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research article 

Biological evaluation of trans- 
dichloridoplatinum(ll) complexes with 3- and 
4-acetylpyridine in comparison to cisplatin 

Lana Filipovic 1 , Sandra Arandelovic 1 , Nevenka Gligorijevic 1 , Ana Krivokuca 1 , 

Radmila Jankovic 1 ,Tatjana Srdic-Rajic 1 , Gordana Rakic 2 , Zivoslav Tesic 2 , Sinisa Radulovic 1 

1 1nstitute for Oncology and Radiology of Serbia, Belgrade, Serbia 
2 Faculty of Chemistry, University of Belgrade, Belgrade, Serbia 

Radiol Oncol 2013; 47(4): 346-357. 

Received 29 March 2013 
Accepted 25 June 2013 

Correspondence to: Sinisa Radulovic, M.D., Ph.D., Scientific Director, Department for Experimental Oncology, Institute for Oncology and 
Radiology of Serbia, Pasterova 14, Belgrade 11000, Serbia. Tel: +38111 2067 434; Fax: +381 11 2067 294; E-mail: 

Disclosure: No potential conflicts of interests were disclosed. 

The paper was presented at the 7 th Conference of Experimental and Translational Oncology, 20-24 th April 2013, Portoroz, Slovenia ( 
coorganised and supported by COST TD1 104 Action ( 

Background. In our previous study we reported the synthesis and cytotoxicity of two frans-platinum(ll) complexes: 
fr"ans-[PrCI 2 (3-acetylpyridine) 2 ] (1) and frans-[PtCI 2 (4-acetylpyridine) 2 ] (2), revealing significant cytotoxic potential of 
2. In order to evaluate the mechanism underlying biological activity of both frans-Pt(ll) isomers, comparative studies 
versus cisplatin were performed in HeLa, MRC-5 and MSI cells. 

Materials and methods. The cytotoxic activity of the investigated complexes was determined using SRB assay. The 
colagenolytic activity was determined using gelatin zymography, while the effect of platinum complexes on matrix 
metalloproteinases 2 and 9 mRNA expression was evaluated by quantitative real-time PCR. Apoptotic potential and 
cell cycle alterations were determined by FACS analyses. Western blot analysis was used to evaluate the effect on 
expression of DNA-repair enzyme ERCC1, and quantitative real-time PCR was used for the ERCC1 mRNA expression 
analysis. In vitro antiangiogenic potential was determined by tube formation assay. Platinum content in intracellular 
DNA and proteins was determined by inductively coupled plasma-optical emission spectrometry. 
Results. Compound 2 displayed an apparent cytoselective profile, and flow cytometry analysis in HeLa cells indicat- 
ed that 2 exerted antiproliferative effect through apoptosis induction, while 1 induced both apoptosis and necrosis. 
Action of 1 and 2, as analyzed by quantitative real-time PCR and Western blot, was associated with down-regulation 
of ERCC1. Both frans-complexes inhibited MMP-9 mRNA expression in HeLa, while 2 significantly abrogated in vitro 
tubulogenesis in MSI cells. 

Conclusions. The ability of 2 to induce multiple and selective in vitro cytotoxic effects encourages further investiga- 
tions of frans-platinum(ll) complexes with substituted pyridines. 

Keywords: angiogenesis; apoptosis; MMPs; MRC-5; trans-platinum(ll) 


Cisplatin (CDDP) represents the basis of combi- 
nation chemotherapy regimens in solid rumors, 
although main drawbacks to its successful ap- 
plication are development of resistance and toxic 
side effects. 12 Search for CDDP analogues with 
improved pharmacological properties by manipu- 

lation of the structure of ligands, has achieved a 
reduction in toxicity, but obtained limited success 
in broadening spectrum of activity. 35 However, 
novel classes of platinum complexes including 
frans-Pt(II) compounds with planar amine ligands 
are able to exert cytotoxicity, equivalent or better 
to that of CDDP, and posses different mechanisms 
of antitumor action. 6-10 Cytotoxicity data of 107 

Radiol Oncol 20 1 3; 47(4): 346-3S7. 

doi: 1 0.2478 raon-20 1 3-0050 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 


platinum compounds from the NCI human tumor 
panel recognized frans-platinum complexes, of 
structural formula [PtCl 2 (L)(L')] with planar amine 
ligands (L and L' may be the same or different), as 
unique group of trans-platinum drugs that had cy- 
totoxicities similar to that of their cfs-isomers and 
CDDP, and possessed a novel cytotoxicity pro- 
file. 11 Search for new platinum compounds with 
the complementary or wider range of activity than 
CDDP, whose actions would be more selective to- 
ward cancer comparing to normal cells, and which 
would possess different targets than the traditional 
CDDP, is always attractive topic. 7 Formation and 
persistence of DNA-adducts are considered vital 
in platinum drug induced cytotoxicity, and type 
of DNA-damage is determined by the nature of 
platinum-coordinating ligands. 41213 However, cel- 
lular sensitivity to platinum complexes is multifac- 
torial, with some of major mechanisms being the 
proficiency of the cellular mechanism for adducts 
recognition and repair, and the ability of cells to 
reduce intracellular platinum, due to deactivation 
by sulphur-containing biomolecules and/or drug 
efflux. 14-16 Numerous studies imply the significance 
of copper transporters in regulating cellular phar- 
macology of CDDP by mediating its uptake and ef- 
flux in different cell lines. 17 

Structure-activity studies up to date demon- 
strated that bulky amine carrier ligands, such as 
pyridine, appear to sterically hinder approach of 
incoming nucleophiles to the axial positions of the 
platinum center, thus reducing deactivation of plat- 
inum by sulphur-containing biomolecules. 18 Trans- 
orientation of planar pyridines seems to contribute 
to greater affinity of complexes for interstrand 
cross-link formation and DNA conformational dis- 
tortion, comparing to c/s-isomers, leading to activa- 
tion of different DNA-repair mechanisms. 19 21 

Recent studies demonstrated that fine tuning of 
the biological activity of trans-platinum pyridines 
may be achieved by different positioning of the 
substituents such as: methyl-group and 3- or 4-hy- 
droxymethyl-group, on the pyridine ring. 19 ' 20 ' 22 

In our previous investigations, we have synthe- 
sized and characterized two frans-platinum(II) com- 
plexes, of structural formula frans-[PtCl 2 (L) 2 ] with 
substituted pyridine ligands, L=n-acetylpyridine, 
(n = 3 or 4) (Figure 1). Cytotoxicity evaluation on the 
panel of tumor cell lines revealed potential of frans- 
[PtCl 2 (4-acetylpyridine) 2 ] to exert activity in low 
micromolar range, with the highest cytotoxicity in 
HeLa cells, comparable to that of CDDP. 23 Aim of 
this study was to investigate the molecular mecha- 
nisms underlying the in vitro biological activity of 


FIGURE 1. Structures of the investigated frans-platinum (II) complexes: 
trans-[PtCI 2 (3-acetylpyridine) 2 ] 1: rrans-[PtCI 2 (4-acetylpyridine) 2 ] 2. 

frans-[PtCl 2 (4-acetylpyridine) 7 ] (complex 2) and 
its less cytotoxic structural isomer frans-[PtCl 2 (3- 
acetylpyridine) 2 ] (complex 1), and to understand 
possible relations to their structural characteristics, 
such as the position of the acetyl substituent on the 
pyridine ring. Mechanistic studies were performed 
in comparison to CDDP in human cervix carcinoma 
cell line (HeLa), and two other cell lines: human 
normal lung fibroblast (MRC-5) cell line, which was 
used as a non-cancerous model system for in vitro 
toxicity evaluation, and murine endothelial cells 
immortalized by infection with a retrovirus encod- 
ing SV40 large T antigen (MSI), as a model system 
for in vitro testing of antiangiogenic effect. 24 In or- 
der to test if the cytotoxic responses produced by 
compounds 1 and 2 in HeLa cells correlated with 
the platinum content in cellular DNA and to evalu- 
ate the mechanism of cytotoxic action, we studied 
the ability of complexes to bind intracellular DNA 
and proteins, and to induce DNA-damage related 
response, cell cycle alterations and apoptosis. 

Based on the literature data reporting inhibitory 
effect of some platinum(II) compounds on matrix 
metalloproteinases (MMP) activity, we assumed 
that frans-[PtCl 2 (n-acetylpyridine) 2 ] (n = 3 or 4), 
may possess ability to modulate diverse cellular 
processes, including those related to the cancer cell 
angiogenic and metastatic behaviour. 25-27 Thus, in 
the final part of our study, we analyzed if the tested 

Radiol Oncol 2013; 47(4): 346-357. 


Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 

complexes affected gelatinolitic activity and mRNA 
expression of secreted forms of MMP-2 and MMP- 
9, or abrogated process of angiogenesis in vitro. 

Materials and methods 


Platinum complexes frans-[PtCl 2 (3-acetylpyridine) 2 ] 
(complex 1) and frans-[PtCl 2 (4-acetylpyridine) 2 ] 
(complex 2) (Figure 1) were synthesized and charac- 
terized by IR and NMR, as previously described. 23 

Cytotoxic activity 
Cell culture 

HeLa and MRC-5 cells cells were maintained as 
monolayer culture in nutrient medium, Roswell 
Park Memorial Institute 1640 medium (RPMI1640), 
(Sigma- Aldrich Co). 28 MSI cells were maintained as 
monolayer culture in nutrient medium, Dulbecco's 
Modified Eagle Medium (DMEM), (Sigma- Aldrich 
Co). Nutrient medium conditions and cell mainte- 
nance procedures were explained previously. 29 

In vitro cytotoxicity assay (SRB) 

Cells were seeded into 96-well plates (Thermo 
Scientific Nunc ™), in number of 7000 cells per well 
(c/w) for MSI and 5000 cells per well for MRC-5, 
and left for 24 h before complexes 1, 2 and CDDP 
were added. Preparation of test solutions was 
performed immediately before experiments by 
dissolving in DMSO. The cells were treated with 
serial dilutions of the studied compounds for 48 
h. Final concentrations achieved per wells were 1 
pM, 3 pM, 10 pM, 30 pM and 100 pM. Each con- 
centration was tested in triplicates, and the final 
concentration of DMSO solvent never exceeded 
0.33%. Cytotoxicity of the investigated platinum 
complexes, and CDDP as a referent compound, 
was evaluated after 48 h of continuous action, us- 
ing sulforhodamine B (Sigma-Aldrich Co.) colori- 
metric assay. 30 The percentages of surviving cells 
relative to untreated controls were determined. 
The IC 50 value, defined as the concentrations of the 
compound causing 50% cell growth inhibition, was 
estimated from the dose-response curves. 

Flow cytometric analysis of cell cycle 
phase distribution 

Quantitative analysis of cell cycle phase distribu- 
tion was performed by flow-cytometric analysis of 
the DNA content in fixed HeLa cells, after stain- 

ing with propidium iodide (PI). 31 Cells were seed- 
ed at density of 2x1 0 5 into 6-well plates (Thermo 
Scientific Nunc™), and grown in nutrient medium. 
After 24 h cells were exposed to the investigated 
compounds 1, 2 and CDDP for 24 h, at concen- 
trations corresponding to IC 50 or 1.5xIC 50 . The de- 
tailed procedure was previously described. 29 Cell 
cycle phase distribution was analyzed using a 
fluorescence activated cell sorting (FACS) Calibur 
Becton Dickinson flow cytometer and Cell Quest 
computer software. 

Statistical analysis 

Calculations of mean, SD, and p values were per- 
formed on triplicate experiments. The Student t- 
test was used to calculate p-values for comparison. 
The significant statistics was set at a p-value <0.05 
(Stata Software). 

Annexin V-FITC apoptotic assay 

Quantitative analysis of apoptotic and necrotic cell 
death induced by the investigated platinum com- 
plexes and CDDP, as a referent compound, was 
performed by Annexin V-FITC apoptosis detection 
kit, according to the manufacturer's instructions 
(BD Biosciences). Precisely, 2xl0 5 HeLa cells treat- 
ed with lxIC 50 of the tested compounds and CDDP 
for 4 and 24 h and the analysis was performed as 
previously reported. 29 

Measurement of platinum binding to 
intracellular DNA or proteins using 

Binding of platinum(II) to cellular DNA and pro- 
teins was analyzed in HeLa cells, using inductively 
coupled plasma optical emission spectrometry 
(ICP-OES). 6xl0 6 cells were seeded into 75 cm 2 
dish (Thermo Scientific Nunc™) and treated with 
the investigated complexes in concentrations cor- 
responding to 0.5xIC 50 . Following 6 or 24 h, cells 
were harvested by scraping, washed by ice cold 
PBS and cell pellet was collected by centrifuga- 
tion at 2000 rpm, 10 min. DNA and proteins were 
isolated using TRI Reagent® (Sigma-Aldrich Co.) 
according to the manufacturer's procedure and 
concentrations were determined spectrophoto- 
metrically by measuring absorbance at A260 and 
A280 nm respectively (Eppendorf BioPhotometer 
6131). Platinum(II) levels were determined in iso- 
lated DNA and protein fractions according to the 
standard procedure, using Thermo Scientific iCAP 
6500 Duo ICP (Thermo Fisher Scientific). 

Radiol Oncol 20 1 3; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 


Quantitative real-time PCR (qRT-PCR) 

Sample preparation for qRT-PCR; RNA extraction 
and cDNA synthesis 

6xl0 6 HeLa cells were seeded in nutrient medium 
and after 24 h treated with the investigated com- 
plexes 1, 2 or CDDP at concentrations correspond- 
ing to 0.5xIC 50 , for 6 and 24 hours. After treatment, 
cells were washed with ice cold PBS and harvest- 
ed by scraping, while cell pellet was collected by 
centrifugation. Total RNA was isolated using TRI 
Reagent® (Sigma-Aldrich Co.) according to manu- 
facturer's recommendations. RNA extraction in 
details and cDNA synthesis were described ear- 
lier. 29 

Quantitative real-time PCR 

The analysis of gene expression level of several genes 
and GAPDH (endogenous control) was done by us- 
ing TaqMan® Gene Expression Assays for human 
genes (assay ID Applied Biosystem, listed as follow- 
ing: Hs_01012155_gl (ERCC1); Hs_01548727_ml 
(MMP-2); Hs_00234579_m (MMP-9); Hs_00355782_ 
ml (GAPDH)) on ABI PRISM® 7500 PCR instru- 
ment (Applied Biosy stems). PCR was performed as 
previously reported. 29 

Western blot 

HeLa cells were treated with the investigated 
platinum complexes 1 and 2 or CDDP for 6 h, at 
concentrations corresponding to the 0.5xIC 50 val- 
ues obtained for 48 h of continuous treatment. 
Cells maintained in nutrient medium were used 
as the untreated control. Sample preparation and 
the analysis were performed as described in the 
previous study. 2932 Purified mouse anti-human 
ERCC1 monoclonal antibody (1:500 dilution) (BD 
Biosciences Pharmingen) was used, as well as the 
secondary anti-mouse IgG-peroxidase conjugated 
antibody (1:2000 dilution) (Sigma-Aldrich Co.). 

Gelatin zymography 

Effect of the investigated frcms-Pt(II) complexes 
and CDDP on gelatinolitic activity of secreted 
matrix metalloproteinases MMP-2 and MMP-9 in 
HeLa was analyzed by zymography in 10% SDS- 
polyacrylamide gels impregnated with 0.1% gela- 
tin. 33 HeLa cells were treated with the complexes 
(0.5xIC 50 ) for 6 h in serum-free medium and the 
precise conditions, as well as the procedure have 
been given previously. 29 The gelatinolytic activities 
were visualized as clear transparent bands against 

□ HeLa 
■ MRC5 

complex 1 complex 2 


Complex 1 

Complex 2 



. ~< >.•■'■ . 

J&* * 


FIGURE 2. A Diagram presenting cytotoxicity of the tested agents and cisplatin 
in terms of IC 50 values, obtained for 48 h of drug action, by SRB assay. IC 50 values 
present average (±SD) obtained from three or more independent experiments. 
Asterisks denotes p values, when comparing MRC-5 cells to HeLa cells, by ANOVA 
test: 1 (*) p > 0.05: 2 (**) p < 0.001: CDDP (*) p > 0.05: B Micrographs of HeLa cells 
or MRC-5 cells exposed to equimolar (5 mMJ concentration of tested platinum 
complexes 1, 2 or CDDP, following 24 h treatment, versus control (non treated 
cells). Micrographs are one representative experiment selected of three and were 
obtained with Olympus digital camera connected to the inverted microscope (Carl 
Zeiss, Jena, Germany, objective 6.3/0.20). 

the blue background of Coomassie brilliant blue- 
stained gelatin. 

Tube formation assay (in vitro 
angiogenesis assay) 

Potential of traHS-platinum(II) complexes and 
CDDP to inhibit angiogenesis in vitro was analyzed 
by tube formation assay in MSI cells. MSI cells, 
when plated into gel of basement membrane pro- 
teins, rapidly organize into multicellular tube-like 
structures, while antiangiogenic effect of the tested 
compounds is observed as the reduction of tube 
formation. 24 Briefly, 24-well plates were coated 
with collagen and allowed to solidify at 37°C 1 h. 
MSI cells were seeded into wells (1x10 s c/w) in nu- 
trient medium. Complexes 1 and 2 were added 2 h 
after cells settled, at concentrations corresponding 
to 0.03xIC 5(y which was non-toxic to the cells. Tube 
formation was observed periodically over time un- 
der microscope and representative pictures were 

Radiol Oncol 2013; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 





□complex 1 [IC50J 
■ complex 2 [IC50] 
■CDDP [IC50] 



Cell cycle phase 


□ complex 1 [1.5 XIC50] 
■ CDDP[1 5xlC50] 



GO/01 S G2M 

Cell cycle phase 

FIGURE 3. Diagrams presenting cell cycle phase distribution of treated HeLa cells, 
obtained by flow-cytometric analysis of the DNA content in fixed cells, after staining 
with PI. HeLa cells were collected following 24 h treatment with tested complexes 
or cisplatin at concentration corresponding to A IC 50 and B 1 .5xlC 50 . Bar graphs 
represent mean ± SD in at least three independent experiments. Asteriks (*) denotes 
p values < 0.05, calculated by Student t-test, indicating statisticaly significant 
differences (Sfata Software). 

taken after 24 h incubation with Olympus digital 
camera connected to the inverted microscope (Carl 
Zeiss, Jena, Germany, objective 6.3/0.20). 

Morphological examination by light 

HeLa cells (50000 c/w) and MRC-5 cells (125000 
c/w), were seeded into 6-well plates (Thermo 
Scientific Nunc™), in the corresponding nutrient 
medium, and after 24 h of growth cells were ex- 
posed to complexes 1, 2 or CDDP, at equimolar con- 
centrations of 5 mM. Following 24 h of treatment, 
cells were observed under the light microscope 
and photographs were taken with Olympus digital 
camera connected to the inverted microscope (Carl 
Zeiss, Jena, Germany, objective 6.3/0.20).. 

Statistical analysis 

Statistical comparison of IC 50 values in MRC-5 cell 
line versus HeLa and MSI cell line, was performed 

using one way statistical analysis of variance (one- 
way ANOVA - GraphPad Software). IC 50 values 
were determined as mean ± SD (standard devia- 
tion) of three or more independent experiments. 


In vitro cytotoxicity assay (SRB) 

In order to further investigate cytotoxic and cy- 
toselective potential of the two trans-platinum iso- 
mers, in comparison to CDDP, growth inhibitory 
study was performed in MRC-5 cells, which were 
used as non-cancerous model for in vitro toxicity 
evaluation; and MSI cells as in vitro model for test- 
ing of antiangiogenic effect. Cytotoxicity of the 
complexes summarized in terms of IC 50 values, is 
presented in Figure 2A. IC 50 values (mM) obtained 
for 48 h of continuous drug action in MRC-5 cells, 
may be arranged in increasing order as following: 
15.4 ± 3.1 mM, for CDDP; 40.0 ± 11.1 mM for com- 
plex 1; and 56.4 + 5.0 mM for 2, indicating lower 
toxicity of frans-complexes in non-cancerous cell 
model comparing to CDDP. Particularly, complex 
2 exerted less cytotoxicity in MRC-5 cells than in 
HeLa, by a factor of approximately four-fold, in- 
dicating significant cytoselective potential toward 
neoplastic cells (p < 0.001). Both rrans-complexes 
exhibited poor activity, in MSI cells with IC 50 val- 
ues being: 76.3 ± 0.5 mM for complex 2; 34.5 ± 7.8 
mM for complex 1; comparing to CDDP (IC 50 18.6 
± 5.4 mM). 

Morphological examination 

The results of the morphological analysis of HeLa 
and MRC-5 cells are presented in Figure 2B, as mi- 
crographs obtained following 24 h agents action. 
Results indicated that in the presence of 5 mM of 
complex 2, concentration which corresponded to 
IC 50 value in HeLa cells, viability of MRC-5 cells was 
not significantly altered, suggesting cytoselective 
potential toward neoplastic HeLa cells. Oppositely, 
complex 1 haven't exerted cytoselective potential, 
as observed in Figure 2B. Morphological changes 
of MRC-5 cells, such as: cell shrinkage and detach- 
ment, following equimolar treatment with CDDP, 
were indicative for apoptosis. 

Determination of cell cycle perturbation 
by flow cytometry 

The potential of the tested complexes to induce cell 
cycle alterations in comparison to CDDP in HeLa 
cells, was examined by flow cytometry using stain- 
ing with PI. Results are presented as diagrams of 

Radiol Oncol 20 1 3; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 


□ control ncomplexl Bcomplex2 "CDDP 

FIGURE 4. A Dot plot diagrams obtained by flow-cytometric 
analysis of treated HeLa cells after dual staining with Annexin 
V-FITC and PI. Annexin V-FITC/PI staining was monitored 
overtime, following 4 and 24 hours in HeLa cells exposed to 
complex 1 , 2 or CDDP at concentrations corresponding to IC S0 . 
Representative dot plots of three independent experiments are 
given, presenting intact cells at lower-left quadrant, FITC(-)/ 
Pl(-); early apoptotic cells at lower-right quadrant, FITC(+)/ 
Pl(-); late apoptotic or necrotic cells at upper-right quadrant, 
FITC(+)/PI(+); and necrotic cells at upper-left quadrant, FITC(-)/ 
Pl(+). B Apoptosis and necrosis were quanitified by FACS after 
Annexin V-FITC and PI labeling; bar graphs represent mean ± 
SD in at least three independent experiments. 

Apoptosis 4h Necrosis 4h Apoptosis 24h Necrosis 24h 

cell distribution over the cell cycle phases after 24 
h of agent action, where Figure 3A shows effects 
of the complexes at concentration corresponding 
to IC 50 and Figure 3B shows effects of the com- 
plexes at concentration corresponding to 1.5xIC 50 . 
Complex 1, induced arrest in the S phase of cell 
cycle at concentrations corresponding to 1.5 x 
IC 50 , but less than CDDP (Figure 3B). Complex 1 
induced decrease of cell percentage in the G0/G1 
phase in concentration dependent manner, com- 
paring to the non-treated control. CDDP induced 
dose-dependent arrest in the S phase of cell cycle, 
and decrease of cell progression through G2/M 
phase (Figures 3A and 3B). 

The Student t-test showed that the difference is 
considered to be statisticaly significant only for GO/ 
Gl cells when two groups of cell cycle results were 
compared (control cells compared to cells treated 

with 1.5xIC 50 . complex 1, and control cells com- 
pared to cells treated with 1.5xIC 50 . CDDP (p<0.05, 
Stata Software)). 

Quantification of apoptosis by annexin 
V-FITC binding 

Potential of the investigated complex to induce 
apoptosis in HeLa cells was assessed by flow cy- 
tometry using Annexin V-FITC and PI dual stain- 
ing. Dot plots are presented in Figure 4A, while 
Figure 4B reports the results of a representative ex- 
periment as percentages of apoptotic cells (Annexin 
V-FITC positive and PI negative) and necrotic cells 
(Annexin V-FITC negative and PI positive) meas- 
ured periodically at 4 and 24 h. Data obtained indi- 
cated that complex 2 caused 15.5% of apoptosis in 
HeLa cells following 24 h of action, while the per- 

Radiol Oncol 2013; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 

□ complex 1 ■ complex 2 ■ CDDP 




24 h 


6 h 24 h 

FIGURE 5. Diagrams presenting quantitative determination of platinum(ll) content in 
DNA and proteins in HeLa cells, obtained by ICP-OES analysis, following 6 h and 24 
h of action of 1, 2 or CDDP; A Platinum content in cellular DNA; B Platinum content 
in cellular protein fraction (pg Pt/mg proteins). Bar graph represent mean ± SD of 
three independent experiments. 

centage of necrotic cells was negligible. Kinetics, 
as well as the degree of apoptosis induction, was 
comparable to CDDP. Complex 1 at concentration 
of IC 50 initiated early apoptotic cell death after 4 
and 24 h action, where apoptotic cell population 
represented 14.2% and 15.5% of total cells, respec- 
tively. Though, after 24 h of action more than 50% 
of total cell population underwent cell death in ei- 
ther apoptotic or necrotic manner. 

Determination of the platinum(ll) 
binding to intracellular DNA and proteins 

Intracellular platinum(II) distribution among DNA 
and protein fractions in HeLa cells treated with eq- 
uitoxic concentrations of investigated complexes 
for 6 and 24 h, was analyzed using ICP-OES analy- 

sis, and results are presented in Figure 5. Levels 
of platinum(II)-DNA binding (Figure 5A), varied 
between the investigated complexes, especially 
following short-term (6 h) treatment, when plati- 
num content (pg Pt/mg DNA) decreased in order: 
21 ± 2.5 (complex 1); 14.5 ±0.4 (CDDP) and 6.4±1.1 
(complex 2). Both CDDP and 1 seemed to be more 
effcient in promoting cellular DNA binding com- 
paring to complex 2, though differences in double- 
stranded DNA platination affinity between com- 
plex 2 and CDDP were in accordance to the recent 
study. 34 Platinum(II)-DNA content, decreased in 
time-dependent manner, and reached comparable 
levels following 24 h of action. Results of the ICP- 
OES analysis of platinum(II) content in the protein 
fraction (Figure 5B), indicated that 1 exhibited the 
highest affinity for protein binding following both 
6 h and 24 h treatment, while 2 exhibited the lowest 
binding affinity. Time dependent decrease of pro- 
tein binding, indicated reversible nature of interac- 
tions of 1 and 2, oppositely to CDDP. 

Protein and mRNA expression of ERCC1 

DNA excision repair protein ERCC1 is an important 
component of NER (Nucleotide Excision Repair) 
which is primarily induced in the repair of bulky 
platinum-DNA adducts. 35 In order to evaluate 
whether investigated complexes induce ERCC1- 
dependent cell response as the result of cytotoxic 
DNA lesions, we investigated mRNA and protein 
expression level of ERCC1. Data obtained on HeLa 
cells after 6 h of continuous treatment with equi- 
toxic concentrations of tested frcms-platinum com- 
plexes or CDDP indicated negative modulation of 
ERCC1 expression on both mRNA and protein lev- 
els (results presented in Figure 6). Complexes 1, 2 
and CDDP decreased ERCC1 mRNA level for 45%, 
40% and 36%, respectively, comparing to the non 
treated control (Figure 6A). Western blot analysis 
(Figure 6B) showed reduction of ERCC1 protein 
levels, following both frcms-complexes 1 and 2 ac- 
tion, while there were no obvious changes associ- 
ated with CDDP treatment. 

Tube formation assay (in vitro 
angiogenesis assay) 

In order to determine the potency of the investigat- 
ed complexes to restrict the angiogenesis of cancer 
cells, we performed an in vitro tube formation assay 
in mouse endothelial cells MSI. In our experiment, 
MSI endothelial cells were treated with sub-toxic 
concentrations of the investigated complexes in or- 

Radiol Oncol 20 1 3; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 


der to distinguish among growth inhibitory effect 
and their potential to inhibit the formation of tube- 
like structures. Antiangiogenic effect was observed 
for both tested trans-platinum complexes, and re- 
sults are presented in Figure 7 A. Trans-complexes, 
particularly complex 2, showed inhibitory effect 
on the formation of cell-cell contact and tube-like 
structures, at very low sub-toxic concentration cor- 
responding to 0.03xIC 50 , while CDDP did not ex- 
hibit any significant effect in this assay. 

Gelatin zymography and determination 
of MMP-9 and MMP-2 expression on 
mRNA level 

We investigated whether tested complexes were 
able to modulate mRNA expression of MMP-2 and 
MMP-9 or affect their gelatinolitic activity in vitro. 
The effect of 1 and 2 on the activity of the secreted 
forms of MMP-2 and MMP-9 in HeLa cells, was 
examined following 6 h action, by gelatin zymog- 
raphy and the results are presented in Figure 7B. 
Quantitative analysis of the gelatin zymography 
was performed by Image J software, and is present- 
ed in Figure 7B. Results obtained indicated that 
complex 1 induced moderate decrease of gelati- 
nolitic activity of MMP-2 and MMP-9 in compari- 
son to the control (non treated cells), when applied 
at concentration of 0.5xIC 50 , (Figure 7C). CDDP 
failed to show effect in this assay, while complex 2 
induced minor enhancement of metalloproteinases 
activity. Results obtained by qRT-PCR indicated 
that trans-complexes reduced level of MMP-9 mR- 
NA, comparing to the control, following 6 h treat- 
ment (Figure 7D). Complex 2 and CDDP, when ap- 
plied at equitoxic concentrations corresponding to 
0.5xIC 50 , upregulated MMP-2 mRNA, while com- 
plex 1 did not induce obvious alteration of MMP-2 
mRNA expression. 


In our previous study we have reported synthesis, 
structural characterization and cytotoxic potential 
of two trans-platinum complexes of structural for- 
mula frans-[PtCl 2 (n-acetylpyridine) 2 ] (n = 3 or 4, 
complex 1 or 2, respectively), revealing significant 
cytotoxic potential of complex 2 on several tumor 
cell lines, with the highest potential in HeLa cells. 23 
In the current study we investigated the mecha- 
nism underlying in vitro antitumor activity of both 
isomers, in order to understand possible relations 
to their structural differences, such as position of 


Q. « 

<5 8 

5 5 

'»> S 

(A 4) 

— s 

X = 

9= — 

i i 

complex 1 complex 2 

CDDP control 

CDDP control 


M p actin 

FIGURE 6. A Results of the qRT-PCR analysis of ERCC1 mRNA presented as diagrams 
showing relative expression level of ERCC1 mRNA, normalized with the GAPDH; Bar 
graph represent mean ± SD of three independent experiments; B Protein expression 
levels of ERCC1 determined by Western blot, and normalized with b-actin. Tested 
agents 1, 2 and CDDP were applied at concentration of 0.5xlC 50 . Western blot results 
show one representative experiment selected of three. 

the acetyl substituent on pyridine ligand. Study 
was performed in comparison to CDDP as referent 

Cytotoxicity evaluation in MRC-5 cells, which 
were used as in vitro non-cancerous cell model, 
showed feature of the tested trans-platinum iso- 
mers to exert less toxicity in MRC-5, than in HeLa. 
Particularly complex 2 with 4-acetylpyridine, ex- 
hibited significant cytoselective potential toward 
neoplastic cells (HeLa) relative to normal cells 
(MRC-5), cells (p < 0.001), comparing to CDDP (p 

According to the results of flow cytometry, an- 
tiproliferative action of complex 1, was associated 
to minor cell cycle arrest in the G0/G1 and S phase, 
and consequent initiation of cell death. When 
tested at equitoxic concentrations (IC 50 ), complex 
1 induced significant percentage of necrotic cells, 
comparing to complex 2 and CDDP, observed al- 
ready after 4 h of action. Complex 2 exhibited the 
rate and kinetics of apoptosis induction similar to 
that of CDDP. 

Mechanism of anticancer activity of platinum 
drugs is believed to be associated with their bind- 
ing with cellular DNA. The level of DNA-binding 

Radiol Oncol 2013; 47(4): 346-357. 


Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 



Complex 2 

Complex 1 




* MMP-9 
A MMP-2 

□complex 1 ■complex2 "CDDP □control 


FIGURE 7. A Micrographs of tube formation of MSI cells, taken following 
24 h treatment with sub-toxic concentrations (0.03xlC 50 ) of complexes 
1, 2 or CDDP, or without treatment (control cells); B Results of the 
gelatin zymography following 6 h treatment with 0.5xlC 50 of 1, 2 or 
CDDP. Supernatants were collected and equal amounts of proteins 
were analysed by zymography. Representative of three independent 
experiments; C Diagram presenting quantification of catalytic activity 
of secreted MMP-2 and MMP-9 in treated HeLa cells, obtained by 
quantitative analysis of zymograms, using Image J software. Data 
are expressed in arbitrary units as mean ± standard deviation of three 
independent experiments; D Results of the qRT-PCR analysis of the MMP- 
2 and MMP-9 mRNA expression level in HeLa cells after 6 h treatment 
with 1, 2 or CDDP, at concentration 0.5xlC 50 . Each bar represents the 
average (±SD) of three independent experiments. 

Ocomplexl lcomplex2 ■CDDP Hconlrol 

is considered to provide more meaningful informa- 
tion (related to activity) than total cellular uptake 
especially as platinum drugs may undergo compl- 
exation with cellular platinophiles, so that only a 
very small fraction of the drugs actually binds with 
DNA. 36 

The results of the present work, demonstrat- 
ing nuclear DNA binding affinity of compounds 1 
and 2, are consistent with the hypothesis that the 
minor structural modifications of carrier ligands 
in frans-mononuclear platinum complexes could 
modulate the DNA binding afinity, resulting in 
the altered biological (pharmacological) activity 
of these new platinum complexes in tumor cells, 
relative to CDDP. 37 Results of the analysis of the 

actual platination of DNA, presented in terms of 
pg Pt/mg DNA, showed that 2 exhibited the lowest 
DNA binding following 6 h treatment, with plati- 
num content being less by a factor of approximately 
threefold comparing to 1, though reaching similar 
level of DNA binding as 1 and CDDP following 24 
h treatment. Structure-activity correlation suggests 
that the acetyl-group in the para-position on the 
planar pyridine rings (4-acetylpyridine) in complex 
2, may additionaly hamper positioning of the non- 
leaving moieties in the adducts of this analogue, 
that would be entirely favorable for its interaction 
with the double helix. 37 It should be noted that com- 
plex 2 platinum-DNA level just slightly decreased 
during 24 h treatment comparing to complex 1 and 

Radiol Oncol 20 1 3; 47(4): 346-357. 

Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 


CDDP, suggesting on the other side sustainable na- 
ture of platinum-DNA lesion. Different studies sup- 
port the assumption that long-lived DNA-adducts, 
formed by mononuclear frans-platinum(II) com- 
plexes containing planar ligands such as quinoline 
or pyridine constitute potential cytotoxic lesions. 38 
The higher cytotoxicity of 2 comparing to 1, may 
be attributed to its ability to form different DNA 
conformational distorsions and lesions which are 
differentially processed by DNA damage recogni- 
tion/repair proteins, and to the ability of compound 
to induce different cellular response. 21 Lower level 
of unfavorable interactions with proteins may be 
additional determinant of enhanced cytotoxicity of 
complex 2 in comparison to 1. 

On the other side, meta-position of the acetyl 
substituent on the pyridine ring (3-acetylpyridine) 
in complex 1, allowed reactivity with intracellular 
DNA and proteins, though in reversible manner. 

Complex 1 platinum levels in cellular DNA de- 
creased from 6 h to 24 h time points, indicating that 
1 treated cells partially recovered from the initial 
cytotoxic stress, which may be due to an early 
DNA damage response and removal of the plati- 
num-DNA lesions. 13 

In order to evaluate role of DNA-damage re- 
pair in mediating differences in cytotoxicity of the 
tested complexes, we further evaluated expression 
of ERCC1 on mRNA and protein level. Nucleotide 
excision repair is one of the DNA-repair mecha- 
nisms primarily activated in response to cisplatin 
induced genotoxic stress. 39 42 In the case of CDDP, 
cycle cycle arrest in the phase G0/G1 (also caused 
by investigated complex 1) and G2/M arrest, may 
be indicative for activation of NER repair pro- 
teins. 20 Nevertheless, western blot and gene expres- 
sion analysis in the current study, revealed reduc- 
tion of ERCC1 mRNA and protein levels, following 
short-term treatment with 1 and 2, while there was 
discordance in mRNA and protein levels following 
CDDP treatment. It is likely that ERCC1 might not 
play fundamental role in mediating sensitivity to 
fraws-platinum complexes, as well as to the inves- 
tigated complexes in the current settings, but ad- 
ditional studies need to be directed toward under- 
standing of the molecular mechanism underlying 
expression status of ERCC1 (mRNA and protein), 
and its correlation to frans-platinum-based drug 
sensitivity. 15 - 43 

In the separate part of our study we investi- 
gated the potential of tested complexes to modu- 
late processes related to angiogenic and metastatic 
potential of tumor cells in vitro. Pathological an- 
giogenesis is a hallmark of cancer and represents 

an important step in the development of metasta- 
sis. 44 " 46 Significant efforts in the area of anticancer 
drug research are focused on the development of a 
drug, which would be able to limitate angiogenesis 
of cancer cells. Study in MSI cells revealed poten- 
tial of 2 to inhibit formation of tube-like structures 
and tumor cell-cell contacts, at very low subtoxic 
concentration (0.03 IC 50 ), while CDDP failed to 
show effect in this assay. Our results indicated po- 
tential of complex 2 to act on multiple processes in 
cancer cells, and that exposure to DNA-damaging 
agents at subtoxic concentrations may alter tumor 
cell behavior. More direct experiments would be 
required to confirm the observed antiangiogenic 
potential of 2 in vitro. 

Matrix metalloproteinases 2 and 9, which are 
frequently over expressed in tumor cells, play a 
critical role in modulation of extra cellular matrix, 
and its role in tumor cell migration, formation of 
tumor cell contacts and angiogenesis transition is 
extensively investigated. Thus, MMPs represent 
a promising target for antitumor drug design. 25 - 45 
Our investigations of the effect of tested complexes 
on gelatinolitic activity of secreted matrix metallo- 
proteinases MMP-2 and MMP-9 and their mRNA 
expression levels, showed that both 1 and 2 caused 
decrease of MMP-9 mRNA, for 6 h action, though 
for the time point observed (6 h), inhibitory effect 
on the enzyme activity level was minor. 44 Only 
complex 1 showed moderate inhibitory effect on 
MMP-2 and MMP-9 activity. Reduction of func- 
tional levels of MMP-9 by 1 and 2 was in correla- 
tion to the reduction of the enzyme mRNA-level, 
which might represent the indirect effect of geno- 
toxic stress. 

Although two investigated complexes of struc- 
tural formula frans-[PtCl,(n-acetylpyridine) 2 ] (n 
= 3 or 4) represent close structural isomers, their 
interactions with cellular targets and consequently 
induced cellular responses are different. Results 
obtained suggest that structural settings play a 
fundamental role, since they are responsible for 
the interaction between frans-Pt drug and cellular 
DNA and proteins and the consequent biological 
effects. Higher cytotoxicity of 2 compared to the 1 
analogue, may be attributed to its ability to form 
different DNA-lesions, produce different cellular 
effects related to damage-precessing and signal ac- 
tivation pathways, and induce multiple cellular re- 
sponses. Our study demonstrated that complex 2 is 
particularly interesting since it exhibited cytotoxic 
and apoptotic potential in HeLa cells comparable 
to that of CDDP, though showing differences in 
terms of reactivity to DNA and proteins, cytoselec- 

Radiol Oncol 2013; 47(4): 346-357. 


Filipovic L et al. / Biological evaluation of trans-Pt(ll) complexes 

tivity toward tumor cells and potential for in vitro 
angiogenesis inhibition. Further explorations are 
needed to determine a possible differential mecha- 
nism of action and elucidate antitumor potential of 
complex in vivo. 

Altogether, properties of complexes of structur- 
al formula trans-[PtCl 2 (n-acetylpyridine) 2 ] (n = 3 or 
4), encourage further investigation of substituted 
frans-platinum pyridines in search for compound 
with different modalities of action toward cancer 
cells in comparison to CDDP. 


This work was supported by the Ministry of 
Science, Republic of Serbia, Grant, No. Ill 41026 and 
Grant, No 172017 (URL address: http://www.mpn. 


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