![The inhibitory effects of cisplatin on the proliferative capacity of cisplatin resistant NSCLC cells. Parent (PT) and cisplatin resistant (CisR) cell lines were treated with increasing concentrations of cisplatin for 72 h. Proliferation was measured using the MTT assay. While cisplatin inhibited the growth of both PT and CisR cell lines, the inhibitory effect of cisplatin was greatly reduced in CisR cells relative to parent cells. Data are expressed as Mean 6 SEM from three independent experiments (n = 3) (*p,0.001 vs PT untreated, $$ p,0.001 vs CisR untreated, # p,0.001 PT vs CisR, $ p,0.01 vs CisR untreated [A549], $ p,0.01 PT vs CisR, #$ p,0.05 PT vs CisR [MOR]). doi:10.1371/journal.pone.0054193.g003](https://www.researchgate.net/profile/Steven-Gray-5/publication/235372179/figure/fig14/AS:669685125230592@1536676784120/The-inhibitory-effects-of-cisplatin-on-the-proliferative-capacity-of-cisplatin-resistant_Q320.jpg)
![Cisplatin-induced apoptosis is reduced in cisplatin resistant cell lines. Parent and resistant cell lines were treated with cisplatin for 72 h. Apoptotic cells, as measured by the percentage cells in the SubG0 phase of the cell cycle, were measured. Levels of apoptosis induced by cisplatin were significantly increased in parent cells while cisplatin-induced apoptosis was significantly reduced in the corresponding resistant cell line. Data are expressed as Mean 6 SEM from three independent experiments (n = 3) (*p,0.01 vs PT untreated, **p,0.001 vs PT untreated, $$ p,0.001 vs CisR untreated, $ p,0.01 vs CisR untreated [A549, H460], $ p,0.05 vs CisR untreated [MOR], $ p,0.05 PT vs CisR, # p,0.001 PT vs CisR, * $ p,0.05 vs PT untreated). doi:10.1371/journal.pone.0054193.g004](https://www.researchgate.net/profile/Steven-Gray-5/publication/235372179/figure/fig15/AS:669685125222404@1536676784372/Cisplatin-induced-apoptosis-is-reduced-in-cisplatin-resistant-cell-lines-Parent-and_Q320.jpg)
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Generation and Characterisation of Cisplatin-Resistant
Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-
Like Signature
Martin P. Barr
1
*, Steven G. Gray
1
, Andreas C. Hoffmann
2
, Ralf A. Hilger
2
, Juergen Thomale
3
,
John D. O’Flaherty
1
, Dean A. Fennell
4
, Derek Richard
5
, John J. O’Leary
6
, Kenneth J. O’Byrne
1
1Thoracic Oncology, Institute of Molecular Medicine, Trinity Centre for Health Sciences St. James’s Hospital & Trinity College Dublin, Dublin, Ireland, 2Molecular Oncology
Risk-Profile Evaluation (M.O.R.E.), Department of Medical Oncology, West German Cancer Centre, University Hospital Essen, Essen, Germany, 3Department of Cell Biology
(Cancer Research), University Duisburg-Essen, Essen, Germany, 4Thoracic Medical Oncology, University of Leicester & Leicester University Hospitals, Leicester, United
Kingdom, 5Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, Australia, 6Department of Histopathology, St. James’s Hospital &
Trinity College Dublin, Dublin, Ireland
Abstract
Introduction:
Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-
small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the
development of novel agents to enhance the sensitivity of cisplatin.
Methods:
An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR,
H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent
cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out
between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by
FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and
b-catenin. Cisplatin-DNA adduct formation, DNA damage (cH2AX) and cellular platinum uptake (ICP-MS) was also assessed.
Results:
Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to
cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell
cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with
increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The
stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and b-catenin.
While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA
adduct formation and significantly decreased cH2AX foci were observed compared to parental cell lines.
Conclusion:
Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature,
providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.
Citation: Barr MP, Gray SG, Hoffmann AC, Hilger RA, Thomale J, et al. (2013) Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer
Cell Lines Displaying a Stem-Like Signature. PLoS ONE 8(1): e54193. doi:10.1371/journal.pone.0054193
Editor: Pan-Chyr Yang, National Taiwan University Hospital, Taiwan
Received February 7, 2012; Accepted December 10, 2012; Published January 17, 2013
Copyright: ß2013 Barr 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: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mbarr@stjames.ie
Introduction
More than one million cases of lung cancer are diagnosed each
year. The disease is the leading cause of cancer-related death in
men and women [1]. Despite intensive efforts to control morbidity
and mortality from lung cancer, the overall five-year survival rate
remains poor.
Cisplatin, cis-Diamminedichloro-platinum(II), is one of the most
commonly used chemotherapeutic agents in the treatment of
cancer, in particular non-small cell lung cancer (NSCLC) [2]. The
cytotoxic effects of cisplatin are mediated by its interaction with
DNA, resulting in the formation of DNA adducts which activate
several signal transduction pathways and culminate in the
activation of apoptosis [3]. While 20–40% of patients with
metastatic NSCLC experience a partial response to newly
developed combination therapies [4], most responders relapse
within six months [5]. Within the population of patients that
relapse, the selection of pre-existing resistant cells and/or
acquisition of resistant cells during treatment with chemotherapy
has been proposed. Therefore, a better understanding of the
molecular basis of cisplatin resistance is warranted in order to
elucidate the mechanisms and markers underlying this drug-
resistant phenotype, which at present radically limits the clinical
utility of this drug in lung cancer patients.
Recently, the cancer stem cell (CSC) theory was proposed to
explain tumour heterogeneity and carcinogenesis [6]. According
PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54193
to this model, tumours may be viewed as a result of abnormal
organogenesis driven by CSC’s. These are self-renewing tumour
cells that are able to initiate and maintain tumour growth through
subpopulations of tumour cells with stem or progenitor cell
characteristics. Using in vitro systems and in vivo models of human
primary lung cancer xenografts in mice, recent research has
demonstrated that lung tumour cells expressing specific CSC
markers were highly tumourigenic, endowed with stem-like
features and spared by treatment with cisplatin [7].
In this study, we have generated and characterised a panel of
cisplatin resistant NSCLC cell lines, providing a valuable tool with
which to investigate the molecular pathways and putative stem
cells markers that may be associated with this resistance phenotype
in lung cancer.
Materials and Methods
Cell Lines
The human large cell lung cancer cell line, NCI-H460
(hereafter referred to as H460) and its resistant variant was kindly
donated by Dr Dean Fennell, Centre for Cancer Research and
Cell Biology, Queen’s University Belfast [8]. The human
adenocarcinoma cell line, MOR [9], and its corresponding
cisplatin resistant variant was obtained from the American Type
Culture Collection (ATCC) (LGC Promochem, Teddington, UK).
A549 (adenocarcinoma) and SKMES-1 (squamous carcinoma) cell
lines were also purchased from the ATCC [10,11]. MOR and
H460 cells were grown in Roswell Park Memorial Institute
(RPMI-1640) medium. A549 cells were cultured in Ham’s F12
media supplemented with 4 mM L-glutamine while SKMES-1
cells were cultured in EMEM media supplemented with 2 mM L-
glutamine and 1% non-essential amino acids (NEAA). For all cell
lines, media was supplemented with 10% heat-inactivated fetal
bovine serum (FBS), penicillin (100 U/ml) and streptomycin
(100 mg/ml) (Lonza, United Kingdom). All cells were grown as
monolayer cultures and maintained in a humidified atmosphere of
5% CO
2
in air at 37uC.
Drugs
Cisplatin [cis-diammineplatinum(II) dichloride] was obtained
from Sigma-Aldrich and dissolved in 0.15 M NaCl. Aliquots were
stored at 220uC for up to a maximum of three months and
thawed immediately before use.
Induction of Cisplatin-Resistance in NSCLC Cells
Cisplatin-resistant (CisR) variants of each cell line were derived
from each original parental (PT) cell line by continuous exposure
to cisplatin (Sigma-Aldrich, UK) following initial dose-response
studies of cisplatin (0.1 mM–100 mM) over 72 h from which IC
50
values were obtained. Initially, each CisR subline was treated with
cisplatin (IC
50
) for 72 h. The media was removed and cells were
allowed to recover for a further 72 h. This development period
was carried out for approximately 6 months, after which time IC
50
concentrations were re-assessed in each resistant cell line. Cells
were then maintained continuously in the presence of cisplatin at
these new IC
50
concentrations for a further 6 months. While A549
cells were initially treated with IC
50
concentrations of cisplatin,
cells were sensitive to treatment at this concentration resulting in
cell senescence and delayed growth. For this reason, the cisplatin
concentration was reduced (IC
25
) until such time as cells
demonstrated sensitivity to cisplatin at the appropriate IC
50
concentration.
Drug Sensitivity Assay (MTT)
Cells (2.5610
3
) were seeded in 96-well plates and allowed to
adhere overnight at 37uC. Briefly, following treatment of cells with
cisplatin for 72 h, MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] was added to each well and
incubated for 4 hrs at 37uC. Dimethylsulphoxide (DMSO) was
added to each well and mixed for 5 min on an orbital shaker.
Absorbance was recorded at 595 nm and sensitivity to cisplatin
was calculated based on cell proliferation measurements at 72 h.
Cell Cycle & Apoptosis Analysis
Cells were collected by trypsinisation, pelleted by centrifugation
at 1300 rpm for 3 min and suspended in 1 ml phosphate-buffered
saline (PBS). Cells were subsequently fixed in 90% cold ethanol
and incubated at room temperature for 30 min. Cells were
pelleted and resuspended in 1 ml PBS containing propidium
iodide (25 mg/ml) and DNase-free RNase A (100 mg/ml).
Following incubation at 37uC for 30 min, cell cycle distribution
of PT and CisR cells were analysed using FACS (Becton
Dickinson, UK). Apoptotic cells (SubG0) were measured in
response to increasing concentrations of cisplatin between PT
and CisR cells following treatment for 24 h.
Clonogenic Survival Assay
The sensitivity of NSCLC cells to cisplatin was measured using
the clonogenic assay, the method of choice used to determine the
effectiveness of cytotoxic agents such as chemotherapy [12]. Cells
were allowed to adhere overnight at 37uC and treated with
increasing concentrations of cisplatin for 9–14 days. Colonies were
fixed and stained with methanol (25% v/v) containing crystal
violet (0.05% w/v) for 30 min after which time residual staining
solution was removed and plates were washed with water.
Colonies consisting of 100 cells or more were counted using the
ColCount
TM
colony counter (Oxford Optronix Ltd, Oxford, UK).
Plating efficiencies (PE) were calculated using the formula:
PE = Number of colonies/Number of cells seeded. The surviving
fraction (SF) was calculated using the formula: SF = Number of
colonies/Number of cells seeded 6PE). Survival curves were
constructed for determination of survival ability of cisplatin-
resistant cells relative to parent cells in response to various
concentrations of cisplatin.
Flow Cytometry Analysis of Putative Cancer Stem Cell
Markers
Parent and cisplatin-resistant cells were collected by trypsination
and washed in FACS buffer (2% FBS 0.1% sodium azide in PBS)
and pelleted by centrifugation at 1300 rpm for 3 min. Dual
staining for CD133 and CD44 (epithelial cell marker) was carried
out. Cells (1610
6
) were incubated with either CD133/1 (AC133)
phycoerythrin (PE)-labelled antibody or isotype control antibody
(IgG1) (Miltenyi Biotec GmbH), or anti-human CD44 FITC-
conjugated antibody and corresponding isotype control (IgG2b)
(ImmunoTools GmbH, Germany) for 30 min in the dark at 4uC.
Cells were washed briefly and resuspended in FACS buffer for
subsequent analysis. Samples were acquired and analysed by
FACS. Side scatter and forward scatter profiles were used to
eliminate debris and cell doublets. The percentage CD133+and
CD44+cells was determined in PT and CisR cell lines by flow
cytometry.
Aldefluor Assay
The Aldefluor Kit (Stem Cell Technologies, Vancouver,
Canada) was used to identify cell populations with aldehyde
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PLOS ONE | www.plosone.org 2 January 2013 | Volume 8 | Issue 1 | e54193
dehydrogenase (ALDH1) activity. The assay was carried according
to manufacturer’s instructions. Briefly, cells (1610
6
cell/ml) were
harvested from PT and CisR cell lines and resuspended in
Aldefluor Assay Buffer and incubated for 60 mins at 37uC. The
amount of fluorescent ALDH reaction product that accumulates in
the cells directly correlates to the ALDH activity in these cells.
Active efflux from the cells is inhibited by the special formulation
of the Aldefluor Assay Buffer. For each cell line (PT and CisR),
control cells were stained using identical conditions but included
a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), to
serve as a negative control for each experiment. Such cells are
recognised by comparing the fluorescence in a test sample to that
in a control sample containing DEAB. As only cells with an intact
cellular membrane can retain the Aldefluor reaction product, only
viable ALDH1-positive cells were identified. The brightly fluour-
escent ALDH1-expressing cells (ALDH1-positive) were detected in
the green fluorescent channel (520–540 nm) of a CyAn
ADP
flow
cytometer (Dako, Glostrup, Denmark) and calculated as the
percentage ALDH1-positive cells in each cell line.
Western Blot Analysis
Total protein was extracted from parent and cisplatin resistant
cells using ice-cold RIPA buffer (50 mM Tris HCl, pH 7.4,
150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton-X 100, 0.1% (w/
v) SDS) supplemented with phenylmethylsulfonyl fluoride (PMSF)
and protease inhibitor cocktail (2 mM AEBSF, 1 mM EDTA,
130 mM Bestatin, 14 mM E-64, 1 mM Leupepin, 0.3 mM Apro-
tinin). Protein concentrations were determined using the bicinch-
oninic acid assay as per manufacturer’s instructions (BCA). Protein
(40 mg) from whole cell lysates was fractionated on 12% SDS-
PAGE gels and transferred to a PVDF membrane (PALL
Corporation, FL, USA). Transfer efficiency and loading were
confirmed by reversible staining of the membrane with Ponseau S
solution (Sigma-Aldrich, UK) following protein transfer. Mem-
branes were blocked at room temperature with 5% non-fat dry
milk in Tris-buffered saline (TBS) containing 0.1% Tween-20
(TBS-T) and screened using a human embryonic stem cell marker
panel (Abcam plc, United Kingdom). These included primary
rabbit polyclonal mouse antibodies to Nanog, Oct-4 and SOX-2
(1:1000). Protein expression of c-Met (Millipore) and b-Catenin
(BD Transduction Laboratories) was also examined using mouse
monoclonal antibodies at 1:100 and 1:2000, respectively. Mem-
branes were washed in TBST and incubated with a secondary
horseradish peroxidase (HRP)-labelled antibody for 1 h at room
temperature (1:2000). Membranes were washed in TBST follow-
ing incubation with secondary antibodies. Bound antibody
complexes were detected and visualised using SuperSignal
H
West
Pico enhanced chemiluminescence substrate (Pierce, IL, USA).
Blots were stripped and re-probed with a/bTubulin antibody
(Cell Signalling) to control for loading. Densitometric analysis was
carried out using TINA
TM
software and percentage expression
represented relative to controls (100%).
Immunofluorescence Microscopy and Measurement of
Cisplatin-DNA Adducts
Cells (PT and CisR) were treated with cisplatin (IC
50
) for 0, 4,
12 and 24 h after which time they were collected by trypsinization
and washed twice in PBS. Cells (1610
6
cell/ml) were resuspended
in PBS and spotted (10 ml), in triplicate, onto Superfrost Gold
Slides (ThermoFisher). Slides were allowed to air dry briefly at
room temperature. Immunofluorescence staining and measure-
ment of specific DNA platination products was performed as
previously described [13], with minor modifications. Briefly, cells
were fixed overnight in ice-cold methanol and subjected to
proteolytic digestion with 60 mg/mL pepsin and 40 mg/mL
proteinase K (100 ml per spot for 10 min at 37uC in a humidified
chamber). Upon blockade of non-specific binding sites with 5%
(w/v) non-fat powdered milk in PBS, slides were incubated with
a rat primary antibody that specifically recognises CDDP-GpG
DNA adducts (RC-18) at 37uC for 2 h or 4uC overnight. Primary
antibody binding was detected using an anti-rat Cy3H-labelled
antibody (Dianova, Hamburg). Slides were then incubated in
1mg/ml (w/v) DAPI in PBS for 30 min at RT for nuclear
counterstaining. Images were acquired on an Axioplan fluores-
cence microscope (Carl Zeiss GmbH, Go¨ttingen, Germany)
coupled to a C4880 CCD camera (Hamamatsu Photonics,
Herrsching, Germany). For the quantification of CDDP-GpG
DNA adducts by immunofluorescence microscopy, fluorescence
signals were measured by quantitative digital image analysis using
the ACAS 6.0 CytometryAnalysis System (ACAS II, Ahrens
Electronics, Bargterheide, Germany). Levels of adducts in each
sample were calculated as arbitrary fluorescence units (AFU’s),
upon normalization of integrated antibody-derived fluorescence
from 200 individual nuclei/sample to the corresponding DNA
content. Data are presented as the mean AFU 695% confidence
interval (CI) from three independent experiments.
cH2AX Foci Formation Assay
Cells (5610
3
) were seeded, in triplicate, in 96-well plates and
allowed to adhere overnight. Parent and resistant cells were
treated with cisplatin for 0, 4, 8, 12 and 24 h. At each time-point,
cell culture media was removed from each well and fixed for
10 min in 100 ml formaldehyde (4% v/v in PBS). Cells were then
washed twice in PBS. Blocking buffer (5% goat serum, 3% Triton
X-100 in PBS) was added to each well and incubated for 1 h at
room temperature. Cells were then incubated overnight at 4uC
with a primary rabbit anti-human anti-phospho-histone 2AX
(Ser139) antibody (1:100) (Cell Signalling Technology) in antibody
dilution buffer (1% BSA.0.3% Triton X-100 in PBS). Following
removal of the primary antibody, cells were washed three times in
PBS and incubated with Alexafluor 488-labelled goat anti-rabbit
secondary antibody (Invitrogen) (1:2000) for 1 h at room
temperature in the dark. Secondary antibody was removed and
cells were washed three times in PBS. Cells were then incubated
with Hoechst 33342 nuclear stain (3 mg/ml) for 30 min at 37uC,
followed by three washed in PBS. Cells staining for phosphory-
lated histone 2AX (detected as green fluorescent foci) were imaged
by immunofluroescence using high content analysis (GE Health-
care). Ten fields of view per well were acquired using a 20X
objective. Nuclear staining was detected using an excitation filter
of 360 nm and emission filter of 460 nm, while Alexafluor 488 was
detected at 480 nm and 535 nm, respectively. Mean nuclear
fluorescence intensity was used as a measure of cH2AX using
InCell analyser 1000 image analysis software.
Quantification of Cellular Cisplatin uptake by ICP-MS
For cisplatin uptake studies, cells (1610
7
cells/ml) were seeded
in culture flasks and allowed to adhere overnight. Cells were then
treated with cisplatin for 24 h. Following treatment, cells were
washed in PBS, harvested and counted. For drug uptake analysis,
cells (1610
6
) were suspended in 1% HNO
3
for 24 h at 70uC.
Lysed cells were analysed by inductively coupled plasma mass
spectrometry (ICP-MS). ICP-MS provides a quantitative analysis
of the concentration of an element in aqueous solution and has
a sensitivity of 5 PPT or better for Platinum products. The analyte
concentration is proportional to the number of ions of a specific
element that reach the mass spectrometer from the vaporised
solution at 6000uC. A single ICP-MS measurement represents the
Cisplatin Resistant NSCLC Cells
PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e54193
average of 20 scans per replicate within five replicates from the
same liquid sample, with a very small error (,5%). Cisplatin
concentrations reported were averaged across four series of
cultures, ensuring that the values are correctly scaled to account
for cell population differences and dilutions.
Standard curves were generated by using aqueous serial
dilutions of stock solutions traceable back to the standard reference
material (SRM) from NIST (National Institute of Standards and
Technology). The coefficients of variation ranged from 1 to 4%
(intra-assay) and from 5 to 10% (inter-assay).
Statistical Analysis
Statistical comparison between groups was carried out using
analysis of variance (ANOVA). Where the means of two data sets
were compared, significance was determined by a two-tailed
Students t-test. Differences were considered to be statistically
significant where p#0.05. Data is graphically represented as mean
6standard error of the mean (SEM). All data was analysed using
GraphPad InStat
TM
(version 3) statistical software.
Results
Generation of IC
50
Concentrations and Development of
NSCLC Cells with a Cisplatin-resistant Phenotype
In order to determine IC
50
values with which to treat parental
cell lines in the generation of cisplatin resistant cell lines, cells were
treated with increasing concentrations of cisplatin ranging from
0.1 mM to 100 mM. The H460 CisR cell line was previously
generated and maintained with 5 mM cisplatin. The sensitivity of
each original (PT) cell line to increasing doses of cisplatin was
demonstrated, where cisplatin significantly (p,0.001) inhibited
proliferation of A549, SKMES-1 and MOR cells at 10 mM–
100 mM over 72 h (Fig. 1A). Dose-response curves were generated
and IC
50
concentrations were calculated for all cell lines (Fig. 1B).
Cisplatin concentrations (IC
50
) varied between all four cell lines
(A549 5.95 mM, SKMES-1 2.65 mM, MOR 3.3 mM, H460 5.0 mM)
and were subsequently used to treat each parent cell line in order
to generate corresponding age and passage-matched cisplatin
resistant cell lines. In the case of H460 cells, maintenance of the
resistant subline was continued at 5 mM. Treatment of A549 cells
with cisplatin (IC
50
) resulted in significant growth delay, with slow
recovery periods. Cells were therefore treated with IC
25
concen-
trations for several weeks prior to selection of a cisplatin resistant
subline at the IC
50
concentration.
Cisplatin resistant sublines were treated with cisplatin for 72 h
after which time media was removed and cells were allowed to
recover and re-populate. During this time, cell survival/pro-
liferation was assessed between PT and CisR cells every 4 weeks to
determine changes in sensitivity to cisplatin. At 6 months, IC
50
values were re-evaluated and deduced from dose-response curves
between PT and CisR cells. A significant fold increase was
observed in the concentration of cisplatin required to inhibit cells
by 50% in cisplatin resistant cells relative to their corresponding
parent cells (Fig. 2). Cells were subsequently maintained in
cisplatin at these concentrations for a further 6 months. In A549
cells, the IC
50
concentration of cisplatin resistant cells was
determined as 23.60 mM compared to 1.58 mM in the original
parent cell line, a 15-fold increase in the concentration of cisplatin
required to obtain a 50% inhibition in cell growth. A significant
increase in IC
50
concentrations was also observed in SKMES-1
cells (16.0 mM vs 4.09 mM), MOR cells (31.98 mM vs 6.39 mM)
and H460 cells (30.40 mM vs 5.72 mM), demonstrating a 4-fold
(SKMES-1) and 5-fold (MOR, H460) increase between CisR and
PT cell lines. Taken together, these initial data demonstrated
a cisplatin-resistant phenotype in four NSCLC cell lines following
continuous in vitro exposure to cisplatin.
Upon characterisation of cells at 52 weeks following exposure of
cells to cisplatin, a significant difference in the proliferation
capacity between PT cell lines and their corresponding cisplatin
resistant sublines was observed (Fig. 3) indicating the emergence of
a resistant phenotype in the resistant sublines relative to the parent
cell lines. While A549 and H460 cells showed significant
differences in their proliferative ability between parental and
corresponding CisR cells at concentrations ranging from as low as
0.1 mM(A549, 65.5660.73 vs 102.5060.87; H460, 87.6660.67 vs
114.0661.57, p,0.001) to 100 mM(A549, 16.5660.29 vs
41.4460.94, p,0.001; H460, 20.8961.22 vs 34.3261.17,
p,0.01), a significant difference was also observed between parent
and CisR SKMES-1 and MOR cells at concentrations of cisplatin
ranging from 10 mM(SKMES-1, 54.3861.56 vs 79.0062.25;
MOR, 64.3362.33 vs 76.8762.77, p,0.01) to 100 mMin
SKMES-1 and MOR cells, respectively (SKMES-1, 32.7961.55
vs 59.3365.20, p,0.01; MOR, 34.3362.50 vs 45.3362.33,
p,0.05).
Cisplatin-induced Apoptosis is Significantly Abrogated in
Resistant Cells Relative to their Parent Counterparts
Levels of cisplatin-induced apoptosis, as determined using the
SubG0 (apoptotic) fraction of cells, were assessed in PT and
corresponding CisR cell lines following treatment of cells with
increasing doses of cisplatin. While there was a significant increase
in lung tumour cell apoptosis of PT cells in response to cisplatin at
concentrations between 10 mM and 100 mM, cisplatin-induced
apoptosis of CisR cells was significantly decreased across all cell
lines (Fig. 4), in particular A549, SKMES-1 and H460 cells. In
A549 and SKMES-1 cells, significant cell death was observed only
at higher concentrations between 40 mM(A549,p,0.01; SKMES-
1,p,0.01) and 100 mM(p,0.001). More significantly however,
H460 CisR cells displayed greater resistance to cisplatin-induced
death at higher concentrations of cisplatin compared to other cell
lines, where significant induction of apoptosis was seen in response
to cisplatin at concentrations as high as 80 mM(p,0.01) and
100 mM(p,0.001). In all lung tumour cell lines, a significant
difference in the cellular response to cisplatin-induced apoptotic
cell death was observed between CisR and PT cells. Significant
differences in the levels of apoptosis between H460 PT and CisR
cells were seen in response to cisplatin at all concentrations
ranging from 10 mM to 100 mM, thereby highlighting a greater
cisplatin resistant phenotype in this CisR cell line.
Cisplatin Resistant NSCLC Cells Accumulate in G0/G1 of
Cell Cycle
At basal levels, and in response to increasing concentrations of
cisplatin, an increased accumulation of cells in the G0/G1 phase
was observed in all CisR cell lines relative to their respective
parental cell lines (Fig. 5A). Representative histograms are shown
for SKMES-1 PT and CisR cells in response to increasing
concentrations of cisplatin (Fig. 5B). In cisplatin resistant cell lines,
treatment with cisplatin induced a significant accumulation of cells
in the G0/G1 phase, relative to PT cells treated at the same
concentrations. Such observations were concomitant with a de-
crease in the S phase of the cell cycle. The basal fractions of cells
between G0/G1, S and G2/M phases of the cell cycle were also
studied between PT and CisR cell lines. A significant increase in
the G0/G1 fraction was found in SKMES-1 CisR (61.6062.734,
p,0.05) and MOR CisR (60.2060.872, p,0.05) cells relative to
their parent counterparts (47.0161.549, 42.4461.351, p,0.05).
Cisplatin Resistant NSCLC Cells
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Cisplatin Resistant NSCLC Cells
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In A549 and H460 cisplatin-resistant cells, greater significance was
seen in the G0/G1 fraction relative to their parent counterparts
(A549, 85.1961.763 vs 52.8362.234, H460, 69.1261.987 vs
39.6162.00, p,0.001). At basal levels, the fraction of cells in the S
and G2/M phases did not change significantly between PT and
CisR cell lines. These alterations in cell cycle distribution may play
an important role in the cisplatin resistant phenotype of the
NSCLC cell lines generated.
Chemoresistant NSCLC Cells Demonstrate Enhanced
Clonogenic Survival Ability
The survival ability of PT and CisR NSCLC cells following
treatment with cisplatin was assessed using the clonogenic survival
assay. All cell lines showed variable resistance between PT and
CisR cells. In the majority of cell lines examined, there was
a significantly higher fraction of surviving colonies of A549,
SKMES-1 and H460 CisR cells relative to parent cells at 1 mM
and 10 mM of cisplatin (Fig. 6). The H460 cell line demonstrated
greater resistance however, with a surviving fraction of 0.6760.04,
0.4760.03 and 0.2660.02, at 0.1 mM, 1 mM and 10 mM,
respectively, relative to the parent cell line (0.4560.06,
0.2760.02 and 0.0360.01). While MOR CisR cells showed
a significant survival of colonies relative to parent cells at 10 mM
(0.3260.03 vs 0.0260.01), this was not significant at 1 mM
(0.4560.05 vs 0.3760.03). These clonogenic survival data further
confirm the cisplatin resistant phenotype of A549, SKMES-1,
MOR and H460 sublines derived from each parent cell line.
Cisplatin-resistant Cell Lines Exhibit Enriched Fractions of
CD133+CD44+Cells
Because the cancer stem-cell compartment comprises of a very
small fraction of the total cancer cell population, it is necessary to
utilise specific cell surface markers for cancer stem cells. The
expression profile of putative stem cell surface markers, CD133
and CD44, were examined between parent and corresponding
cisplatin resistant cell lines. Using double staining by flow
Figure 1. Cisplatin inhibits proliferation of lung cancer cells in a dose-dependent manner. (A) NSCLC cells were treated with increasing
concentrations of cisplatin (0.1 mM–100 mM) for 72 h. Cell survival was measured using the MTT assay. Cisplatin significantly reduced proliferation of
A549, SKMES-1 and MOR NSCLC cells. (B) Dose-response curves were generated from which IC
50
values were deduced. Data are expressed as Mean 6
SEM from three independent experiments (n = 3) (*p,0.001 vs untreated).
doi:10.1371/journal.pone.0054193.g001
Figure 2. Cells exhibit increased fold changes in IC
50
concentrations following long-term exposure to cisplatin. Following
maintenance of cisplatin treated sublines in culture for 6 months with cisplatin, IC
50
concentrations were re-assessed for each cell line using dose-
response curves generated by GraphPad Prism software. A significant increase in IC
50
concentration was determined for each cisplatin resistant cell
line relative to that for the corresponding age-matched parental cell line.
doi:10.1371/journal.pone.0054193.g002
Cisplatin Resistant NSCLC Cells
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cytometry, we examined whether the established cisplatin resistant
cell lines displayed an enrichment of CD133+cells and whether
cells expressing CD133 were also CD44 positive. A549, MOR and
H460 cell lines exposed to cisplatin showed an increased
enrichment of CD133+cells relative to each matched parent cell
line (Fig. 7A). A 5-fold enrichment in the CD133+population in
CisR A549 (2.8960.58 vs 0.5360.11) and MOR (0.5160.02 vs
0.0960.04) cells was observed compared to parent cells, while
a greater than 12-fold increase was observed in H460 CisR cells
(6.2060.40 vs 0.5060.30). The cell surface marker, CD44, was
also expressed on each of these cell lines (Fig. 7B). Levels of CD44
were similar in A549 PT and CisR cells, while a significant
increase in expression of CD44+cells was observed MOR CisR
cells relative to PT cells (5.5260.31 vs 1.0960.15). Similarly,
a significant increase was detected in H460 CisR cells (94.4260.52
vs 38.2266.08), with much greater levels of CD44+cells also seen
in MOR CisR cells relative to parent cells (5.52560.305 vs
1.08560.145). SKMES-1 cells contained a greater than 2-fold
enrichment of CD133+cells within the cisplatin resistant
population relative to the parent cell line (5.1061.27 vs
2.1660.69). Both PT and CisR SKMES-1 cell lines had similar
levels of CD44, similar to that observed in A549 cells,
demonstrating a cell progeny with the same CD44 expression
profile. For CD133 and CD44 markers, there was no statistically
significance between PT and CisR SKMES-1 cells. Interestingly
however, all cisplatin-resistant NSCLC cell lines with increased
CD133+fractions also exhibited increased numbers of CD44+
cells (CD133+/CD44+).
Identification of Increased ALDH Activity in Cisplatin
Resistant Cell Lines
Using the Aldefluor assay to assess the presence and size of the
cell population with ALDH enzymatic activity in our panel of four
NSCLC cell lines, a significant increase in ALDH activity was
demonstrated within each population of CisR cells relative to
parent cells (Fig. 8A) as illustrated by representative dot plots and
mean fluorescence intensity histograms (Fig. 8B). Relative to PT
cells, A549, MOR and H460 CisR cells had significantly increased
levels of ALDH+cells (A549, 34.0463.10 vs 6.0860.60, MOR,
50.2461.63 vs 18.4063.79, H460, 36.3962.34 vs 8.8960.75).
However, while there was a trend towards an increase in the
ALDH+fraction in the SKMES-1 CisR cell line, this was not
significant relative to the parental cell line (3.9661.16 vs
1.6260.32). This pattern of expression was similar to that
observed for CD133 in SKMES-1 CisR cells, where only a modest
increase in CD133+cells was also found.
Figure 3. The inhibitory effects of cisplatin on the proliferative capacity of cisplatin resistant NSCLC cells. Parent (PT) and cisplatin
resistant (CisR) cell lines were treated with increasing concentrations of cisplatin for 72 h. Proliferation was measured using the MTT assay. While
cisplatin inhibited the growth of both PT and CisR cell lines, the inhibitory effect of cisplatin was greatly reduced in CisR cells relative to parent cells.
Data are expressed as Mean 6SEM from three independent experiments (n = 3) (*p,0.001 vs PT untreated,
$$
p,0.001 vs CisR untreated,
#
p,0.001
PT vs CisR,
$
p,0.01 vs CisR untreated [A549],
$
p,0.01 PT vs CisR,
#$
p,0.05 PT vs CisR [MOR]).
doi:10.1371/journal.pone.0054193.g003
Cisplatin Resistant NSCLC Cells
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Cancer Stem Cell Marker Expression Profile of Nanog,
Oct-4 and SOX-2 Proteins
In order to further define a distinct cisplatin resistant stem cell
population of NSCLC cells within our panel of cell lines, a number
of key human embryonic cancer stem cell markers were examined
at the protein level between parent and cisplatin resistant cell lines.
Three distinct cancer stem cell markers, Nanog, Oct-4 and SOX-2
were assessed (Fig. 9). Differential expression was observed across
all cell lines. Significantly increased expression of Nanog was
observed in H460 (239.6764.055), A549 (22062.517) and
SKMES1 (19867.00) cisplatin resistant cell lines compared to
controls or parent cells (100%). Little, if no difference, was
observed in MOR cisplatin resistant cells relative to parental cells.
Protein levels of Oct-4 were significantly upregulated in MOR
(12060.8819), H460 (12962.082) and A549 (140.6662.963) cell
lines but not in SKMES1 (105.3361.453) cells. Significant
increases in the levels of SOX-2 protein expression were observed
across all cisplatin resistant cell lines relative to parent cells.
Resistant Cells Demonstrate Increased Expression of the
EMT Regulators, c-Met and b-catenin
The epithelial to mesenchymal transition (EMT) is a key step in
the progression of tumours towards metastasis and invasion.
Moreover, cancer cells undergoing EMT have been found to show
increased resistance to apoptosis and certain chemotherapeutic
drugs and acquire traits reminiscent of those expressed by stem
cells. In a preliminary analysis, expression levels of two important
EMT regulators, c-Met and b-catenin, were examined at the
protein level in our panel of cisplatin resistant and parent cell lines
(Fig. 10). While H460 (260.6268.426), A549 (155.2569.357) and
SKMES1 (145.6266.741) cisplatin resistant cell lines showed
significantly higher levels of c-Met protein compared to that
observed in their parent counterparts (100%), b-catenin was
significantly upregulated in A549 (193.3364.269) and SKMES1
(138.2767.679) cells only.
Cisplatin Resistant Lung Cancer Cells Show Decreased
Cisplatin-GpG DNA Adduct Formation
To determine the level of DNA adducts in the nuclear DNA of
cisplatin resistant cells, we established a quantitative immunocy-
tological assay using a monoclonal antibody-based immunocyto-
Figure 4. Cisplatin-induced apoptosis is reduced in cisplatin resistant cell lines. Parent and resistant cell lines were treated with cisplatin
for 72 h. Apoptotic cells, as measured by the percentage cells in the SubG0 phase of the cell cycle, were measured. Levels of apoptosis induced by
cisplatin were significantly increased in parent cells while cisplatin-induced apoptosis was significantly reduced in the corresponding resistant cell
line. Data are expressed as Mean 6SEM from three independent experiments (n = 3) (*p,0.01 vs PT untreated, **p,0.001 vs PT untreated,
$$
p,0.001 vs CisR untreated,
$
p,0.01 vs CisR untreated [A549, H460],
$
p,0.05 vs CisR untreated [MOR],
$
p,0.05 PT vs CisR,
#
p,0.001 PT vs CisR,
*
$
p,0.05 vs PT untreated).
doi:10.1371/journal.pone.0054193.g004
Cisplatin Resistant NSCLC Cells
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Cisplatin Resistant NSCLC Cells
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logical measurement of DNA intrastrand cross-links. Following
treatment of parent and cisplatin resistant NSCLC cells with
cisplatin over a time-course of 24 h, cells were stained for the
quantitative analysis of Pt-(GpG) cross-links in DNA using
a specific antibody RC-18. In our panel of parental cell lines, an
increase in cispatin-DNA adduct formation was readily detectable
in the nuclei. This was in contrast to that observed in
corresponding cisplatin resistant cells where there was significantly
less adduct formation in all resistant cell lines, most notably in
MOR cells (Fig. 11A). The measurements of integrated immuno-
fluorescence signals from individual nuclei by quantitative image
analysis revealed a distinct pattern of adduct levels between each
parent and cisplatin resistant cell line. The accumulation of Pt-
DNA lesions 24 h post treatment was significantly higher in all
resistant cell lines (Fig. 11B).
Enhanced DNA Double-strand Break Repair Ability of
Resistant Sublines
To investigate DNA double strand break (DSB) repair capacity
in our panel of cell lines, the H2AX foci formation assay was used
following treatment of parent and cisplatin resistant sublines over
a period of 24 h. At 4, 8, 12 and 24 h post treatment, resistant cells
repaired DNA-DSB’s more efficiently than parent cells, as
indicated by the significantly lower amount of phosphorylated-
cH2AX foci (Fig. 12A). While exposure of parental cells to
cisplatin resulted in a gradual accumulation of c-H2AX foci with
significant increases as early as 4 h post cisplatin treatment, this
effect was most pronounced by 24 h. In contrast, the number of
foci was significantly lower in resistant sublines following treatment
with cisplatin (MOR 68.50961.72 vs 82.64560.73, p,0.01; H460
47.8160.65 vs 74.4861.62, p,0.001; A549 39.4061.26 vs
Figure 5. Cisplatin resistant cells accumulate in the G0/G1 phase of the cell cycle. Parent and Cisplatin resistant NSCLC cells were treated
with cisplatin for 24 h. Cell cycle distribution of PT and CisR cells was examined by propidium iodide staining and measured by FACS (A). A significant
accumulation of CisR cells was observed in the G0/G1 phase of the cell cycle across the panel of cell lines. Representative histograms showing cell
cycle distribution of SKMES-1 CisR cells and PT counterparts in response to increasing concentrations of cisplatin are shown (B). Data are expressed as
Mean 6SEM from three independent experiments (n = 3) (
#
p,0.001,
*
p,0.05).
doi:10.1371/journal.pone.0054193.g005
Figure 6. Clonogenic survival ability of cisplatin resistant cells is increased with increasing doses of cisplatin. A549, SKMES-1, MOR and
H460 PT and CisR cells were seeded in 6-well plates using optimised seeding densities. Following treatment with cisplatin for 72 h, media was
removed and cells were allowed to recover for between 9–14 days after which time surviving colonies were stained using crystal violet stain and
counted. The survival ability of CisR cells was significantly increased at various concentrations of cisplatin between cell lines relative to their parental
counterparts, based on the number of colonies on plate following incubation with cisplatin. Data are expressed as Mean 6SEM from three
independent experiments (n = 3) (
$
p,0.05,
#
p,0.01,
*
p,0.001).
doi:10.1371/journal.pone.0054193.g006
Cisplatin Resistant NSCLC Cells
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Cisplatin Resistant NSCLC Cells
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69.1160.93, p,0.001; SKMES-1 72.0960.98 vs 98.6661.52,
p,0.001) suggesting a increase in the DNA repair capacity of
these chemoresistant cell lines. Within each cell line, differences in
foci number varied at each time point. In A549 cisplatin resistant
cells, little difference in phosphorylated-H2AX between time
points was observed, with an significant increase at 12 h post
treatment only.
Cellular Uptake of Cisplatin is Reduced in Chemoresistant
Cells
To determine differences in sensitivity to the growth inhibitory
effects of cisplatin between parent and resistant lung tumour cells
were accompanied by differences in whole cell platinum accumu-
lation, as is commonly observed in cells selected for such platinum
resistance, our panel of cell lines were treated with cisplatin for
24 h and intracellular cisplatin levels were quantified by ICP-MS
(Fig. 13). Across all four parental cell lines, findings from ICP-MS
analysis demonstrated a increased uptake of cisplatin after
treatment for 24 h relative to untreated parental cells (MOR
24,319.20 ng vs 98.45 ng; H460 18,890 ng vs 74.57 ng; A549
26,417.60 ng vs 183.63 ng; SKMES-1 16,184.90 ng vs 105.10 ng)
Resistant cells however, had significantly reduced uptake of
cisplatin following exposure to cisplatin for a similar time period,
compared to that observed in their parental counterparts (MOR
5,217.8 ng vs 23,319.20 ng; H460 6,984.30 ng vs 18,890 ng; A549
20,204 ng vs 26,417.60 ng; SKMES-1 9,781.80 ng vs
16,184.90 ng).
Figure 7. Enrichment of CD133+and CD44+fractions in cisplatin resistant sublines. Antibody staining of PT and CisR cell lines for CD133
cell surface expression was carried out by flow cytometry using a CD133/1 (AC133) phycoerythrin (PE)-labelled antibody and IgG1 isotype control
antibody. The percentage CD133+cells were plotted for all cell lines (A). Differential expression of the CSC marker CD44 was examined using an anti-
human CD44 FITC-conjugated antibody and corresponding IgG2b isotype control antibody. Expression levels of CD44 were determined for all cell
lines and plotted as a percentage of the tumour cell population expressing CD44 (B). Data are expressed as Mean 6SEM from three independent
experiments (n = 3) Data are expressed as Mean 6SEM from three independent experiments (n = 3) (
#
p,0.01,
*
p,0.001).
doi:10.1371/journal.pone.0054193.g007
Figure 8. Chemoresistant cell lines display increased aldehyde dehydrogenase (ALDH1) activity. ALDH1 activity was measured between
PT and CisR NSCLC cells using the Aldefluor assay. Cells were incubated with ALDH1 substrate that converts intracellular ALDH1 into a negatively
charged reaction product, preventing diffusion from the cells. A control sample was also included for each parent and resistant cell line that consisted
of a specific inhibitor of ALDH1, diethylaminobenzaldehyde (DEAB), in order to establish baseline fluorescence. The reaction was measured in the
green fluorescence channel of a flow cytometer. The percentage ALDH1 activity between parent and cisplatin resistant cells was calculated (A). Dot
plots and histograms showing mean intensity fluorescence (MFI) between parent (red) and cisplatin resistant (black) cell lines are represented (B).
Data are expressed as Mean 6SEM from three independent experiments (n = 3) (
*
p,0.001).
doi:10.1371/journal.pone.0054193.g008
Cisplatin Resistant NSCLC Cells
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Discussion
Since its introduction into clinical trials, cisplatin has had
a major impact in the treatment of cancer, changing the course of
treatment for several tumours such as those of the ovary, testes,
head, neck and lung (2). To date, the most effective systemic
chemotherapy for NSCLC is platinum-based combinations that
remain the standard first-line chemotherapy for this cancer type.
While an understanding of the mode of action is desirable in
refining therapeutic approaches that further enhance the anti-
tumour activity of this platinum drug, cisplatin poses a number of
major problems, one of which is the acquisition of cisplatin
resistance that undermines its curative potential. This understand-
ing is also critical for elucidating mechanisms underlying the drug-
resistant phenotype associated with cisplatin resistance, particu-
larly in NSCLC. An example highlighting this limitation is with
ovarian cancer which generally responds well to cisplatin-based
therapy. Unfortunately, the initial response rate of up to 70% is
not durable and results in a 5-year patient survival rate of only 15–
20%, primarily as tumours become resistant to therapy [14].
Likewise, in small cell lung cancer, the relapse rate can be as high
as 95% [15]. The onset of resistance creates a further therapeutic
complication in that tumours failing to respond to cisplatin are
cross-resistant to diverse unrelated anti-tumour drugs [16]. This
suggests that cisplatin and other agents likely share common
mechanisms of resistance. In this respect, it is noteworthy that
cisplatin-resistant tumours are fully cross-resistant to the platinum
analogue carboplatin [17,18]. As cisplatin-based chemotherapy for
NSCLC appears to have reached a plateau and a better un-
derstanding of the mechanisms of cisplatin resistance are slowly
unravelling, there is an urgent need for a better understanding of
the molecular mechanisms underlying the cisplatin resistant
phenotype.
We have generated a clinically-relevant, isogenic model of
cisplatin resistance in a panel of NSCLC cell lines from original,
age-matched parent cell lines and characterised these in terms of
their proliferative and apoptotic potential, cell cycle distribution,
clonogenic survival ability and stem-like properties. Using IC
50
concentrations, cisplatin resistant cell lines were established over
time through chronic in vitro exposure to the drug after which time
IC
50
values were re-assessed in cisplatin treated cell lines and
found to be significantly higher, demonstrating a more resistant
phenotype. Changes in the proliferative and apoptotic properties
of cisplatin resistant cell lines relative to their corresponding parent
Figure 9. Cancer stem cell markers, Nanog, Oct-4 and SOX-2, are upregulated in cisplatin resistant cells. Total proteins were isolated
from parent and corresponding cisplatin resistant sublines and subjected to SDS-PAGE gel electrophoresis and transfer by Western blot. Using a panel
of human embryonic stem cell markers, Nanog, Oct-4 and SOX-2 protein expression was examined between parent and resistant cell lines. While
H460, A549 and SKMES-1 resistant cells exhibited increased expression of Nanog and SOX-2 proteins, Oct-4 expression was significantly upregulated
in MOR, H460 and A549 cells only. Cisplatin resistant MOR cells demonstrated increased levels of SOX-2 and Oct-4 proteins relative to parental cells.
Data are expressed as Mean 6SEM from three independent experiments (n = 3) (
*
p,0.001).
doi:10.1371/journal.pone.0054193.g009
Cisplatin Resistant NSCLC Cells
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cell lines in response to increasing concentrations of cisplatin
suggested increased resistance to the chemotherapeutic drug with
significant differences observed between parent and resistant cells
at various concentrations. A549 and H460 cell lines, in particular,
were found to be most resistant to cisplatin in terms of their
proliferative and apoptotic response to cisplatin. Differences in the
cell cycle distribution of PT and CisR cell lines, at basal levels,
were also observed, with cisplatin resistant cells having a higher
accumulation of cells in the G0/G1 phase of the cell cycle and
a corresponding decrease in the number of cells in S phase. This
difference was most notable in A549 and H460 cells.
One of the most common cellular checkpoints affected in
response to cisplatin treatment is G2/M arrest where p21
WAF1
,
one of several genes transactivated by p53 as a result of exposure
to cisplatin, is involved in both inducing and sustaining this cell
cycle arrest [19]. However, the accumulation of p21
WAF1
following
DNA damage has been classically associated with a G0/G1 arrest
[20]. It is of interest that A549 and H460 cell lines were most
resistant of the four cell lines characterised, while the squamous
cell carcinoma cell line, SKMES-1, was the least resistant of these.
Possible molecular factors that may influence, in part, the
resistance phenotype observed between our panel of cell lines
may be attributable to their p53 status. While A549, MOR and
H460 cells have wild-type p53, SKMES-1 cells are p53 mutant.
Activation of p53 by cisplatin-induced DNA damage has been
reported to have various effects on cellular sensitivity to cisplatin.
In some studies, activation of p53 has been shown to provide
cytoprotection against cisplatin [21,22] In contrast, increased
resistance to cisplatin with disruption of normal WT p53 function
has also been demonstrated [23].
Since the discovery of cancer stem cells in haematopoietic
cancers and other solid tumours, little is known to date regarding
the biology of lung cancer stem cells. The existence of cancer stem
cells within a lung tumour cell population may explain the
ineffectiveness of current treatments in consistently eradicating
tumour cells. Therapies may target the majority of cancer cells
while residual lung cancer stem cells may regenerate a cancer cell
population resulting in tumour relapse following chemotherapy.
As such, there is an increasing need to identify and develop new
therapeutic targets for specifically eradicating this cell population.
While the marker profile of lung cancer stem cells remains to be
explored, some commonly used strategies that have been used to
date include the cell surface stem cell markers, CD133 and CD44,
in addition to aldehyde dehydrogenase activity. Recent studies
using NSCLC cell lines and fresh lung tumour tissues suggest
CD133 as the lung CSC marker of choice [7,24,25,26] while
cytometric analysis and sorting of marker-positive cells is currently
the standard method used [27]. In a recent study by Bertolini et al.,
cisplatin treatment of lung cancer cells resulted in the enrichment
of a CD133+fraction of cells with a cisplatin resistant phenotype
following acute cytotoxic exposure to cisplatin. Likewise, in vivo
subpopulations of CD133+cells were spared by cisplatin treatment
Figure 10. EMT marker expression, c-Met and b-catenin. Total proteins from parent and corresponding cisplatin resistant sublines were
subjected to SDS-PAGE gel electrophoresis and transfer by Western blot. Expression levels of the EMT markers, c-Met and b-catenin, were examined
across all cell lines. c-Met protein levels were significantly upregulated in H460, A549 and SKMES-1 resistant cell lines, while b-catenin levels were
significantly upregulated in A549 and SKMES-1 cells only. Data are expressed as Mean 6SEM from three independent experiments (n = 3) (
*
p,0.001).
doi:10.1371/journal.pone.0054193.g010
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Cisplatin Resistant NSCLC Cells
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of lung tumour xenografts established from primary lung tumours.
Exposure of A549 lung tumour cells to cisplatin using IC
80
concentrations resulted in an 8-fold enrichment of CD133+cells.
In support of these findings, cisplatin resistant A549 cells
generated in our study, showed a greater than 5-fold increase in
CD133+expressing cells (IC
50
concentration) relative to parent
cells, highlighting and further confirming CD133 as a potential
marker of cisplatin resistance in NSCLC. Biochemical studies
demonstrating a functional role for CD133 in cell cycle regulation
and proliferation have been reported [28], consistent with some of
the functional studies highlighted in the cisplatin resistant NSCLC
cell lines established in this study. Chemoresistant cells expressing
increased levels of CD133 also showed a significant arrest in the
G0/G1 phase of the cell cycle relative to parent cells.
The membrane-bound glycoprotein, CD44, is found expressed
in many tumour cell types and is an important factor in tumour
growth, invasion and metastasis. Recent studies have provided
support for its role as CSC marker. In colorectal cancer, the clonal
expansion and xenograft initiation capacity of CD44+CSCs could
be inhibited by CD44 knockdown [29]. In small cell lung cancers,
it was shown that activation of CD44-MAPK-PI3K signalling
results in the increased expression of urokinase plasminogen
activator and its receptor, uPAR, and MDR1, resulting in
enhanced invasive and multi-drug resistant cancer phenotypes
[30]. In our panel of cisplatin-resistant NSCLC cell lines, an
enrichment of CD44-expressing subpopulations was demonstrat-
ed. Such findings are in agreement with recent studies examining
the identification of lung CSCs in a series of in vitro and in vivo
studies [31]. However, differences in CSC marker profile
expression do exist between studies. In the study reported by
Leung et al., 0% and 95.90% of CD44+cells was observed in A549
and H23 cell lines, respectively, while in a study by Stuelten et al.
[32], 84.41% and 30.95% were detected. Our findings using A549
cells are in agreement with those of Stuelten et al. where 97.69%
CD44+cells were found within the cisplatin-resistant population
and 98.71% in parent cells. Such variations in expression between
studies may be explained by individual variation among different
cell lines or differences in the composition or functional
characteristics of the cancer stem cell populations. Determining
the true percentage of CSC’s within tumours or established cell
lines remains controversial in the absence of a specific CSC
marker, particularly in lung cancer.
The aldehyde dehydrogenase family of enzymes belong to
a family of intracellular enzymes involved in cellular detoxification
and oxidisation of intracellular aldehydes, resulting in drug
resistance [33,34]. Its function and clinical significance in relation
to stem cell function is still under investigation in lung cancer.
There is however, documented evidence to support ALDH as
a marker for lung cancer stem cells. In a study by Jiang et al., high
levels of ALDH protein expression correlated with poor prognosis,
consistent with the idea that ALDH+lung tumour cells are
enriched with lung cancer stem cells [35]. It is of interest that in
our panel of cisplatin resistant NSCLC cell lines that displayed
a significant increase in the number of CD133+cells, there was
a significant corresponding enrichment of the cancer stem cell
marker, CD133, relative to that seen in parent cells. This was
significantly increased in A549, MOR and H460 cells, with the
exception of the squamous cell carcinoma cell line, SKMES-1.
Such findings are in agreement with a more resistant cell
phenotype. The histological and regional diversity found in lung
cancer may, in part, be attributed to the presence of diverse pools
of self-renewing stem cells in the adult lung epithelium [36].
Evidence that cisplatin resistant subpopulations of cells within
our panel of cell lines display characteristics of putative cancer
stem cells is further supported in this study using a panel of cancer
stem cell markers which were differentially upregulated across our
panel of cell lines. Nanog, Oct-4 and SOX-2 stem cell markers
were significantly upregulated in a number of cisplatin resistant
cell lines compared to their corresponding parental counterparts.
However, while such increases in expression of Nanog, Oct-4 and
SOX-2 represent a pluripotency regulation network, significantly
elevated levels of SOX-2 protein were found compared to that
found Nanog and Oct-4. Recent studies demonstrate that CSC’s
have higher tumorigenic properties than those of differentiated
cancer cells and that the transcription factor, SOX-2, plays a vital
role in maintaining the unique properties of CSC’s [37]. However,
the function and underlying mechanism of SOX-2 in carcinogen-
esis of lung cancer are still elusive. In a study by Chen et al,
expression of SOX-2 in human lung tissues of normal individuals
as well as patients with adenocarcinoma, squamous cell carcino-
ma, and large cell carcinoma demonstrated specific overexpression
of SOX-2 in all types of lung cancer tissues. This finding supports
the notion that SOX-2 contributes to the tumorigenesis of lung
cancer. In addition, higher expression of the oncogenes c-MYC,
WNT1,WNT2 and NOTCH1 was detected in side population (SP)
cells than in non-side population (NSP) cells of A549 lung cancer
cells, indicating a possible mechanism for the tumorigenic
potential of CSC’s. Silencing of the SOX-2 gene reduced the
tumorigenic properties of A549 cells with subsequent attenuated
expression of c-MYC,WNT1,WNT2, and NOTCH1 in xenografted
NOD/SCID mice. These results provide evidence that SOX-2
may regulate the expression of oncogenes in CSC’s to promote the
development of human lung cancer [38].
The progression of many cancer types is often accompanied by
changes in the pattern of gene expression of neoplastic cells,
resulting in a highly tumorigenic and invasive cell phenotype.
Some of these changes are reminiscent of an epithelial to
mesenchymal transition (EMT), a process characterised by loss
of epithelial features and gain of mesenchymal properties. While
loss of E-cadherin has emerged as one of the common indicators of
EMT, this has been shown to result in the release of b-catenin in
addition to its cytoplasmic accumulation and further translocation
to the nucleus where it can activate LEF/TCF (lymphoid
enhancer factor/T cell factor) transcription. We show in this
preliminary analysis of EMT marker expression that b-catenin in
significantly upregulated in two of our cisplatin resistant cell lines.
Dysregulation of the c-Met receptor, or overexpression of its
ligand, hepatocyte growth factor (HGF), has also been associated
with an aggressive cancer cell phenotype and the EMT process.
Our data highlight the potential involvement of this EMT
regulator in NSCLC cells with a cisplatin resistant phenotype
with increased protein expression of c-Met in three of four
Figure 11. Cisplatin-DNA adduct formation and immunofluorescence. Lung cancer cell lines were treated with cisplatin for up to 24 h and
fixed on Superfrost Gold Slides using ice-cold methanol. Cells were stained overnight at 4uC using a primary antibody that specifically recognizes
CDDP-GpG DNA adducts (RC-18). Antibody binding was detected using an anti-rat Cy3H-labelled antibody and counterstained using DAPI (1 mg/ml
(w/v). Images were acquired on an Axioplan fluorescence microscope (A). Adducts were quantified and measured as arbitrary fluorescence units
(AFU’s) upon normalisation of integrated antibody-derived fluorescence from 200 individual nuclei of the corresponding DNA content. Data are
presented as the mean AFU 695% confidence interval (CI) from three independent experiments (B).
doi:10.1371/journal.pone.0054193.g011
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PLOS ONE | www.plosone.org 16 January 2013 | Volume 8 | Issue 1 | e54193
resistant sublines. Further studies using inhibitors to EMT
signalling pathways may be warranted to circumvent the resistance
conferred by certain cancer cells to chemotherapeutic agents.
The anti-cancer activity of cisplatin is based on the formation of
platination products in the nuclear DNA [39]. Several of these
adducts have been identified, of which the guanine-guanine
Figure 12. Measurement of cH2AX foci formation and DNA damage. Following treatment of parent and chemoresistant cells with cisplatin
for 4, 8, 12 and 24 h, cells were fixed in formaldehyde and incubated with a primary rabbit anti-human anti-phospho-histone 2AX (Ser139) antibody.
Cells were subsequently labelled with an Alexafluor 488-labelled goat anti-rabbit secondary antibody and Hoechst 33342 nuclear stain prior to
analysis by high content analysis using the InCell Analyser 1000 (A). Data are expressed as Mean 6SEM from three independent experiments (n = 3)
(
#
p,0.05,
$
p,0.01,
*
p,0.001) (B).
doi:10.1371/journal.pone.0054193.g012
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PLOS ONE | www.plosone.org 17 January 2013 | Volume 8 | Issue 1 | e54193
intrastrand cross-link, cis-Pt(NH
3
)
2
d(pGpG) [Pt-(GG)], represents
.70% of total DNA platination. Persistence of such lesions within
the nuclear DNA can ultimately result in impaired replication and
transcription, thereby triggering apoptosis. The nucleotide exci-
sion repair (NER) pathway has been suggested to be one of the
main cellular defense mechanisms against cisplatin-induced
intrastrand cross-links [40]. Up until recently, the measurement
of platinum concentrations was based predominantly on spectro-
scopic methods [41]. In this study, we used an adduct-specific
monoclonal antibody in combination with digital image analysis to
visualise and quantify levels of distinct DNA platination products
within the nuclei of individual cells. The degree of DNA adduct
formation by cisplatin is cell-type specific and may likely depend
on a number of pharmacokinetic parameters such as drug export
by membrane transporters [42] or cytoplasmic detoxification [43].
We investigated the effects of cisplatin on the repair of cisplatin-
induced double strand breaks (DSB’s) by immunofluorescence
imaging of cH2AX foci. Given that cH2AX appears rapidly at
DSB’s and disappears as repair proceeds [44], it serves as
a sensitive and specific marker for unrepaired DNA damage.
These findings, together with our observation that chemoresistant
cells displayed decreased cisplatin-GpG DNA adducts following
exposure to cisplatin compared to parent cells, are indicative of
potential key mechanisms that may be implicated in the process of
cisplatin transport and/or repair in our panel of NSCLC cell lines.
Data from ICP-MS analysis demonstrated a significant accumu-
lation of cisplatin in parent cells upon treatment with cisplatin
compared to that measured in cisplatin resistant cells. Upon
treatment, platinum drugs have been shown to be extensively
sequestered into subcellular compartments which in turn limit
their access to critical targets. While in some cell types, this
sequestration process is accompanied by enhanced drug export
[45], others have shown enhanced storage of the drug inside the
cell, most likely in a non-toxic form [46]. In the latter of these
studies, forced expression of the copper transporters ATP7A and
ATP7B rendered cells resistant to cisplatin and other platinum
drugs. Future studies warrant investigation as to the expression of
these copper transporters in cisplatin resistant lung cancer cells
and to verify whether this resistance mechanism is independent of
copper efflux transporters.
We have generated an isogenic model of cisplatin resistance in
a panel of NSCLC cell lines and characterised these based on
a number of functional cellular parameters relative to their original
parental cell line. The presence and enrichment of stem-cell
Figure 13. Quantification of cisplatin uptake by lung cancer cells using ICP-MS. Exponentially growing cells were treated with cisplatin for
24 h after which time they were washed in PBS, harvested and counted. Digestion of cells (1610
6
) in 1% nitric acid for 24 h at 70uC was carried out
prior to ICP-MS analysis. Platinum determination was performed using Inductively Coupled Plasma Mass Spectrophotometry. Instrumental settings
were optimised in order to yield maximum sensitivity for platinum.
doi:10.1371/journal.pone.0054193.g013
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PLOS ONE | www.plosone.org 18 January 2013 | Volume 8 | Issue 1 | e54193
markers support the presence of a chemoresistant population of
lung cancer cells with a stem-like signature that may be useful as
a clinically relevant in vitro model for studying mechanisms of
cisplatin resistance in NSCLC. Moreover, we have identified
differences in cisplatin-DNA adduct formation and DNA repair of
cisplatin-induced DSB’s between parent and chemoresistant cells
following uptake of cisplatin. These findings provide a rationale for
more specific therapeutic targeting in the treatment of this disease.
Acknowledgments
Thanks to Dr Michael F. Gallagher, Department of Histopathology,
Trinity College Dublin, for reading this manuscript.
Author Contributions
Provision of support and advice on cisplatin resistance: DR DF JJO.
Reading of the Manuscript: JDO. Conceived and designed the
experiments: MPB KJO. Performed the experiments: MPB. Analyzed
the data: MPB ACH JT RAH JJO. Contributed reagents/materials/
analysis tools: MPB SGG DF DR. Wrote the paper: MPB.
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