas a pro-drug to deliver hydrogen peroxide to tissuserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 102 no 38 pp 13604ndash13609 2005

[52] A M Evens P Lecane D Magda et al ldquoMotexafin gadoliniumgenerates reactive oxygen species and induces apoptosis insensitive and highly resistant multiple myeloma cellsrdquo Bloodvol 105 no 3 pp 1265ndash1273 2005

[53] M Lopez-Lazaro ldquoDual role of hydrogen peroxide in cancerpossible relevance to cancer chemoprevention and therapyrdquoCancer Letters vol 252 no 1 pp 1ndash8 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


during catalysis Many electrophilic compounds selectivelyand irreversibly modify the active site amino acids of TrxR[18ndash22] Since GPx has cysteine and selenocysteine residuein its active site the inactivation mechanism of GPx withmetal complexes resembles that of TrxR [23] Inactivation ofCat having histidine aspartic acid and phenyl alanine aminoacids in its active site might be carried out via coordinationof these amino acids to metal complexes or coordination ofother amino acids outside the catalytic site [24]

The cytotoxic activities of Ru(II) Pd(II) and Pt(II)phosphine complexes on A549 and K562 cell lines and theinactivation of the GPx Cat and TrxR enzymes of these cellsvia the metal phosphine complexes have been investigated inthis study

2 Material and Method

21 Synthesis All reactions were carried out under puri-fied nitrogen using standard Schlenk techniques Solventswere purified by standard methods and distilled undernitrogen prior to use [PdCl

2((CH2OH)2PCH2)2NCH3] (C1)

[PtCl2((Ph2PCH2)2NCH3) (timin)

2] (C3) was prepared

according to the procedure described in the literature [2526] [RuCl

2(((CH

2OH)2PCH2)2NCH3)2] was synthesized

according to the new method for this study NMR spectrawere recorded on a BrukerARX-300 spectrometer usingD

2O

and CDCl3as solvents In the NMR spectra the 1H and 13C

chemical shifts are reported in ppm downfield from the inter-nal standard SiMe

4 The 31P NMR (1215MHz) spectra were

recorded with 85 H3PO4as an external standard and pos-

itive chemical shifts lie downfield of the standard Elementalanalyses were performed by the Inonu University ResearchLaboratory in Malatya Turkey All reagents were purchasedfrom Aldrich Chemical Co and were used without furtherpurification

211 Synthesis of [RuCl2(((CH

2OH)2PCH2)2NCH3)2] (C2)

An aqueous solution (10mL) of [((CH2OH)2PCH2)2NCH3]

(2mmol) was added dropwise to the ruthenium precursor[Ru(COD)Cl

2] (095mmol) in toluene (10mL) at 40∘C with

constant stirring The mixture was further stirred for 48 hand the aqueous layer was separated from the organic layerThe aqueous solution was concentrated to 5mL in vacuumand evaporated slowly at room temperature to afford thegreen colored complex C2 at 78 yield

Anal Calcd for [RuCl2(((CH

2OH)2PCH2)2NCH3)2]

(C2) C 256H 58 N 43 Found C 271H 65 N409 1HNMR (D

2O 25∘C) 120575 36 (s 16H PCH

2OH) 120575 32

(s 8H PCH2N) 120575 25 (s 6H NCH

3) 31P-[1H]NMR (D

2O

25∘C) 120575 65 ppm (s Ru-P) 13C NMR (d-DMSO 25∘C) 12057559 ppm (s ndashPCH

2OH) 120575 47 ppm (m ndashNCH

3) 120575 245 (s

PCH2N) FT-IR (KBr cmminus1) 1250 (CndashOH) 1050 (NndashC) 1460

(CndashH) 1150 (PndashCndashN(R)ndashCndashP) 3200ndash3400 (OndashH)

22 Cell Culture In order to examine the anticancer activitiesof metal phosphine complexes two different human cancercell lines were used a K562 cell line provided by CukurovaUniversityrsquosHematologyClinic and anA549 cell line supplied

byGaziantepUniversityrsquos Cell Culture LaboratoryThe cancercells weremaintained in the logarithmic phase at 37∘C in a 5carbon dioxide atmosphere using a culture medium con-taining 10 fetal bovine serum 1 penicillin and 1 strep-tomycin RPMI-1640 (Sigma) (developed by Roswell ParkMemorial Institute)

23 MTT Test (Cytotoxicity Test) The growth inhibitoryeffect towards cancer cell lines was evaluated by means ofMTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide a yellow tetrazole) assay [27] Briefly 3 times 103 cellswell dependent upon the growth characteristics of the cellline were seeded in 96-well microplates in the growthmedium (100120583L) and then incubated at 37∘C in a 5 carbondioxide atmosphere After 24 h the medium was removedand replaced with a freshmedium containing the appropriateconcentrations (4762 3801 2857 1905 952 and 476iusdotmLminus1) of the phosphine metal complexes being studiedTriplicate cultures were established for each treatment After48 h each well was treated with 10 120583L of a 5mgsdotmLminus1 MTTsaline solution and following 5 h of incubation 100 120583L ofdimethyl sulfoxide (DMSO) was addedThe inhibition of cellgrowth induced by the phosphine metal complexes wasdetected after incubation (30 minutes) by measuring theabsorbance of eachwell at 570 nmusing aBio-Rad 680micro-plate reader The IC

50values represent the concentrations of

phosphinemetal complexes that reduce themean absorbanceat 570 nm to 50 of those in the untreated control wellscontaining only culture medium Thus cytotoxicities of thecompounds were determined All data were from at leastthree independent experiments and are expressed as mean plusmnstandard deviation

24 Catalase Activity Assay Catalase activity was measuredas described by Claiborne [28] 3 times 103 cellswell were platedin 96-well microplates in a growthmedium (100 120583L) and thenincubated at 37∘C in a 5 carbon dioxide atmosphere After24 h the medium was removed and replaced with a fresh onecontaining the IC

50values of the metal complexes for each

cell for 48 hThe cells werewashed twice in a phosphate buffersolution (PBS) and then collected a protease inhibitor cock-tail purchased from Sigma was added into the cell suspensionand sonicated in a 50mM potassium phosphate buffer (PBpH 70) on ice for 25 s (using Bandelin SONOPULS HD 2200ultrasonic homogenizer) at a 10 output and 80 duty cycleAfter the sonication procedure the protein concentrationwas determined by Lowry at alrsquos method for Cat and otherenzyme activity assays [29]

Cell extracts (200ndash400mg) were added to 3mL of a10mM H

2O2in 50mM potassium phosphate buffer (pH 78)

and the disappearance of H2O2(extinction coefficient (120576) of

00396 cm2sdot120583molminus1)was immediatelymeasured at 240 nm for60 s at 15 s intervals Catalase activity was expressed in unitsper grams of protein

25 Glutathione Peroxidase Activity Assay After applying thesame sonicating procedure to the cells each 5 120583L sample ofthe cell content was incubated for 10min at 37∘C in a 495 120583L




TrxRControl

C1C2C3


TrxRControl

C1C2C3


GPxControl

C1C2C3


GPxControl

C1C2C3


CatControl

C1C2C3


CatControl

C1C2C3




2O2The decrease




50values of



31 Results





complex [RuCl2(((CH



2(((CH



3 Consequently when




2ndashP protons





C1

PdCl

Cl

P

P

N

HOH2C

HOH2C

H3C

CH2OH

CH2OH

(a)

P

N

P

Ru

P

P

N

Cl

Cl

C2

HOH2CHOH2C

HOH2C

HOH2C

H3C

CH2OHCH2OH

CH3

CH2OH

CH2OH

(b)

N

PPt

HN

O

N

O

N

NH

O O

C3

H3C

CH3

(H3C)3

PPh2

Ph2

(c)





50








P

N

P

Ru

P

PP

N

P

RuCl

ClCl

+ N

Cl

HOH2C

HOH2C

CH2OH HOH2C HOH2C

HOH2CHOH2C

CH2OH CH2OH

CH2OHCH2OHCH2OH

H3C H3C CH3

48h40

∘C


2OH)2PCH2)2NCH

3)2] (C2) complex

C1 C2 C3

120 saat

48 saat72 saat

Metal complexes

0

4

2

6

8

10

IC50

valu

es (m

M)

7981 7436

46254575

5302

0158

Figure 3 IC50



50inhibited the


0

1

2

3

4

5

C1 C2 C3

2625

3352

005

2396

4918

24 h48 h72 h

Metal complexesIC

50

valu

es (m

M)


cells





H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)





H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)






0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




TrxRControl

C1C2C3


TrxRControl

C1C2C3


GPxControl

C1C2C3


GPxControl

C1C2C3


CatControl

C1C2C3


CatControl

C1C2C3




2O2The decrease




50values of



31 Results





complex [RuCl2(((CH



2(((CH



3 Consequently when




2ndashP protons





C1

PdCl

Cl

P

P

N

HOH2C

HOH2C

H3C

CH2OH

CH2OH

(a)

P

N

P

Ru

P

P

N

Cl

Cl

C2

HOH2CHOH2C

HOH2C

HOH2C

H3C

CH2OHCH2OH

CH3

CH2OH

CH2OH

(b)

N

PPt

HN

O

N

O

N

NH

O O

C3

H3C

CH3

(H3C)3

PPh2

Ph2

(c)





50








P

N

P

Ru

P

PP

N

P

RuCl

ClCl

+ N

Cl

HOH2C

HOH2C

CH2OH HOH2C HOH2C

HOH2CHOH2C

CH2OH CH2OH

CH2OHCH2OHCH2OH

H3C H3C CH3

48h40

∘C


2OH)2PCH2)2NCH

3)2] (C2) complex

C1 C2 C3

120 saat

48 saat72 saat

Metal complexes

0

4

2

6

8

10

IC50

valu

es (m

M)

7981 7436

46254575

5302

0158

Figure 3 IC50



50inhibited the


0

1

2

3

4

5

C1 C2 C3

2625

3352

005

2396

4918

24 h48 h72 h

Metal complexesIC

50

valu

es (m

M)


cells





H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)





H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)






0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


C1

PdCl

Cl

P

P

N

HOH2C

HOH2C

H3C

CH2OH

CH2OH

(a)

P

N

P

Ru

P

P

N

Cl

Cl

C2

HOH2CHOH2C

HOH2C

HOH2C

H3C

CH2OHCH2OH

CH3

CH2OH

CH2OH

(b)

N

PPt

HN

O

N

O

N

NH

O O

C3

H3C

CH3

(H3C)3

PPh2

Ph2

(c)





50








P

N

P

Ru

P

PP

N

P

RuCl

ClCl

+ N

Cl

HOH2C

HOH2C

CH2OH HOH2C HOH2C

HOH2CHOH2C

CH2OH CH2OH

CH2OHCH2OHCH2OH

H3C H3C CH3

48h40

∘C


2OH)2PCH2)2NCH

3)2] (C2) complex

C1 C2 C3

120 saat

48 saat72 saat

Metal complexes

0

4

2

6

8

10

IC50

valu

es (m

M)

7981 7436

46254575

5302

0158

Figure 3 IC50



50inhibited the


0

1

2

3

4

5

C1 C2 C3

2625

3352

005

2396

4918

24 h48 h72 h

Metal complexesIC

50

valu

es (m

M)


cells





H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)





H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)






0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


P

N

P

Ru

P

PP

N

P

RuCl

ClCl

+ N

Cl

HOH2C

HOH2C

CH2OH HOH2C HOH2C

HOH2CHOH2C

CH2OH CH2OH

CH2OHCH2OHCH2OH

H3C H3C CH3

48h40

∘C


2OH)2PCH2)2NCH

3)2] (C2) complex

C1 C2 C3

120 saat

48 saat72 saat

Metal complexes

0

4

2

6

8

10

IC50

valu

es (m

M)

7981 7436

46254575

5302

0158

Figure 3 IC50



50inhibited the


0

1

2

3

4

5

C1 C2 C3

2625

3352

005

2396

4918

24 h48 h72 h

Metal complexesIC

50

valu

es (m

M)


cells





H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)





H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)






0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


H2O 72h

(a)

C1 72 h

(b)

C2 72 hC2 72 hC2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)





H2O 72h

(a)

C1 72 h

(b)

C2 72 h

(c)

DMSO 48 h

(d)

C3 48 h

(e)






0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

669

48824173

5526

2961

69746039

3794

7074

TrxRGPxCat

Metal complexes

Inhi

bitio

n of

enzy

mes

()


50values





0

20

40

60

80

100

C1 (Pd) C2 (Ru) C3 (Pt)

44553233

4296

9167100 100

6284 6565 6934

TrxRGPxCat

Inhi

bitio

n of

enzy

mes

()

Metal complexes


50values










2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






2O2production



2O2generat-



4 Conclusion




Acknowledgments


References










































2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





























2(o-C6H4O)-














3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







3E)xAgBrO

3(E = P As Sb 119909 =

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article antioxidant enzyme inhibitor role...

Documents