Differential Cytotoxic Activity of a Novel Palladium-
Based Compound on Prostate Cell Lines, Primary
Prostate Epithelial Cells and Prostate Stem Cells
Engin Ulukaya1*., Fiona M. Frame2., Buse Cevatemre3, Davide Pellacani2, Hannah Walker2,
Vincent M. Mann4, Matthew S. Simms5, Michael J. Stower6, Veysel T. Yilmaz7, Norman J. Maitland2
1Department of Medical Biochemistry, Medical School, Uludag University, Bursa, Turkey, 2Department of Biology, YCR Cancer Research Unit, University of York,
Heslington, York, North Yorkshire, United Kingdom, 3Department of Biology, Faculty of Arts and Sciences, Uludag University, Bursa, Turkey, 4Hull York Medical School,
University of Hull, Hull, United Kingdom, 5Department of Urology, Castle Hill Hospital (Hull and East Yorkshire Hospitals NHS Trust), Cottingham, United Kingdom, 6 York
District Hospital, York, United Kingdom, 7Department of Chemistry, Faculty of Arts and Sciences, Uludag University, Bursa, Turkey
Abstract
The outcome for patients with advanced metastatic and recurrent prostate cancer is still poor. Therefore, new
chemotherapeutics are required, especially for killing cancer stem cells that are thought to be responsible for disease
recurrence. In this study, we screened the effect of a novel palladium-based anticancer agent (Pd complex) against six
different prostate cancer cell lines, and primary cultures from seven Gleason 6/7 prostate cancer, three Gleason 8/9 prostate
cancer and four benign prostate hyperplasia patient samples, as well as cancer stem cells selected from primary cultures.
MTT and ATP viability assays were used to assess cell growth and flow cytometry to assess cell cycle status. In addition,
immunofluorescence was used to detect cH2AX nuclear foci, indicative of DNA damage, and Western blotting to assess the
induction of apoptosis and autophagy. The Pd complex showed a powerful growth-inhibitory effect against both cell lines
and primary cultures. More importantly, it successfully reduced the viability of cancer stem cells as first reported in this
study. The Pd complex induced DNA damage and differentially induced evidence of cell death, as well as autophagy. In
conclusion, this novel agent may be promising for use against the bulk of the tumour cell population as well as the prostate
cancer stem cells, which are thought to be responsible for the resistance of metastatic prostate cancer to chemotherapy.
This study also indicates that the combined use of the Pd complex with an autophagy modulator may be a more promising
approach to treat prostate cancer. In addition, the differential effects observed between cell lines and primary cells
emphasise the importance of the model used to test novel drugs including its genetic background, and indeed the
necessity of using cells cultured from patient samples.
Citation: Ulukaya E, Frame FM, Cevatemre B, Pellacani D, Walker H, et al. (2013) Differential Cytotoxic Activity of a Novel Palladium-Based Compound on Prostate
Cell Lines, Primary Prostate Epithelial Cells and Prostate Stem Cells. PLoS ONE 8(5): e64278. doi:10.1371/journal.pone.0064278
Editor: Kamyar Afarinkia, Univ of Bradford, United Kingdom
Received April 19, 2012; Accepted April 15, 2013; Published May 10, 2013
Copyright: 
 2013 Ulukaya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: E. Ulukaya was funded by the Council of Higher Education and the Research Fund of Uludag University. http://www.uludag.edu.tr/. This work was also
funded by a Yorkshire Cancer Research Core Grant (F.M. Frame, D. Pellacani and N.J. Maitland). http://www.yorkshirecancerresearch.org.uk/. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: eulukaya@uludag.edu.tr
. These authors contributed equally to this work.
Introduction become of increasing interest within the last 15 years. As such,
different Pd complexes with promising activity against varying
Prostate cancer is the most commonly diagnosed cancer in kinds of tumor cell lines from both solid tumors and hematological
males and is the second highest cause of male cancer-related death malignancies have been synthesized and tested over the years
[1,2]. Although new drugs have recently been introduced into the [9,10,11,12,13,14,15]. Their lipophilicity or solubility seems to
clinic, the response to therapy for metastatic prostate cancer is still provide satisfactory cytostatic activity [16]. The increased solubil-
poor [3,4,5]. Therefore, there is an urgent need for more efficient ity of Pd complexes, compared to platinum, also makes Pd
or different kinds of drugs specifically targeting radio-recurrent complexes more attractive. For example, Pd complexes of
and hormone-resistant prostate cancer, as well as prostate cancer
glyoxylic oxime were found to have higher aqueous solubility
stem cells (CSCs) [3,4,6]. New metal-based agents like palladium
than platinum(II) (Pt) complexes of glyoxylic oxime [17].
(Pd) complexes are promising for the development of improved
There are only a few studies on the effect of newly-synthesized
chemotherapeutic drugs. There is a significant similarity between
palladium(II) complexes on prostate-derived cell lines: for example,
the coordination chemistry of Pd and platinum (Pt) compounds as
palladium(II) has been complexed with different ligands such as
antitumor drugs [7].
triazole [10], triphenylphosphines [18], dithiocarbamate [19], or
Although the synthesis of Pd complexes with anti-fungal, anti-
hydrazine [20]; and even curcumin, which is a well-known plant-
viral, anti-cancer, and anti-bacterial activities dates back to more
than 30 years [8], the anti-cancer activities of Pd complexes have
PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64278
Palladium’s Cytotoxicity on Prostate Cancer Cells
based compound with apoptosis-inducing activity on cancer cells Prostate epithelial cells were cultured as previously described
[21]. [26] in stem cell media (SCM) consisting of keratinocyte serum-
In addition to the ligands above, the bioorganic and medicinal free media (KSFM) with bovine pituitary extract (BPE) and
chemistry of 2,29:69,20-terpyridine (terpy) complexes of Pd(II) and epidermal growth factor (EGF), glutamine, stem cell factor (SCF),
Pt(II) is also an active and growing area of interest [13,22]. Taking granulocyte macrophage colony stimulating factor (GM-CSF),
into account the promising activity of Pd complexes against leukaemia inhibitory factor (LIF) and cholera toxin. Cells were
cancer, we have therefore synthesized new Pt and Pd complexes; grown with STO feeder cells (irradiated at 60 Gy) on collagen I-
[Pd(sac)(terpy)](sac)?4 H2O, [Pt(sac)(terpy)](sac)?5 H2O, [PdCl(ter- coated plates. The detailed information of the primary cells used is
py)](sac)?2 H2O, [PtCl(terpy)](sac)?2 H2O (sac = saccharinate, and given in Table 2.
terpy = 2,29:6920-terpyridine) [23]. Among these, the Pd complex-
es, but not the Pt complexes, were found to exhibit considerable Isolation of Cancer Stem Cells, Transit Amplifying Cells
anti-growth effect against non-small cell lung cancer cells in vitro and Committed Basal Cells
[24]. The [Pd(sac)(terpy)](sac)?4 H2O complex was further inves- Epithelial cells from human tumour or BPH materials were
tigated against breast cancer cells both in vitro and in vivo and cultured for several weeks and the cells treated at very low
showed powerful anti-growth activity against this cancer type [25]. passages. The cultured basal cell population were trypsinized,
In the present paper, we have investigated the cytotoxic activity resuspended in SCM and then plated on BSA-blocked collagen I-
of our formulation of Pd complex, formulated as [PdCl(terpy)](- coated plates. After 30 min, cells that did not attach to the
sac)?4 H2O, against prostate cancer cells. The Pd complex was substratum were collected, consisting of the committed basal cells
found to exhibit powerful growth-inhibiting activity, against cell (CBs), which are a2b1integrinlo. The cells that attached to
lines and primary cultures, as well as prostate CSCs. The substratum were trypsinised, resuspended in MACs buffer and
induction of apoptosis in cell lines by this compound indicates its incubated with CD133-microbeads (Cat no. 130-050-801, Milte-
potential as a new cytotoxic agent. However, the induction of nyi Biotec Inc., Auburn, CA, USA). MACs MS columns (Cat
autophagy but not apoptosis in primary prostate cells suggests that no. 130-042-201, Miltenyi Biotec Inc., Auburn, CA, USA) were
a combination of the complex with autophagy inhibitors may be a used to select the CD133+ and CD1332 cells. Finally, the three
preferred treatment strategy. Significantly, we have shown a cell populations were obtained: stem cells (SCs) - a2b1integrinhi/
differential effect of the compound, which is dependent on genetic CD133+, transit-amplifying cells (TAs) - a2b1integrinhi/CD1332
background of cells that could also influence treatment choice. In and committed basal cells (CBs) - a2b1integrinlo.
addition, to our knowledge, this is the first study showing anti-
growth activity of the Pd complexes against CSCs and it thereby Chemicals
warrants further investigation as a chemotherapeutic for prostate The palladium (Pd) complex was synthesized in the Chemistry
cancer. Department of the Science and Art Faculty of Uludag University.
The synthesis, characterization and crystal structure of the
Methods palladium(II) complex has been reported elsewhere [23].
[PdCl(terpy)](sac)?2 H2O was synthesized by the direct addition
Culture of Cell Lines of an equimolar amount of sac ions to [Pd(terpy)Cl]Cl?2 H2O in
In this study, six different prostate cell lines (PNT1A, PNT2-C2, solution in high yield. The orange crystals of the compound were
BPH-1, PC-3, LNCaP, P4E6) were used (Table 1). These cell lines obtained and its molecular structure was confirmed by X-ray
encompass the spectrum of cellular differentiation status (basal, diffraction. The chemical structure is shown in Figure 1A. Stock
intermediate and luminal phenotypes), as well as the spectrum of and final concentrations of the Pd complex were prepared in the
normal, early cancer and late cancer. BPH-1 is derived from appropriate culture medium. The Pd complex was used at
benign prostate hyperplasia (BPH), while PNT2-C2 and PNT1A concentrations ranging from 0.39 to 100 mM. Cisplatin
are derived from normal prostate. PC-3, LNCaP and P4E6 are (sc200896, Santa Cruz Biotechnology, Santa Cruz, CA, USA)
cancer cell lines. LNCaP, PNT2-C2, PNT1A were grown in (Figure 1B) and Etoposide (E1383, Sigma-Aldrich, Saint Louis,
RPMI medium with 10% FCS (foetal calf serum); PC-3 was grown MO, USA) (Figure 1C), were used as positive controls for cytotoxic
in Ham’s F-12 medium with 7% FCS; BPH-1 was grown in RPMI activity at doses of 25 mM and 12 mM or 6 mM, respectively.
medium with 5% FCS; P4E6 was grown in KSFM (Keratinocyte
serum free medium) with 2% FCS. No antibiotics were used in any MTS Assay
media. The cells were incubated at 37uC in a humidified This assay was performed for the initial screening of the effect of
atmosphere containing 5% CO2. the Pd complex on the cell lines and the whole cell population of
primary cultures. The CellTiter 96H Aqueous One Solution Cell
Culture of Primary Prostate Epithelial Cells Proliferation Assay kit (G3580, Promega, Madison, WI, USA) was
Primary prostate epithelial cells were isolated from human tissue used and the manufacturer’s instructions were followed. Briefly,
samples. The samples were collected with ethical permission from after treating cells that were seeded at a density of 5,000 cells per
York District Hospital (York, UK) and Castle Hill Hospital well in a 96-well plate in triplicate for a desired period (24 h, 48 h,
(Cottingham, UK). Benign prostatic hyperplasia (BPH) and 72 h), 20 mL of reagent was added to each well. Following 2.5 h
prostate cancer samples were obtained from TURP (transurethral incubation at 37uC, the absorbance was read at 485 nm using a
resection of the prostate), radical prostatectomy (laparoscopic and plate reader (PolarStar Optima, BMG Labtech, UK). Percent
open) and cystectomy operations. All patients gave written consent viability was calculated using the formula (% Viability = [(Sample
for their tissue to be used for research and all patient samples were Absorbance/Control Absorbance)]6100.
anonymised. Permission was approved by the Local Research
Ethical Committees, associated with York District Hospital and ATP Assay
Castle Hill Hospital. Permission was administered by the This assay was employed for the bioluminescent determination
Yorkshire and Humber Research Ethics Committee. of the adenosine 59-triphosphate (ATP) released from fractionated
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Table 1. Cell lines.
Cell line Diagnosis Source/Reference
PNT1A Normal prostate epithelium immortalized with SV40 Kind gift to the lab of Norman Maitland from P. Berthon Currently
available from Health Protection Agency Culture Collections.
PNT2-C2 Normal prostate epithelium immortalized with SV40 Obtained from ECACC (no longer available from ECACC)
BPH-1 Primary epithelial culture from benign prostatic hyperplasia Obtained by Norman Maitland, with kind permission from Simon
immortalized with SV40 Hayward [49]. Not commercially available.
P4E6 Epithelial culture established from well-differentiated prostate cancer/E6 Derived in York [50]. Currently available from Health Protection
gene from human papillomavirus introduced by retroviral insertion Agency Culture Collections.
PC-3 Prostate adenocarcinoma/bone metastasis ATCC
LNCaP Prostate carcinoma/lymph node metastasis ATCC
doi:10.1371/journal.pone.0064278.t001
living cells (cancer stem cells, transit amplifying cells and collagen-coated 96 well plate in triplicate. After treating cells for
committed basal cells). As ATP is rapidly degraded in dead cells, 72 h, 150 mL of medium was removed from each well. 50 mL of
high intracellular levels provide a selective assay for living cells. cell extraction reagent was added into each original well.
The ATP-bioluminescent somatic cell assay kit (FLASC, Sigma- Following 20 min incubation at RT, 50 mL of cell extract was
Aldrich, Saint Louis, MO, USA) was used with protocol transferred to a white 96-well plate. Finally, 50 mL of ATP assay
modifications. Briefly, 50–500 cells per well were seeded in a mix solution was added into the wells and luminescence was read
using a plate reader (PolarStar Optima, BMG Labtech, UK).
Percent viability was calculated using the formula (% Viabili-
Table 2. Primary epithelial cells. ty = [(Sample RLU/Control RLU)]x100 where RLU refers to
relative light units.
Sample Passage Operation Diagnosis Immunofluorescence
01409 6 C benign cH2AX staining: Cells were seeded onto 8-well collagen I-
03108 6 T benign coated chamber slides. Briefly, following 48 h treatment with the
+ Pd complex or etoposide, cells were washed with PBS and fixed01608 2 3 T benign
with 2% paraformaldehyde in PBS with 0.2% Triton X-100,
08109 4 T benign
pH 8.2 for 20 min, and then permeabilised with 0.5% NP40 in
00409 4 T benign PBS for 20 min at RT followed by three washes with PBS. After
07611 2 T benign blocking of non-specific binding with 2% BSA in PBS with 1%
22612 3 T benign goat serum for 1 hour at RT, primary antibody (anti-phospho-
05908 2 T benign histone H2A.X (Ser139), clone JBW301, Millipore, Cat no. 05–
636) at 1:1000 dilution was added in 3% BSA in PBS at 4uC
07011la 3 R Cancer on hormones Gleason 7
overnight followed by three washes in 0.5% BSA in PBS with
07011lb 3 R Cancer on hormones Gleason 7 0.175% Tween 20. Following incubation with secondary antibody
05411rb 5 R Cancer Gleason 7 (Goat anti-mouse Alexa Fluor 568, Invitrogen, Cat no. A-11004)
07311ra 3 R Cancer Gleason 7 at 1:1000 dilution for 45 min in 3% BSA in PBS and three more
06711rb 6 R Cancer Gleason 6 washes in the same washing buffer, the slides were mounted using
Vectashield with DAPI (Vector Laboratories, Cat no. H-1200).
06211rb 4 R Cancer Gleason 7
LC3-B staining: Cells were treated as above then fixed with 4%
06611lb 5 R Cancer Gleason 7 paraformaldehyde, followed by an incubation in 0.3% Triton X-
04811rb 5 R Cancer Gleason 6 100. After blocking with 10% normal goat serum, cells were
06411ra 3 R Cancer Gleason 7 incubated in anti-LC3B 1:200 (Ab51520, abcam) diluted in 0.1%
06411lb 3 R Cancer Gleason 7 Triton X-100 in PBS. Secondary antibody was Alexa Fluor
568 goat anti-rabbit IgG 1:1000 (Invitrogen A11011).
25212ra 3 R Cancer Gleason 7
23912ra 6 R Cancer Gleason 9
Flow Cytometry
22412 2/3 chT Cancer Gleason 7
Following drug treatment, floating cells in the media were
22112 4 R Cancer Gleason 7 pooled with adherent cells, which were collected by trypsinisation.
22012ra 5 R Cancer Gleason 7 Following centrifugation cells were resuspended in 0.5 ml PBS.
07311la 7 R Cancer Gleason 7 Cells were fixed in 2 ml ice cold 70% ethanol, which was added in
16312 5 chT Cancer Gleason 8 a dropwise fashion while vortexing. Cells were incubated on ice for
30 min then washed in 5 ml PBS and resuspended in 0.4 ml PBS.
22912 2 chT Cancer Gleason 8/9
Following storage at 4uC overnight, 50 ml of RNAse (1 mg/ml)
14912 3 chT Cancer Gleason 9 and 50 ml of propidium iodide (1 mg/ml) were added to the cells.
Following incubation at 37uC for 30 min the cells were analysed
C=Cystectomy/T = Transurethral resection of the prostate/R = Radical
Prostatectomy/chT = channel TURP. for 2N and 4N DNA content on a flow cytometer (Cyan ADP
doi:10.1371/journal.pone.0064278.t002 Analyser, Beckman Coulter).
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 1. Chemical structures. (A) Palladium complex [PdCl(terpy)](sac)?2 H2O, (sac = saccharinate, and terpy = 2,29:69,20-terpyridine).
M= Palladium(II) (B) Cisplatin (C) Etoposide.
doi:10.1371/journal.pone.0064278.g001
Western Blotting Results
Following drug treatment, cell lysates were harvested using
Cytobuster Protein Extraction Reagent (71009, EMD Millipore, Effect of Pd Complex on Cell Lines
Darmstadt, Germany) with protease inhibitors (cOmplete, EDTA- The anti-growth effect of the Pd complex was tested against six
free Protease Inhibitor Cocktail Tablets, Roche Applied Science, different cell lines at three different time points, 24 h, 48 h and
UK). 20 mg of protein extract were loaded on 12% SDS-PAGE 72 h (Figure 2). The Pd complex inhibited the growth of all cell
gels and wet-transferred to a PVDF membrane. Antibodies used lines almost completely at 100 mM concentration at 72 h. A
include: monoclonal anti-b-actin 1:5000 (A5316, Sigma-Aldrich), comparison was made to etoposide (25 mM) and to cisplatin
anti-LC3B 1:3000 (Ab51520, abcam), cleaved caspase-3 (Asp175) (12 mM), used as known cytotoxic agents. At 72 h, the lowest IC50
1:1000 (9661S, Cell Signaling Technology) and secondary value, 0.1399 mM, was for the BPH-1 cell line, with PNT1A cells
antibodies were Rabbit anti-mouse-HRP 1:10000 (P0260, Dako) having a similarly low IC50 of 0.1064 mM, while PNT2-C2 cells
and anti-rabbit IgG HRP-linked 1:5000 (Cell Signaling Technol- were more resistant, with an IC50 value of 0.9033 mM (Table 3).
ogy Inc. 7074S). Kaleidoscope protein marker was run on each gel The well differentiated early stage prostate cancer cell line P4E6,
(161-0324, Bio-Rad). and LNCaP, which is from a lymph node metastasis with the most
luminal phenotype (androgen-positive) had IC50s of 4.372 mM and
Statistics and Calculations 3.433 mM, respectively, whereas the cancer cell line from a bone
metastasis, PC-3, had an IC value of 26.79 mM. There was a less
MTS and ATP assays were performed in triplicate and data 50
+ dramatic effect on PNT2-C2 cells compared to the other normalpresented as the mean /2 standard deviation. IC50 values
and benign cell lines (Figure 2A(iii)). However, the cancer cell line
(Table 3 and Table 4) were calculated from graphs of transformed
from the bone metastasis is least susceptible to the drug, with a
data following application of the nonlinear regression (curve fit)
significantly higher IC (Figure 2B(iii)). Overall, the Pd complex
that represents the log(inhibitor) ‘v’ normalized response (Graph- 50
successfully reduced viability of all cell lines tested with some
Pad Prism software) (Supplementary Figures S1 and S2). For
variation in response.
significance calculations, the Wilcoxon rank sum test was used
(Sigmaplot). Flow cytometry analysis was carried out in triplicate
and results are presented as an average with error bars indicating Effect of Pd Complex on Primary Cultures from Benign
the standard error. and Malignant Samples
The most complete dose response was observed at the 72 h
time-point, and so this time point was used to explore the anti-
Table 3. IC50 values of the Pd complex in cell lines.
Cell lines 24 h IC50 (mM) Cell lines 48 h IC50 (mM) Cell lines 72 h IC50 (mM)
PNT1A 27.97 PNT1A 1.258 PNT1A 0.1064
PNT2-C2 1.732 PNT2-C2 17.17 PNT2-C2 0.9033
BPH-1 17.62 BPH-1 1.881 BPH-1 0.1399
AVERAGE 15.774 AVERAGE 6.769666667 AVERAGE 0.3832
P4E6 21.80 P4E6 9.660 P4E6 4.372
PC-3 98.91 PC-3 49.15 PC-3 26.79
LNCaP 105.5 LNCaP 11.90 LNCaP 3.433
AVERAGE 75.40333333 AVERAGE 23.57 AVERAGE 11.53166667
doi:10.1371/journal.pone.0064278.t003
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Table 4. IC values of the Pd complex in primary cells. stem cells (SCs) were explored. SCs were isolated from primary50
cultures derived from three benign and five prostate carcinoma
(Gl6/7) patient samples. In addition to SCs, TA cells, and CB cells
BPH primary cells IC (mM) New were also isolated. The SCs are a rare population of cells, and as50
such the anti-growth effect was measured by the ATP assay, since
7611 6.974 it significantly more sensitive than the MTS assay and can
1608 5.969 accurately measure low cell numbers (Figure 4A). The Pd complex
3108 7.326 was tested at two different doses (6.25 and 25 mM) on the basis of
8109 14.43 previous experiments where 6.25 mM was the lowest concentra-
tion inducing a significant anti-growth effect and 25 mM caused a
AVERAGE 8.67475
dramatic reduction in cell viability (80%–100% in BPH and
PCa Primary cells (Gl7) IC (mM) New Gleason 6/7 cancers). It is clearly shown that 25 mM Pd complex50
was significantly more cytotoxic in stem cells, compared to 25 mM
06411ra 3.778 etoposide (Figure 4B). (Using a Wilcoxon rank sum test to measure
22412 6.303 the effect of 25 mM etoposide versus 25 mM PD003, the latter is
significantly more cytotoxic with a P value = 0.004 in all three
06411lb 5.196
tests, comparing each population separately). The 6.25 mM Pd
07011la 5.357 complex resulted in an anti-growth effect that was less than 25 mM
07011lb 6.025 Pd complex but still more cytotoxic than 25 mM etoposide. (Using
07311ra 5.371 a Wilcoxon rank sum test to compare 6.25 mM Pd complex versus
06211rb 11.06 25 mM Pd, there is a significant difference in cytoxicity with a P
value =,0.001 in all three tests, comparing each population
06711rb 8.241
separately). Cisplatin also appeared to be significantly cytotoxic to
06611lb 13.12
all cell populations; 6 mM of cisplatin was equivalently cytotoxic to
AVERAGE 7.16122 6 mM Pd complex (P values showed no significant difference
SC‘v’SC=0.073, TA‘v’TA=0.4, CB‘v’CB= 0.533). SCs ap-
PCa Primary cells (Gl8/9) IC50 (mM) peared to have increased viability compared to TA and CB cells
following etoposide treatment (although this was not statistically
16312 70.41
significant), which was not the case following Pd complex
22912 46.68
treatment.
14912 64.07
AVERAGE 60.38666667 DNA-damaging Effect of Pd Complex
Since the mechanism of action of the Pd complex has not been
doi:10.1371/journal.pone.0064278.t004
fully characterized, the DNA-damaging effect was assessed. 10,000
cells per well in 8-well chamber slides were treated for 48 h with
growth effect of the Pd complex on primary cultures from patient Pd complex. Nuclei with evidence of cH2AX nuclear foci,
tissue to assess a model closer to the disease state. The Pd complex indicative of DNA damage, were counted on 10 randomly chosen
was tested on primary cultures derived from patients with benign fields at the highest (63x) magnification and the mean number of
prostate hyperplasia (BPH, n= 4 from four patients) (Figure 3A), positively-stained nuclei was calculated. At least 100 cells per well
prostate carcinoma with low Gleason grades (6/7) (n = 9 from were counted. Both etoposide and Pd complex at the same dose
seven patients) (Figure 3B) and with high Gleason grades (8/9) yielded similar levels of DNA damage (Figure 5A). 3.12 mM of the
(n = 3 from three patients) (Figure 3C). The dose response curve Pd complex did not induce a significant level of DNA damage.
was strikingly similar between BPH and Gleason 6/7 samples,
with concentrations higher than 6.25 having a significant anti-
Effect of Pd Complex on the Cell Cycle
growth effect on all samples. Compared to etoposide, the Pd
m Following on from the observation that the Pd complex causedcomplex at the same concentration (25 M) was found to
DNA damage, we explored its effect on cell cycle status, since
significantly reduce cell viability at least 5.6-fold in benign samples
DNA damage can lead to activation of cell cycle checkpoints and
(P = 0.029) and 10.66-fold in malignant samples (P =,0.001)
cell death (Figure 5B–C, Supplementary Figure S3). Etoposide
(median values used to calculate fold difference). Significantly, the
caused an S phase arrest in PNT2-C2, PC3 and LNCaP cell lines
Pd complex had a less pronounced effect in high Gleason grade
and also in primary prostate epithelial cells (measured at 48 h post-
(8/9) prostate cancer (Figure 3C). For the benign and Gleason 6/7
treatment), which is not unexpected since etoposide treatment
cancer cultures the average IC50 was 8.67 mM and 7.16 mM leads to DNA damage in the S phase of the cell cycle [27].
respectively, while for the high Gleason grade cancers (8/9) the
Following treatment with the Pd complex, the cell lines showed an
IC50 was 60.39 mM (Table 4), indicating that these cultures are increase in cells with sub-G1 DNA content, indicative of cell death
more resistant, or less susceptible to the complex. Considering the
in all cases, except PC3 cells where there was almost no change.
need for new drugs to treat high Gleason grade tumours that are
Of the other cell lines, the PNT2-C2 cells were the least
often radiorecurrent and hormone-resistant, this is a significant
susceptible. Generally, at lower concentrations (6 mM and
observation regarding these samples, and one that could have been 12 mM), the Pd complex showed similar levels of cell death to
missed if using only cell lines. the cisplatin and etoposide controls. In the normal cells (BPH-1,
PNT2-C2), the increase in cells with sub-G1 DNA content was
Effect of Pd Complex on Cancer Stem Cells from Primary accompanied by a reduction in the S and G2 peaks, indicative of
Epithelial Cultures either a G1 arrest followed by cell death, or a cell replication
Prostate tumours are heterogeneous, and so the anti-growth failure preceding cell death. In primary cells, treatment with the
effects of the Pd complex specifically on benign and malignant Pd complex gave a clear dose response showing increase in sub-G1
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 2. Anti-growth effect of the Pd complex on cell lines. Anti-growth effect was measured by the MTS assay at 24 h, 48 h and 72 h.
PNT1A, PNT2-C2, and BPH-1 have either normal tissue or benign prostatic hyperplasia tissue origin (Ai–iii), while PC-3, LNCaP and P4E6 cell lines have
malignant origin (Bi–iii). IC50s are presented in Table 3. Transformed graphs are presented in Supplementary Figure S1.
doi:10.1371/journal.pone.0064278.g002
content, and also induced an increase in cells with more than 4N C2 cells, LNCaPs and PC3 cells as well as primary cells. Overall, it
DNA content, potentially indicative of induction of aneuploidy. appears that the Pd complex had a different effect on the cell cycle
Similarly to etoposide, cisplatin caused an S phase arrest in PNT2- status than either etoposide or cisplatin.
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 3. Anti-growth effect of the Pd complex on primary cultures. Anti-growth effect was measured by the MTS assay at 72 h using cells
derived from patients with (A) benign prostate hyperplasia, (B) prostate carcinoma with Gleason grade 6/7 and (C) prostate carcinoma with Gleason
grade 8/9. IC50s are presented in Table 4. Transformed graphs are presented in Supplementary Figure S2.
doi:10.1371/journal.pone.0064278.g003
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 4. Anti-growth effect of the Pd complex on cancer stem cells (CSC). (A) MTS assay and ATP assay were compared to assess anti-
growth effect using small cells numbers. (B) Anti-growth effect was measured by the ATP assay using SCs, TA cells and CB cells derived from three
patients with benign prostate hyperplasia (white-filled shapes) and five patients with prostate carcinoma (black-filled shapes). White bar represents
the median value of all the samples.
doi:10.1371/journal.pone.0064278.g004
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 5. DNA-damaging effect and effect on Cell Cycle Status of Pd complex. (A) Primary cultures isolated from two patients with prostate
carcinoma were assessed. Cells were stained and scored for nuclear foci indicative of DNA damage Representative examples of cells negative and
positive for nuclear foci are shown. (B) Normal (PNT2-C2) and benign (BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) and (C) four
primary cultures derived from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or
cisplatin as control treatments. Cell cycle phase was measured using propidium iodide staining and flow cytometry analysis.
doi:10.1371/journal.pone.0064278.g005
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Induction of Apoptosis and Autophagy by the Pd Nadeem et al [18] it varied from 5.80 to .100 mM. The reason
Complex for this broad range may be related to the nature of ligands
The indication of reduced cell growth resulting from the cell attached to the palladium metal core. In another study, in which
proliferation assays, along with the increase in sub-G1 content of different cell lines from different cancer types (central nervous
the treated cell populations, led to the investigation of cleaved system, colon, breast, leukemia, and prostate) were used, the Pd
caspase-3 activity to assess induction of apoptosis (Figure 6A(i), complexes resulted in broad range (13 to.100 mM) of IC50 values
Supplementary Figure S3). There was a clear induction of cleaved depending on both cell types and the mono- and dinuclear Pd
caspase-3 in BPH-1, PNT2-C2 and P4E6 cells when treated with complexes [31]. Taken together, the great variability in IC50
Pd complex, etoposide and cisplatin. However induction of values seems to depend on both cell type and the ligand attached.
cleaved caspase-3 in LNCaP cells was only observed after In the study of Mukherjee et al using palladium complexes,
treatment with 25 mM Pd complex, and there was no evidence prostate cancer (PC-3) cells died by apoptosis following cell cycle
of cleaved caspase-3 in PC3 cells that present as a very resistant arrest at G2/M phase [20]. Ultimately, it would be hoped that a
cell line. This also correlated with the much lower percentage of new drug preferentially killed cancer cells over normal cells.
PC3 cells with a sub-G1 DNA content. More significantly, there Although it was first encouraging that one of the normal cell lines,
was no evidence of cleaved caspase-3 in two primary samples PNT2-C2, appeared to be less susceptible to the Pd complex than
(Figure 6B(i)), and only a positive result with the BPH sample at a the other normal (PNT1A) and benign (BPH-1) cell lines, once
low dose. Since the sub-G1 content increases in primary cells in compared to the cancer cell lines it became apparent that the
response to Pd complex but with no corresponding caspase cancer cell lines are overall less susceptible to the Pd complex with
activity, this may mean that the apoptotic kinetics differ between average IC50 values at 72 h being 11.53 mM, whereas the normal
the primary cells and cell lines or that the sub-G1 content in the cell lines had an average of 0.38 mM. Therefore, more drug is
primary cells could be attributed to necrosis. To investigate the required to reduce viability of the cancer cells. This is disappoint-
mechanism of death in the cell lines and indeed the absence of ing but unfortunately not surprising, and new approaches to
apoptosis in the primary cells, levels of LC3-I and LC3-II were modify the compound in order to target it to the tumour while
measured, to assess autophagy. The ratio of LC3-II to LC3-1 used sparing the normal tissue would be desirable. Significantly, this
to be the standard measurement of autophagy, however it is now study shows the importance of using a panel of cell lines, and not
accepted that levels of LC3-II alone should be assessed relative to a just one ‘normal’ and one ‘cancer’ cell line. There is variability
typical control such as actin [28,29]. There was a clear increase in between the normal versus cancers, just as there is variability
the expression of LC3-II in BPH-1, PNT2-C2 and P4E6 cells between the different normal cell lines and different cancer cell
following increasing doses of Pd complex (Figure 6A(ii). Treatment lines.
with etoposide or cisplatin did not significantly change levels of Cell lines are very commonly used for initial high throughput
LC3-I and LC3-II. The levels of LC3-I and LC3-II in LNCaP and screening of cytotoxic anti-cancer compounds. However, it is
PC3 cells did not change dramatically or in a dose-dependent physiologically more relevant to use primary cultures to obtain
fashion with any treatment. In all primary cells there was a clear results that are closer to the patient. Therefore, in addition to the
increase in LC3-II, the modified version of LC3-I that is present many cell lines used in this study, we studied the anti-growth effect
on the autophagosomes and indicative of autophagy [29], with of the Pd complex on primary cultures from patient tumour
increasing Pd complex treatment (Figure 6B(ii)). This was also samples (Gleason grade 7). We found that the Pd complex had a
observed using immunofluorescence and autophagosomes were powerful growth-inhibiting effect on these primary cancer cells.
observed in primary cells following treatment with Pd complex Most of the IC50 values had quite a narrow range of around 3.778
(Figure 6C). Again, there was no significant change in LC3-I or to 13.12 mM depending on the patient from whom the cells were
LC3-II levels following etoposide or cisplatin treatment. This isolated. This was comparable to the cancer cell line IC50 values.
provided further clear evidence that the mechanism of action of Interestingly, when the Pd complex was tested against the cells
the Pd complex is different to etoposide or cisplatin, and indeed isolated from benign prostatic hyperplasia patients the results were
different between the cell types studied. quite similar to those found in the malignant samples with IC50
values ranging from 5.969 mM to 14.43 mM. Therefore, once
Discussion again the Pd complex did not preferentially kill cancer cells, but
importantly did not preferentially kill benign primary cells (unlike
Recurrent prostate cancer eventually results in the death of the the normal cell lines). However, more significantly, when the drug
patient due to resistance to chemotherapy and ineffective was tested against cultures from high Gleason grade tumours
chemotherapy, almost inevitably within 2 years from the failure (Gleason 8/9) the IC50 range for these samples was 46.68–
of hormone treatment [4]. Therefore, more efficient drugs/ 70.41 mM. Therefore, around ten times higher concentration of
approaches are required. In this study, we investigated the anti- the drug is required (using median values) to reduce the viability of
growth effect of a novel palladium complex, which is a growing these aggressive cancers compared to the lower grade cancers.
area in anti-cancer drug development. In vitro studies on different This is a statistically significant difference, P= 0.016. This is the
kinds of palladium complexes recently synthesized by both our first study testing this novel compound on primary epithelial cell
group and others have produced promising results [19,20,30]. In cultures of prostate and clearly highlights the utility of both cell
addition to in vitro studies, our in vivo study on breast cancer cell lines and primary cells when assessing a new drug.
lines also resulted in considerable cell death by inducing apoptosis There is now increasing evidence that cancer stem cells are
via cell death receptors, as well as inhibition of angiogenesis [25]. responsible for the recurrence of disease, due to their resistance to
In this study, we have found that the Pd complex had a current chemotherapy [32,33,34]. Therefore, we investigated the
significant growth-inhibiting activity against both prostate cancer effect of the Pd complex on cancer stem cells isolated from
cell lines and cell lines derived from normal and benign prostate. malignant samples and stem cells isolated from benign prostate
IC50 values have ranged from 0.1064 mM to 26.79 mM (72 h), hyperplasia samples. In addition to CSCs, TA and CB cells from
depending on the cell line. In the literature, IC50 values of the same cultures were also isolated and studied. We found that
palladium compounds also have a broad range. In the study of the Pd complex had much more potent cytotoxic activity than
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Palladium’s Cytotoxicity on Prostate Cancer Cells
Figure 6. Expression of apoptosis and autophagy-related proteins following treatment with Pd complex. Normal (PNT2-C2) and benign
(BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) (A) and four primary cultures (B) one derived from patient with BPH and three derived
from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or cisplatin as control treatments.
Lysates were harvested and Western blotting was carried out staining for cleaved caspase-3 indicative of apoptosis induction or LC3B protein,
indicative of autophagy. (C) Images of primary cells (sample 23912) treated with Pd complex and stained with LC3-B antibody. Shown are three
example images of untreated cells and cells treated with 12 mM and 25 mM Pd complex. Autophagosome vesicles are clearly visible in red.
doi:10.1371/journal.pone.0064278.g006
etoposide at the same concentration (25 mM) but had comparable repairable, but the damage caused by the Pd complex may not,
toxicity to cisplatin at the 6 mM range. This is also the first report and is therefore more lethal. Future studies will develop this area
in the literature, to our knowledge, on the effect of Pd complexes and will investigate the types of DNA damage occurring as well as
on cancer stem cells. Eradication of cancer stem cells is an aim of the active DNA repair mechanisms. It is known that there can be
most novel strategies, but with the potential for these cells to resistance to platinum-based drugs and that this can be related to
display increased resistance, it is likely that they may need more the DNA repair mechanisms in the cell [35]. It will therefore be
rigorous treatment. In fact, in our study we also used the interesting to determine if cells also acquire resistance to palladium
concentration of 50 mM of the complex, which killed all cells (data complexes, and whether this is through similar mechanisms, or
not shown). However, this concentration may be too toxic to the indeed if they are less likely to acquire resistance to these
other cells of the body, and thus not be tolerated by patients. compounds. Following increase of the sub-G1 population in the
Indeed, although we observed significant anti-growth effects of cell cycle analysis, induction of apoptosis was confirmed with
cancer cells, this Pd complex also efficiently killed normal and Western blotting of cleaved caspase-3. Interestingly apoptosis was
benign cells. The overall toxicity of the drug will be an important induced in all of the cell lines (only at the highest Pd complex
future consideration. concentration for LNCaP), except PC3. No apoptosis was induced
To investigate the mechanism of cell death and the effect of the in the primary cancer cells.
Pd complex on the cell cycle, we first studied the DNA-damaging In both cell lines and primary cells, autophagy was measured by
effect of the Pd complex. Platinum compounds are known to assessing levels of LC3-I and LC3-II. Increased levels of LC3-II
induce DNA adducts, which block replication and transcription protein typically hints at induction of autophagy, although future
resulting in DNA damage and cell death [35]. Interestingly, we studies would have to incorporate autophagic flux to elucidate the
found that both Pd complex and etoposide caused the same level complete response [28,36]. Increased levels of LC3-I may occur
of DNA damage, measured as cH2AX foci (indicative of double- prior to an increase in LC3-II or could indicate that there is a
strand breaks) although the Pd complex resulted in a more block in autophagy at an early stage. Autophagy is a dichotomy in
powerful cytotoxic effect than etoposide. This implies that some of cancer because in some circumstances it can be a cell-protective
the DNA damage resulting from etoposide treatment may be survival mechanism responding to hypoxia, nutrient deprivation
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Palladium’s Cytotoxicity on Prostate Cancer Cells
or stress, whilst in other circumstances it can be a prelude to lines and primary cells, including cancer stem cells, and provide
autophagy-induced caspase-independent cell death [37,38]. In- first indications of a complex cell death mechanism. Although we
deed, autophagy and apoptosis are activated by similar stimuli but have shown that this is a drug with high toxicity, the potential to
can be mutually exclusive or simultaneous [37]. In terms of using use it at lower doses in a combination strategy with autophagy
autophagy as a treatment strategy in cancer, this has been modulators, is worth further exploration. Finally, we think that a
approached in two ways. In CML, the combination of a TK key message of this study lies in the use of a panel of cell lines
inhibitor with an autophagy inhibitor increased cytotoxicity alongside patient samples. We have shown that depending on the
[39,40]. Secondly, use of temolozide, a pro-autophagic drug, in genetic background of the cells as well as the aggressiveness of the
glioblastoma in combination with an mTOR inhibitor induced cancer, there are different outcomes to drug treatment. This
autophagy and cell death [41]. Therefore, it is imperative to should be taken into account when testing other palladium drugs
understand the biology of the cancer cells under treatment before and drugs in general, such that one cell line is not overly relied
deciding on the best strategy. Indeed, within this dichotomous upon, and that patient samples are included in any study of this
role, it seems that autophagy is less active in early stage cancers kind.
with its cell-protective role, only coming to the fore in later stage
cancers [41,42,43]. In terms of prostate cancer, there have already Supporting Information
been studies indicating that autophagy protects against hormone
ablation therapy, and combining androgen deprivation with Figure S1 Graphs of transformed data from Figure 2 following
autophagy inhibition led to synergistic cell death suggesting a application of the nonlinear regression (curve fit) that represents
new potential strategy to overcome hormone therapy resistance the log(inhibitor) ‘v’ normalized response, from which the IC50s
[44]. There are also clinical trials underway combining docetaxel were calculated (GraphPad Prism software).
(standard chemotherapy for prostate cancer) with hydroxyquinone (TIF)
autophagy inhibitor [45]. From our results, the presence or
Figure S2 Graphs of transformed data from Figure 3 following
absence of PTEN may contribute to the outcome of these
strategies. In PC3 and LNCaP cells the palladium compound did application of the nonlinear regression (curve fit) that represents
not induce autophagy and only induced apoptosis at high the log(inhibitor) ‘v’ normalized response, from which the IC50s
concentrations (LNCaP) or not at all. Both cell lines contain were calculated (GraphPad Prism software).
inactive PTEN [46] and since mTOR is a key negative regulator (TIF)
of autophagy, the absence of PTEN, a negative regulator of Figure S3 Images of cells treated for flow cytometry cell cycle
mTOR, could result in lack of induction of autophagy. In prostate analysis and for protein lysates.
cancers around 40% of patients lack PTEN activity, a proportion (TIF)
that increases in castration-resistant prostate cancer [47,48]. The
other cell lines tested (BPH-1, PNT2-C2 and P4E6) are PTEN- Acknowledgments
positive and were susceptible to the drug. Thus, there may be
differential efficacy of this combination strategy between patients, The authors would like to thank Urology surgeons Mr(s) L Coombes, G
depending on the genetic background of the tumour. Cooksey and J Hetherington (Castlehill Hopsital, Cottingham) for
In conclusion, the Pd complex had a considerable anti-growth assistance in supplying prostate tissue. Great appreciation goes to the
patients from York District Hospital, York and Castlehill Hospital,
effect on most prostate cancer cell lines and primary cultures.
Cottingham for providing prostate tissue. We would like to thank Michael
Importantly, it also successfully inhibited the viability of cancer
Fraser for the immunofluorescence protocol. Thank you to S. Hayward for
stem cells, implying that this Pd complex may be used for the the gift of the BPH-1 cells and P. Berthon for the PNT1A cells.
treatment of metastatic prostate cancer that is extremely resistant
to conventional therapy. Previous work has shown that Palladium
Author Contributions
complexes can cause cell death by necrosis or apoptosis. This study
showed that the Pd complex induced autophagy in some cases, Conceived and designed the experiments: EU FMF VTY NJM. Performed
and therefore points to a new area of investigation. Here we the experiments: EU FMF BC. Analyzed the data: EU DP FMF.
present a comprehensive overview of the effects of a novel Contributed reagents/materials/analysis tools: VTY FMF HW VMM
MSS MJS. Wrote the paper: EU FMF.
Palladium complex on cell viability in an extended panel of cell
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