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ATR-FTIR spectroscopy shows changes in ovarian

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Cite this: Analyst, 2018, 143, 6087

cancer cells after incubation with novel
organoamidoplatinum(II) complexes†
Khansa Al-Jorani,a Anja Rüther,a Rukshani Haputhanthri, a Glen B. Deacon, b

Hsiu Lin Li,b Carleen Cullinanec,d and Bayden R. Wood *a

Received 12th August 2018,
Accepted 2nd November 2018
DOI: 10.1039/c8an01558a
rsc.li/analyst
Attenuated Total Reflection Fourier Transform Infrared (ATR-FT-IR) spectroscopy has been applied to
compare the effect of the new organoamidoplatinum(II) complexes [Pt{NH(p-HC6F4)CH2CH2N( p-HC6F4)}
( py)(O2CR)] (R = C6F4 or 2,4,6-Me3C6H2) with cisplatin on cells from one cisplatin-sensitive ovarian
cancer cell line (A2780) and one cisplatin-resistant ovarian cancer cell line (A2780R). After incubation of
the cells with cisplatin, 1 and 2 for 48 hours, distinct changes were found in the ATR-FT-IR spectra.
Comparison of the second derivative spectra suggests that 1 and 2 induce similar chemical changes in
both cell lines, A2780 and A2780R, while cisplatin had a slight effect on A2780 and A2780R cells.
Furthermore, drugs 1 and 2 result in changes to the phosphodiester and polysaccharide bands in the
spectra. This work shows how ATR-FT-IR can be applied to monitor the effects of organoamidoplatinum
(II) complexes on cisplatin-sensitive and cisplatin-resistant cell lines providing potential information on
how drugs affect the cellular metabolism.

Introduction chloridoplatinum(II)) by Rosenberg in 1965 resulted from the

Ovarian cancer ranks as the most common cancer causing
death amongst women and is the most common cancer of the
female reproductive tract,1 and early detection is not readily
achieved.2,3 Chemotherapy is one of the major treatments for
ovarian cancer. In order to improve the efficacy of chemo-
therapy, it is necessary to understand the mechanism of action
of the chemotherapeutic drugs and how they interact with bio-
logical molecules. Such knowledge could further contribute to
understanding drug resistance.4,5 Platinum-based drugs have a
major role in chemotherapy.6,7 Different platinum-based com-
plexes have different effects on each type of tumour due to the
different sensitivity of each cancer cell type and differences in
their proliferation rate.8
Platinum coordination complexes are active as anticancer
drugs due to their ligand-exchange kinetics,9 which lead to
their capacity to disrupt the replication of cancer cells.10,11 The
discovery of the biological activity of cisplatin (cis-diaminedi-
a
Centre for Biospectroscopy and School of Chemistry, Monash University, Clayton,
Victoria, 3800, Australia. E-mail: Bayden.Wood@monash.edu; Tel: +61-399-055-721
b
School of Chemistry, Monash University, Clayton, Victoria, 3800, Australia
c
The Sir Peter MacCallum Department of Oncology Department, Melbourne,
Australia
d example through the delivery mechanism, through different
Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8an01558a
ability of cisplatin to inhibit cellular division.12 From that time,
it has become the major treatment for different types of solid
tumours such as testicular, ovarian, bladder, head and neck
cancers.13–15 The inspiration of developing new platinum drugs
partly resulted from the great efficacy of cisplatin on testicular
cancer where the cure rates reached >80%.16 The challenge is to
ensure a similar success rate for other cancers but with reduced
side effects and without the development of resistance.
Despite the fact that platinum anticancer drugs are success-
ful in cancer therapy, they have some dose limiting side effects
such as nephrotoxicity, neurotoxicity, ototoxicity, alopecia and
emetogenesis.17–20 Another drawback is the drug resistance of
tumours. This occurs for many reasons such as an increase in
drug efflux from the cell, the activation of detoxification
systems, an increase in repairing DNA damage, a loss of tumour
suppressor function and altered gene expression.21,22 Thus,
huge efforts have been made to improve the function of plati-
num drugs by reducing side effects and resistance.23 Resistance
in ovarian cancer is broad and tumours can become refractory
to alternative antitumour drugs.24,25 One focus in the search for
new chemotherapeutic drugs has been on “rule breakers”.25,26
These compounds do not conform to the structure/activity rules
to which current clinical platinum drugs conform.27,28
These offer prospect of a different mode of action, for
behaviour towards competing targets, and a different inter-
action with DNA.29,30

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Polyfluorophenyl substituted organoamidoplatinum(II)
complexes [Pt{N(R)CH2}2( py)2] (R = polyfluorophenyl; py = pyri-
dine) are active against a number of tumours in vitro and
in vivo,31,32 and have different uptake mechanism from cispla-
tin and it has recently been shown that the initial target is
adenine in DNA.
The compounds are sterically hindered and air and moist-
ure stable. The stability is due to the electron-withdrawing
nature of the polyfluorophenyl group. The crystal structures of
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these complexes reveal that the stereochemistry around the
amide nitrogens moves towards trigonal rather than tetra-
hedral and the bond between the nitrogen and the ipso carbon
of the polyfluorophenyl ring is shorter than the single bond
between the same nitrogen amide and the carbon of a methyl-
ene group.33 This bond is similar in length to an aromatic N–C
bond, indicating the delocalization of negative charge away
from the amido nitrogen thereby stabilizing the complex
towards hydrolysis.33 The organoamidoplatinum(II) com-
pounds are unusual for active species in having four nitrogen
donor atoms, and are “rule breakers” because of the absence
of H substituents on any N donor atom.31,32 We have now
begun derivatisation of these complexes in an attempt to
understand and extend their activity. Thus, the lead drug, [Pt
{N( p-HC6F4)CH2}2( py)2] (Pt103) reacts with pentafluorobenzoic
acid and 2,4,6-trimethylbenzoic acid by a photo-induced acido-
lysis and substitution to afford the complexes [[N,N′-bis(2,3,5,6-
tetrafluorophenyl)ethane-1,2-diaminato(1-)](pentafluorobenzoato)
(pyridine)platinum(II)] (1) and [[N,N′-bis(2,3,5,6-tetrafluorophenyl)
ethane-1,2-diaminato(1-)](trimethylbenzoato)(pyridine)platinum(II)]
(2) (Fig. 1).34 The compounds now have an NH functionality
unlike Pt103 but the amine ligands ( py and –CH2NHR) are
trans to each other in contradiction to the usual structure/
activity rules.27,28,32,35
Spectroscopic methods can be used to investigate the inter-
action between platinum complexes and cancer cells and to
differentiate between drug sensitive and resistant cancer cell
lines in terms of drug accumulation and drug efflux.36
Infrared (IR) spectroscopy offers a promising means to
determine cancer biomarkers and to monitor the effect of
drug interaction at the cellular level. The application of
Fourier Transform (FT-IR) spectroscopy to study drug–cell
interactions was recently reviewed.37 The νasPO2− and νsPO2−
Fig. 1 Platinum(II) complexes 1 and 2.
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stretching vibrations occurring at ca. 1125 & 1085 cm−1
respectively from the OvPvO− linkages of the polynucleotide
chains in polymers and ribonucleic acids have been shown to
be sensitive to DNA conformation, drug interaction and
apoptosis.38–41 FT-IR spectroscopy has also been applied to
discriminate between benign and malignant cells. It was
found that the phosphodiester bands are generally more
intense in the malignant cells.42 The identification and assign-
ment of major spectral features by functional group analysis is
especially helpful for the qualitative analysis of pure organic
molecules since the FT-IR spectrum of every molecule is a
unique fingerprint with bands from proteins, nucleic acids,
and sugars. Attenuated Total Reflection (ATR) FT-IR spec-
troscopy can be used to identify characteristic vibrational
modes of the cells that are specific to molecular bonds, func-
tional groups, and intra- and intermolecular interactions such
as hydrogen bonding, cation–π interactions, π–π interactions,
DNA base stacking and dispersion forces.37,43 In this study, we
applied ATR-FT-IR spectroscopy to investigate the effect of
compounds 1 and 2 in comparison to cisplatin on the ovarian
cell line A2780, which is sensitive to cisplatin and on the
A2780R cell line, which is resistant to cisplatin. Using
Principal Component Analysis (PCA) we compare the effects of
each drug on the resistant and sensitive cell lines. This study
provides a methodological approach for the application of
ATR-FT-IR spectroscopy to monitor the effects of drug resis-
tance in terms of changes in the cellular biochemistry.
Experimental
Materials
A2780, A2780R and SKOV-3 ovarian cancer and L1210 and
1210/DDP mouse leukaemia cell lines were obtained from the
Peter MacCallum Cancer Centre (305 Grattan St, Melbourne
VIC 3000). Cells were seeded using RPMI 1640 medium with
10% foetal bovine serum (Sigma Aldrich, Sigma St Louis, MO,
USA) and incubated at 37 °C and 5% CO2. Cisplatin was sup-
plied by the Institute of Drug Technology, Victoria, Australia.
The synthesis and characterization of the platinum complexes
1 and 2 have recently been reported.34
Solutions of platinum complexes
The stock solutions were prepared by dissolving each com-
pound (1 and 2) in a water acetone mixture (10%) to concen-
trations of 6 mM, 0.6 mM and 0.06 mM. Stock solutions were
kept in the dark. Solutions of cisplatin were prepared in saline
to obtain the concentrations 5 mM, 0.5 mM and 0.05 mM. The
control samples were prepared by adding 34 μL of acetone and
40 μL of saline separately to 4 mL of RPMI medium which is
already supplemented with 10% of FBS.
Cell culture
A2780 and A2780R ovarian cancer cell lines were cultivated
using RPMI 1640 medium (Sigma Aldrich) with 10% foetal
bovine serum, 1% L-glutamine (Gibco) and 1% penicillin–
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streptomycin (Sigma Aldrich), under a humidified 5% CO2 Table 1 IC50 values for L1210 (cisplatin sensitive) and L1210/DDP (cis-
atmosphere at 37 °C. platin resistant) and SKOV-3 cell lines

IC50 measurements IC50 (μM) IC50 (μM) in IC50 (μM) Initial solvent
Compound in L1210 L1210/DDP in SKOV-3 for drug
IC50 testing against leukaemia L1210 and L1210/DDP31 and
1 0.7 0.9 6.6 Acetone
SKOV-3 ovarian cell lines was based on an established 2 0.4 0.3 0.7 Acetone
method.44 The IC50 experiments on A2780 and A2780R cell Cisplatin 0.6 6.7 6.3 Saline
lines were performed using a Sulforhodamine B (SRB) assay.
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Preparation of samples for IR measurements

For the IR measurements, 34 µL of 1 & 2 concentration was
added to 4 mL RPMI medium. For cisplatin, 40 µL was added to
4 mL RPMI. Cells were seeded for 48 h before treatment with the
drugs. After addition of the drug solution, they were incubated
again for 48 h before fixation. Cells were fixed using a methanol :
acetic acid (3 : 1) fixative. The fixative solution was freshly pre-
pared. Cells were trypsinyzed and washed 3 times in the fixative
solution. The cell suspension was centrifuged in a 15 mL Falcon
tube (1200 rpm) in fixative and the supernatant discarded. The
fixed cells were kept at 4 °C prior to ATR-FT-IR analysis.
Instrumentation
ATR-FT-IR spectra of drugs and cells were recorded using a
Golden Gate single bounce diamond micro-ATR accessory
coupled to a Bruker IFS Equinox FT-IR system (Bruker Optics
Pty. Ltd, Billerica, MA, USA) with a mercury cadmium telluride
detector. FT-IR spectra were processed using Bruker OPUS soft-
ware, version 6.0 (Bruker Optics Pty. Ltd). The resolution was
8 cm−1 and a zero-filling factor of 2 was chosen. 50 interfero-
grams were co-added for each spectrum. The spectra were cor-
rected for wavenumber dependent penetration depth using the
Extended ATR correction in OPUS.
Data pre-processing
All spectra were pre-processed using the PLS toolbox in
MATLAB (MathWorks, Natick, MA). For PCA the second deriva-
tives were calculated using the Savitzky–Golay algorithm with 9
smoothing points followed by standard normal variate (SNV)
and mean centring.
Data processing
PCA (PLS toolbox in MATLAB) was applied to the second deriva-
tive spectra in the region of 800 to 1300 cm−1. Validation was per-
formed using the leave-out-one cross-validation method.
Results and discussion
In conjunction with the primary study of the interaction of 1
and 2 with cells by infrared spectroscopy, a preliminary evalu-
ation of their in vitro anti-tumour activity has been carried out.
L1210/DDP (cisplatin resistant) and SKOV-3 cell lines and com-
pared with the behaviour of cisplatin (Table 1). The results
show that both 1 and 2 are active in vitro but there is a big
difference in activity between the two drugs when using
SKOV-3 cells. The difference is smaller when the L1210 and
L1210/DDP cell lines were tested. This shows that different cell
lines react differently to the same drug. The activity of 1 and 2
is markedly greater than cisplatin in the cisplatin resistant cell
line (L1210/DDP) indicating that resistance does not extend to
1 and 2. Against SKOV-3, 1 has comparable activity to cisplatin,
whereas 2 is 10 fold more active, a surprising differentiation
on change of carboxylate group.
IC50 values for compounds 1, 2 and cisplatin against the
A2780 cell lines
The IC50 values of 1 and 2 as well as cisplatin were determined
with the A2780 ovarian cancer cell line. The Graph Pad Prism
7 software was used to analyse the dataset and determine the
average IC50 values. The IC50 values (Table 2) show that all
three drugs are active with only marginal differences, though 1
appears to be the most effective. Overall both 1 and 2 are simi-
larly active against L1210, L1210/DDP, and A2780, but show
differences against SKOV-3 ovarian cancer cells where 2 is
much more effective. Both drugs are 8 to 10 times more
effective than cisplatin against the cisplatin-resistant line
L1210/DDP, with a similar differential between 2 and cisplatin
against SKOV-3.
Spectral characterisation of drugs
The two cell lines (cisplatin-sensitive A2780 and the cisplatin-
resistant A2780R cell line) were incubated with 3 different
drugs (1, 2 and cisplatin). Fig. 2 shows the raw FT-IR spectra
and the second derivative spectra of 1, 2 and cisplatin.
FT-IR spectra of the polyfluorophenyl substituted organoa-
midoplatinum(II) complexes show ν(C–F) absorptions of the
polyfluorophenyl group in the range between 1000–900 cm−1.
The absorption of ν(N–H) shown in the region from
3104–3080 cm−1 is not observed in the spectra of Pt103 (not
shown) indicating the presence of a protonated amide group
Table 2 In vitro activity of 1 and 2 in the A2780 cell line

IC50 values for compounds 1, 2 and cisplatin against L1210,

Compound IC50 (μM) in A2780 Initial solvent for drug
L1210/DDP, and SKOV-3 cell lines
1 0.2 10% water : acetone
The activity of these two drugs 1 and 2 was tested by determi- 2 0.3 10% water : acetone
nation of IC50 values against L1210 (cisplatin sensitive) and Cisplatin 0.5 Saline

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Fig. 2 FT-IR raw spectra and second derivative spectra of 1, 2 and
cisplatin.
(resulting in an amine group) in compounds 1 and 2. In
addition, the hydrogen bonding decreases the wavenumber
value of the amide group stretching. The bands assigned to
carboxylate groups in compounds 1 and 2 were strong in the
region from 1660–1610 cm−1 for asymmetric vibration and
from 1360–1350 cm−1 for the symmetric vibration. The bands
at 1317 and 1296 cm−1 in the cisplatin second derivative
spectra are assigned to the symmetric deformation of the NH3
group (δsNH3).
FT-IR spectra of cell lines
To determine the similarities and differences between the
A2780 and A2780R cell lines, FT-IR spectra were recorded of
the untreated cells. A comparison of the spectra reveals that
more dramatic changes induced by drugs occur in the A2780
cell line compared to the A2780R cell line. The changes can be
seen in the average spectra for the A2780 cell line and include
bands at 1234, 1169, 1080, 1053, 1011, 964, 930 and 887 cm−1
(Fig. 4) mainly associated with nucleic acid vibrations.
The differences between spectra are small hence PCA was
performed on the second derivative spectra in the region from
1300–800 cm−1 where the main nucleic acid and carbohydrate
bands reside. PCA reduces the dimensionality of the data set
and can be used to study similarities and differences in the
spectra. The Score Plots show the clusters in the PCA while the
Loading Plots show the variables that are responsible for each
PC as a function of the wavenumber. For PC1 the negative
loadings are associated with positive scores and the positive
loadings are associated with negative score values because
second derivative spectra were used in the PCA model. For PC2
the negative loadings are associated with negative scores and
the positive loadings associated with the positive scores. Fig. 3
shows the PCA Scores and Loading Plots from PCA on second
derivative spectra of A2780 and A2780R. Each spectrum is rep-
resented by a Score point in the PCA PC1 versus PC2 Scores
Plot. The spectra were collected from 3 cultures of cells with 5
to 7 technical replicates acquired for each culture.
Some separation is visible in the Scores Plot, mostly along
PC2, which indicates that the macromolecular chemistry is
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Fig. 3 PCA Scores Plot of the untreated cell lines A2780 (blue) and
A2780R ( purple) and loadings of PC1 (left panel) and PC2 (right panel).
quite different between A2780 and A2780R cell lines. However,
PC1 shows more variance for cells from the A2780R cell line.
The A2780R cell line is derived or isolated from A2780 cells
after treatment with cisplatin. It was found that resistant cells
are able to alter the metabolism of saccharides in order to
form colonies. Strong negative PC1 loadings observed at 1277,
1234, 1219, 1134, 1088, 1065, 1030 and 975 cm−1 associated
with mainly nucleic acid bands are probably indicative of cells
in different states of division. Strong positive PC1 loadings are
observed at 1257, 1157, 1045, 1007, 948 and 883 cm−1, which
are more characteristic of C–O stretching vibrations of carbo-
hydrates and indicative of metabolic differences between the
cells. The separation of resistant and sensitive cell lines occurs
along PC2. Strong PC2 positive loadings at 1227, 1084, 1041
and 964 cm−1 are mainly associated with nucleic acid modes
and are associated with the A2780 sensitive cell line. The PC2
negative loadings are associated with the A2780R resistant cell
line and show strong loadings for bands at 1269, 1196, 1065,
1014 and 979 cm−1. The 1269 cm−1 band is difficult to assign,
while the other bands are tentatively assigned to C–O stretch-
ing vibrations from polysaccharides.
FT-IR spectra of cell lines inoculated with cisplatin, 1 and 2
For each drug treatment, three concentrations were prepared:
0.5, 5 and 50 µM. However, only concentrations of 5 µM and
50 µM of 1 and 2 seemed show an effect on the cells, and no
effect was noticed using 0.5 μM. Treatment with 5 μM 1 and 2
showed an effect but it was weak and hard to interpret, there-
fore only data from the treatment with 50 μM 1 and 2 is shown
in this study. Only a concentration of 50 μm cisplatin had an
effect on the tested cells. Both the A2780 and the A2780R
ovarian cell lines were treated with each drug. Three sets of
replicates were prepared for each experiment. Each replicate
comprised of 30 spectra, which were averaged for each drug
concentration. These were compared to the untreated control
cells. Fig. 4 (upper panels) shows the FT-IR absorbance spectra
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The assignment of FT-IR bands in biological samples is

generally challenging due to the complex biochemical compo-
sition of cells including e.g. proteins, lipids and carbohydrates
producing many overlapping bands in the FT-IR spectra. By
calculating the second derivative, hidden spectral features can
be resolved (Fig. 4, lower panels). Here, the A2780 cells treated
with 1 and 2 show a blue shift in the bands at 1234, 1053,
1011, 930, 887 and 829 cm−1, a red shift in the bands at
1170 cm−1 and a decrease in intensity of the 964 cm−1 band.
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Bands at 1230, 1052 and 964 cm−1 are assigned to asymmetric

and symmetric phosphodiester group vibrations of nucleic
Fig. 4 A: FT-IR absorbance (upper panel) and second derivative (lower acids. Changes in these bands indicate DNA conformational
panel) spectra of A2780 cells treated with acetone (blue), 1 (dark red), 2
(red), saline (green) and cisplatin ( purple). B: FT-IR absorbance (upper
changes possibly from denaturation and would be expected in
panel) and second derivative (lower panel) spectra of A2780R cells response to the drug interacting with DNA. There is an
treated with acetone (blue), 1 (dark red), 2 (red), saline (green) and cis- increase of intensity in all the spectral features from drug-
platin ( purple). treated cells except for bands at 1230, 964, 884 and 827 cm−1.
The treated A2780R cells show a blue shift of the bands at
1234, 1053, 1011, 964, 887 and 829 cm−1, while the bands
of the A2780 (cisplatin sensitive) and A2780R (cisplatin resist- 1170 and 930 cm−1 show a red shift. There is an increase in
ant) cell lines, untreated (blue), treated with 50 µM 1 (green), the absorbance of bands at 1234 and 1169 cm−1, assigned pre-
with 50 µM 2 (red) and with 50 µM cisplatin in the spectral dominantly to C–O stretching modes from carbohydrates and
region from 1300 to 800 cm−1. The major bands are assigned a decrease in absorbance for bands at 965, 930, 885 and
in Table 3 and the changes on treatment with drugs are sum- 827 cm−1 assigned to mainly RNA vibrational modes. The
marised in Table 4. bands 1080 and 1207 cm−1 showed a significant decrease in
absorbance with drugs 1, 2 and cisplatin.
To further investigate the spectra of the non-treated cells
Table 3 FT-IR band assignments for cells and biological
materials40,45,46 and the treated cells, PCA was applied for all samples after pre-
processing. PCA was performed on the second derivative of
Band position infrared spectra from control cells and cells inoculated with
(cm−1) cisplatin, 1 and 2. Fig. 5 shows a PC1 versus PC2 Scores Plot of
1234 νas(PO2) overlapping of proteins and nucleic acids the control cells, inoculated with saline or acetone and cells
1211–1207 νas(PO2) treated with 1 and 2 when using a drug concentration of
1170–1169 νas(CO–O–C) and ν(C–O) from glycomaterials and
50 µM for both the A2780 and the A2780R cell line. The posi-
proteins ν(C–O) RNA ribose
1170 νas(C–O) in esters tive loading bands are correlated with the negative scores and
1122, 1121 νs(PO2−) in RNA and DNA νs(PO2−)RNA vice versa.
1053 νs(PO2−) in RNA and DNA νs(PO2−)RNA
There is a clear separation between the scores of control
1011 ν(C–O) deoxyribose
965, 964 νas(PO3−) DNA and RNA ribose, ν(C–C) and ν(C–O) cells and the drug inoculated cells along PC1 in both cell lines
in deoxyribose of DNA tumour cells for 1 and 2, while for cisplatin the separation is along PC2.
930 N(COP) phosphorylated protein
This indicates that the separation is not due to spectral fea-
887 C–C, C–O deoxyribose
830, 827 C2′ endo conformation of sugar tures from the drug but rather biochemical changes induced
Symmetric (νs), asymmetric (νas) in the cells affected by the drug. The spectra for each drug

Table 4 Band changes in average spectra of both A2780 and A2780R with 50 μM 1, 2 and cisplatin

2 1 Cisplatin
−1
Band cm A2780 A2780R A2780 A2780R A2780 A2780R

1234 Red shift Red shift Red shift Red shift Red shift Red shift
1211 Disappear Disappear Disappear Disappear Disappear Disappear
1170 More intense More intense More intense More intense More intense More intense
1121 More intense No change More intense No change More intense No change
1052 Red shift Red shift Red shift Red shift Less intense Less intense
1009 Red shift More intense Red shift More intense Less intense More intense
964 Less intense More intense Less intense More intense Less intense Less intense
931 No change Blue shift and less intense No change Blue shift and less intense More intense No change
885 Less intense Less intense Less intense Less intense More intense No change
830 Red shift Red shift Red shift Red shift No change Red shift

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Fig. 5 PCA Scores (left panels) and Loading Plots (right panels) of the
A2780 cell line (A) and A2780R cell line (B) treated with 50 μM cisplatin
( purple), 1 (dark red) and 2 (red) and saline (green) or acetone (blue) as
controls.
treatment and each cell line were individually assessed using
PCA in the fingerprint region (1300 to 800 cm−1) where the
greatest changes were observed. For each PCA Scores Plot the
controls were separated well from spectra of the drug inocu-
lated cells. The separation between treated cells and controls
at the concentration of 50 µM for 1 and 2 occurs mostly along
PC1 demonstrating a profound effect of these drugs on the
cells. However, for cisplatin treated cells, the separation occurs
along PC2, which indicates a weaker effect of cisplatin on the
cells. For PC1, the negative bands in the Loading Plots corre-
late with the positive score values because the second deriva-
tives were used in the PCA decomposition and not the raw
spectra. In this case, the negative bands in the Loadings Plot
refer to the treated cells, while the positive loadings corres-
pond to the control cells. The PCA loadings for each experi-
ment are remarkably similar irrespective of the drug or the cell
type. The drugs appear to be eliciting a similar apoptotic
response in both cell lines. The Loading Plots show that the
bands responsible for the separation of the control cells from
the cells treated with 1 or 2 are associated with changes in
ν(C–O) vibrations in RNA (1169 cm−1), phosphodiester of
nucleic acids DNA and RNA (1097 cm−1), ν(PO2−) and ν(C–O)
at 1020 cm−1. This indicates that both drugs 1 and 2 affect the
nucleic acids in the A2780 cell line. Fig. 5 shows that com-
plexes 1 and 2 have a stronger effect compared to cisplatin on
both cell lines. The spectra of the cisplatin treated cells separ-
ated well along PC2. For PC2, the negative bands in the
Loading Plots correlate with the negative score values.
Therefore, the negative PC scores represent the treated cells
and show characteristic features at 1235, 1159, 1117, 1081,
1027, 983, 927, 903 and 845 cm−1. The positive PC loadings,
on the other hand, represent the control sample and show
characteristic bands at 1195, 1139, 1099, 1046, 1007, 962, 910
and 883 cm−1. Unlike the other drugs, cisplatin had quite a
different effect on the both the A2780 and A2780R cell lines.
The PCA loadings from spectra of cisplatin-treated cells com-
pared to control cells are very different for the A2780 and
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A2780R cell lines, while they are similar for spectra of organoa-
midoplatinum drug-treated cells compared to control cells in
A2780 and A2780R. Compounds 1 and 2 show some similar
effects on both cell lines. However, less separation was
observed when using cisplatin especially in the case of the
A2780R cell line. The results indicate that 1 and 2 have a much
more profound effect and a similar mode of action to each
other and different from cisplatin. The A2780R cells appeared
less affected by cisplatin than the A2780 cells. The bands 1238,
1173, 1080, 1061, 960, 887 and 844 cm−1 are correlated directly
to the second derivative spectra of the inoculated cells with cis-
platin. Some of them are shown as strong bands in the
Loadings Plot, which indicates a stronger effect of cisplatin on
bands such as 1238, 1173, 1080 and 1061 cm−1. They correlate
with 1234, 1169, 1080 and 1053 cm−1 bands in the second
derivative. One characteristic spectral feature that is highly
affected by the drugs is around 1234 cm−1 and assigned to the
asymmetric stretching vibration of phosphodiester functional
groups of DNA and RNA polysaccharide backbone structures.
Other bands around 1170, 1052 and 1009 cm−1 are tentatively
assigned to symmetric stretching vibrations of PO2− groups in
nucleic acids and stretching vibration of C–O–P and C–O–C of
polysaccharides. The changes are consistent with those
observed for apoptosis in other studies. Gao et al.47 combined
FT-IR with fluorescence activated cell sorting (FACS) to analyze
SW620 cells following treatment with 5-fluorouracil for 12, 24
and 48 h. The apoptotic cells had several spectral character-
istics including the peaks at 1240 cm−1 which increased in
wavenumber and a band at 1040 cm−1, associated with poly-
saccharides, appeared at 24 and 48 h, which was blue shifted
and the absorbance 1040/1460 ratio increased at the late stage
of apoptosis. Zhou et al.41 reported a number of differences
between normal HL60 cells, differentiated cells induced by all-
trans retinoic acid (ATRA) and PMA, and apoptotic cells
induced by vircristine (VCR). Their results indicated that the α-
helix content of the membrane protein of differentiated HL60
cells induced by ATRA and PMA increased, while the β-sheet
content of membrane proteins of apoptotic cells induced by
VCR also increased. Analysis of the lipid marker band at
2854–2853 cm−1 showed a higher membrane lipid content in
HL60 cells during differentiation or apoptosis compared with
that of untreated HL60 cells. The increased lipid content
during differentiation and apoptosis was also corroborated by
the higher intensity of the 1456–1455 cm−1 CH2-deformation
band of the lipid methylene groups. Zhou et al.41 also reported
an increase in the DNA/protein absorption ratio (1087 cm−1/
1540 cm−1) during differentiation and apoptosis in leukemic
cell lines. Gasparri and Muzio48 quantified apoptotic cells by
using attenuated total reflection (ATR) on HL60 leukaemic
cells with camptothecin and monitored apoptosis over time.
Several ATR-FTIR spectral changes occurred during the apopto-
tic process and noted the apoptotic index was inversely corre-
lated with the spectral area in the region 1200–900 cm−1,
assigned to the absorption of nucleic acids. Hence the spectral
changes observed in our study are consistent with changes
indicative of apoptosis.
This journal is © The Royal Society of Chemistry 2018

Analyst
Conclusions
In this study we compared our new organoamidoplatinum(II)
drugs with the model compound cisplatin. It was shown that
both 1 and 2 have profound and similar effects irrespective of
the cell line. However, cisplatin generated quite different
Scores and Loadings Plots compared to the other drugs. The
separation on the PC1 versus PC2 Scores Plots were not as dra-
matic compared to the organoamidoplatinum(II) complexes
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especially in the case of the A2780R cell line. Moreover, the
Loading Plots were quite different between the sensitive and
resistant cell line for cisplatin but were almost identical for the
organoamide drugs. Based on the Loadings Plot both organoa-
mide drugs and cisplatin showed significant effects on the
nucleic acid structure. The results indicate that the organoami-
doplatinum(II) complexes have a much more profound effect
on both cisplatin sensitive and resistant cell lines when com-
pared to cisplatin. While the organoamidoplatinum(II) drugs
show potential toxicity, testing and in vivo studies are required
before the compounds can be advanced as anti-tumour drugs.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Mr. Finlay Shanks for the technical support and Ms
Alison Slater and Dr Kelly Waldeck for assistance with cell
culture studies. B. R. W. is supported by an Australian Research
Council (ARC) Future Fellowship grant FT120100926.
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