Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug

Article abstract of DOI:10.1016/j.bcp.2005.12.039

Platinum complexes are widely used in cancer chemotherapy; however, they are associated with toxicity, high "non-specific" reactivity and relatively poor pharmacokinetic profiles. In particular, their low cellular uptake and rapid metabolic inactivation means that the amount of "active" drug reaching the nuclear compartment is low. Our strategy to facilitate nuclear accumulation was to introduce a hydrophobic anthraquinone (1C3) moiety to the Pt-complex. Anthraquinones are known to readily intercalate into DNA strands and hence, the Pt-1C3 complex may represent an effective system for the delivery of the platinum moiety to nuclear DNA. Efficacy of the complex was determined by measuring the extent and potency of cytotoxicity in comparison to cisplatin and an anthraquinone based anticancer drug, doxorubicin. The Pt-1C3 complex generated higher levels of cytotoxicity than cisplatin, with a potency of 19 ± 4 μM in the DLD-1 cancer cell line. However, this potency was not significantly different to that of the 1C3 moiety alone. To examine the reason for the apparent lack of platinum related cytotoxicity, the cellular distribution was characterised. Confocal fluorescence microscopy indicated that the Pt-1C3 complex was rapidly sequestered into lysosomes, in contrast to the nuclear localisation of doxorubicin. In addition, there was negligible DNA associated Pt following administration of the novel complex. Thus, the addition of a 1C3 moiety generated sequestration of the complex to lysosomes, thereby preventing localisation to the nucleus.

Full text of DOI:10.1016/j.bcp.2005.12.039

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 1 3 6 – 1 1 4 5
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Cytotoxic efficacy of an anthraquinone linked platinum
anticancer drug
R.A. Alderden a, H.R. Mellor b, S. Modok b, T.W. Hambley a, R. Callaghan b,
*
a Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, NSW 2006, Australia
b Oxford Drug Resistance Group, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford,
Oxford OX3 9DU, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Platinum complexes are widely used in cancer chemotherapy; however, they are associated
with toxicity, high ‘‘non-specific’’ reactivity and relatively poor pharmacokinetic profiles. In
particular, their low cellular uptake and rapid metabolic inactivation means that the
amount of ‘‘active’’ drug reaching the nuclear compartment is low. Our strategy to facilitate
nuclear accumulation was to introduce a hydrophobic anthraquinone (1C3) moiety to the Pt-
complex. Anthraquinones are known to readily intercalate into DNA strands and hence, the
Pt-1C3 complex may represent an effective system for the delivery of the platinum moiety to
nuclear DNA. Efficacy of the complex was determined by measuring the extent and potency
of cytotoxicity in comparison to cisplatin and an anthraquinone based anticancer drug,
doxorubicin. The Pt-1C3 complex generated higher levels of cytotoxicity than cisplatin, with
a potency of 19 ꢀ 4 mM in the DLD-1 cancer cell line. However, this potency was not
significantly different to that of the 1C3 moiety alone. To examine the reason for the
apparent lack of platinum related cytotoxicity, the cellular distribution was characterised.
Confocal fluorescence microscopy indicated that the Pt-1C3 complex was rapidly seques-
tered into lysosomes, in contrast to the nuclear localisation of doxorubicin. In addition,
there was negligible DNA associated Pt following administration of the novel complex. Thus,
the addition of a 1C3 moiety generated sequestration of the complex to lysosomes, thereby
preventing localisation to the nucleus.
Received 2 December 2005
Accepted 20 December 2005
Keywords:
Platinum
Chemotherapy
Cancer
Lysosomes
Anthraquinone
Cytotoxicity
# 2006 Elsevier Inc. All rights reserved.
1.
Introduction
lining these cellular effects have shed considerable light on
the mechanisms of anticancer drug resistance and consider-
Cisplatin is used for the treatment or palliation of many types
of cancer including non-small cell lung, ovarian and testicular
[1]. However, these cancers frequently develop resistance to
cisplatin and many others display an inherent lack of
sensitivity. The limited effectiveness of cisplatin is due to
many factors including: (a) a poor pharmacokinetic profile,
(b) low accumulation in cells, (c) increased production of
intracellular thiols (e.g. glutathione and metallothionein) and
(d) increased DNA repair capacity [2–4]. Investigations out-
able effort has been directed towards the development of
novel platinum containing compounds that deliver greater
efficacy.
Often, strategies aimed at improving platinum drug
efficacy attempt to increase cellular uptake and facilitate
targeting of the compounds to DNA [5,6]. The latter involves
the incorporation of a functional group into the platinum
complex that will interact or intercalate with DNA. Additional
benefits of this intercalation strategy include a reduction in
* Corresponding author. Tel.: +44 1865 221 110; fax: +44 1865 221 834.
E-mail address: richard.callaghan@ndcls.ox.ac.uk (R. Callaghan).
0006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2005.12.039

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non-specific modification of cellular macromolecules and the
2.2.
Chemical synthesis of platinum complexes
possible generation of novel DNA lesions. The latter may
evade DNA repair processes leading to more effective
disruption of DNA replication. For example, diaminocyclo-
hexane–Pt–DNA adducts generated by oxaliplatin can trigger
apoptosis more efficiently than diammine–Pt–DNA adducts
produced by cisplatin in certain tumour types [7]. Further-
more, a number of Pt-complexes containing the DNA inter-
calating agent 9-aminoacridine have been developed and
these compounds display increased reaction with DNA with
a novel reaction mechanism to generate the adduct [8].
The structure activity relationships and antitumour activ-
ity of several platinum complexes linked to anthraquinone
intercalating moieties by an alkyl amino chain have been
examined [9–13]. A number of these complexes were shown to
have equivalent in vitro and in vivo activity to cisplatin and to
possess higher activity than the components individually (the
free anthraquinone ligand and the analogous platinum
complex) or a 1:1 molar mixture of the two [11].
2.2.1. Instrumentation
Diffuse reflectance infrared Fourier transform spectra
(DRIFTS) were measured on a BIO-RAD FTS-40 or FTS-7
spectrophotometer. Potassium bromide was used as both
the background and the matrix over the range 400–4000 cmꢁ1
.
1H and 13C NMR spectra were recorded at 300 K on a Bruker
AMX 300 MHz spectrometer. All spectra were recorded using
commercially available solvents (Aldrich) of >99.6% isotopic
purity and referenced to TPS (3-(trimethylsilyl)propionic acid)
or solvent isotopic impurities. 195Pt NMR spectra were
recorded at 300 K on a Bruker AMX 400 MHz spectrometer.
Chemical shifts were referenced to Na2[PtCl6]. Elemental
analyses (C, H, N) were performed by the Microanalytical
Service of the Australian National University, Canberra, ACT,
or Chemical and Micro Analytical Services, Belmont, Vic.
2.2.2. 1-[(3-Aminopropyl)amino]-anthracene-9,10-dione
(1C3)
Alkylamino-anthraquinone containing compounds such
as the anthracycline antibiotic doxorubicin are established
and clinically used anticancer drugs. Doxorubicin is known to
intercalate into DNA and this observation sparked the
development of novel DNA intercalators. For example, the
clinically used anticancer drug mitoxantrone was developed
from rational design on data obtained from chemical screen-
ing of anthraquinone-based intercalating agents [14,15].
Mitoxantrone interacts with DNA in a sequence selective
manner and its intercalation leads to cytotoxicity via inhibi-
tion of Topoisomerase II. The high affinity interaction with
DNA is thought to be stabilised by the two cationic (at
physiological pH) alkylamino side chains that form electro-
static interactions and hydrogen bonds with the DNA strand
[16–18]. In addition, a systematic chemical screening inves-
tigation has identified the cationic alkylamino group on mito-
xantrone derivatives as a key factor in the nuclear localisation,
DNA binding and inhibition of DNA synthesis [19].
The following synthetic procedure is based upon that outlined
by Barasch et al. [20]. Propane-1,3-diamine (15 mL, 180 mmol)
was added to a solution of 1-chloroanthraquinone (10 g,
41 mmol) in dry toluene (350 mL, dried over anhydrous
Na2SO4) and refluxed for 18 h. The red suspension was
evaporated to dryness under reduced pressure and resus-
pended in chloroform (500 mL). Gaseous hydrochloric acid
was bubbled through the suspension for approximately 2 days,
or until the mixture became pale pink in colour. The product
was extracted into an aqueous layer with several washings of
water. Using 3 M NaOH, the pH of the aqueous layer was
adjusted to approximately 8, followed by subsequent extrac-
tion of the final product into chloroform. The solution was
evaporated to dryness under reduced pressure, giving a red
solid (7.6717 g, 27.4 mmol, 77% yield). 1H NMR (300 MHz, CDCl3)
d 9.74 (broad, 1H), 8.24 (m, 2H), 7.72 (m, 2H), 7.57 (dd, 1H), 7.51 (t,
1H), 7.07 (dd, 1H), 3.41 (m, 2H), 2.93 (t, 2H), 1.91 (m, 2H), 1.38
(broad, 2H); 13C NMR (300 MHz, CDCl3) d 185.10, 183.89, 151.86,
135.41, 135.11, 134.75, 134.02, 133.13, 133.01, 126.81, 126.75,
117.94, 115.75, 113.06, 40.68, 39.94, 32.92; IR (KBr, cmꢁ1): 3356 m,
3273 m, 3182 m, 2934 m, 2862 m, 1662 s, 1628 s, 1594 s, 1572 s,
1511 s, 1470 m, 1465 m, 1409 m, 1303 s, 1272 s, 1236 m, 1176 m,
1160 m, 1071 m, 1019 m, 972 m, 916 m, 830 m, 806 m, 786 m, 735
s, 709 s, 673 m, 662 m, 598 m, 549 m, 481 m, 419 m.
In the present study a novel Pt(II)-complex containing a
co-ordinated alkylamino-anthraquinone compound was
examined for its efficacy to produce cytotoxicity in a colon
cancer cell line. The efficacy was compared to the anthraqui-
none ligand, cisplatin, a hydrophobic Pt-complex (JM118) and
doxorubicin. The ability of the novel Pt–anthraquinone
complex to generate cytotoxicity was related to its sub-
cellular localisation.
2.2.3. Amminetrichloroplatinate(II) [PtCl3(NH3)]ꢁ
The following synthetic procedure is based upon that outlined
by Cai et al. [21]. Cisplatin (4.0 g, 13.3 mmol), tetraethylam-
monium chloride monohydrate (2.94 g, 16.0 mmol), and
ammonium chloride (0.20 g, 3.7 mmol) were dissolved in
N,N-dimethylacetamide (150 mL). The reaction mixture was
heated at 100 8C, purging with a slow stream of N2 for 8–10 h.
The solution changed from yellow to orange in colour, and its
volume evaporated to approximately 50 mL, during the course
of the reaction. Hexane/ethyl acetate (1:1, 300 mL) was added
to the solution and it was kept overnight at ꢁ20 8C. The
supernatant was decanted off, and the solid extracted using
60 mL of acetonitrile. The remaining solid, assumed to be a
mixture of ammonium chloride and cisplatin, was washed
with water to recover unreacted cisplatin. Water (5 mL) was
2.
Materials and methods
2.1.
Materials
RPMI-1640 + Glutamax
I culture medium, foetal bovine
serum, trypsin–EDTA and penicillin/streptomycin were all
purchased from Life Technologies—Invitrogen (Paisley, UK).
Doxorubicin hydrochloride was obtained from Sigma Labora-
tories (Poole, UK). SYTO21, MitoTracker Green and Lyso-
Tracker Green were all purchased from Molecular Probes—
Invitrogen. All solvents and reagents were of at least
analytical grade and were used as received without further
purification, unless otherwise stated.

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added to the acetonitrile solution and the acetonitrile was
removed under reduced pressure, resulting in an orange
aqueous solution of [PtCl3(NH3)]ꢁ (11.58 mmol, 87% yield).
2.3.
DNA fluorescence titration
Small quantities of a stock solution of calf thymus DNA were
added to 38 mM solutions of 1C3 and Pt-1C3, such that the ratio
of DNA base pairs:anthraquinone compound ranged from 0:1
to 8:1. Following each addition, solutions were mixed and
fluorescence emission spectra recorded within 10 min.
2.2.4. cis-[1-[[(3-Aminopropyl)amino]-anthracene-9,10-
dione]ammine]dichloro-platinum(II) (Pt-1C3)
The following synthetic procedure is based upon that outlined
by Gibson et al. [11]. A solution of 1C3 (1.6535 g, 5.9 mmol) in
MeOH (approximately 2 L) was added to an aqueous solution of
[PtCl3(NH3)]ꢁ (6.0 mmol) over a period of approximately 1 h.
Additional MeOH was added, as required, to ensure that
minimal precipitation occurred. The solution was stirred at
room temperature for approximately 3 weeks, during which
time a dark red precipitate formed. The solid was filtered and
washed with MeOH. The resulting red solid was ground to a
fine powder, resuspended in MeOH (500 mL), stirred for several
days, then filtered and washed with MeOH. The process of
resuspension was repeated until elemental analysis revealed
that the product did not contain unreacted 1C3. The final
product was obtained as a red solid (0.8309 g, 1.47 mmol, 25%
yield). 195Pt NMR (DMF/HCl): d ꢁ2157 ppm. Analysis calculated
for PtC17H19N3O2Cl2: C, 36.25; H, 3.40; N, 7.46. Found: C, 36.37; H,
3.29; N, 7.32.
2.4.
Cell culture
The human colon adenocarcinoma cell line DLD-1 was kindly
provided by Dr. Roger Phillips (University of Bradford, UK). The
DLD-1 cells were grown in RPMI-1640 medium containing
Glutamax I and 25 mM HEPES supplemented with 10% (v/v)
heat inactivated foetal bovine serum and penicillin/strepto-
mycin (100 IU/mL and 100 mg/mL, respectively). Cells were
grown at 37 8C (5% CO2) as monolayer cultures for a maximum
of 20 passages and routinely harvested with trypsin–EDTA.
2.5.
Drug cytotoxicity assays
DLD-1 cells were seeded in 96-well plates at a density of 3 ꢂ 103
cells per well in 100 mL supplemented medium. The cells were
Fig. 1 – Structures of drugs used in the investigations. Structures of: (a) cisplatin, (b) JM118, (c) 1-[(3-aminopropyl)amino]-
anthracene-9,10-dione (1C3), (d) cis-[1-[[(3-aminopropyl)amino]-anthracene-9,10-dione]ammine]dichloro-platinum(II) (Pt-
1C3) and (e) doxorubicin.

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1139
allowed to adhere for 24 h prior to drug addition. Drugs were
added in a 100 mL aliquot in culture medium at twice the
desired final concentration. Stocks of cisplatin and JM118 were
made at 0.5 mM in 100 mM KCl and added to cells in the
concentration range 5 ꢂ 10ꢁ7 to 2 ꢂ 10ꢁ5 M. The relative
insolubility of the platinum complex containing the anthra-
quinone moiety (Pt-1C3) and the organic moiety alone (1C3)
precluded storage in 100 mM KCl. Stock solutions of 0.5 mM
were made in 100 mM KCl and 60% (v/v) dimethylformamide
(DMF). Administration of the solvent DMF to the cells was
maintained below 1% (v/v). Doxorubicin was stored as a 50 mM
stock in DMSO and addition of this solvent to cells was
maintained at <0.1% (v/v) and final drug concentrations were
in the range 5 ꢂ 10ꢁ9 to 2 ꢂ 10ꢁ5 M.
Cells were exposed to drugs for 2 or 4 h (unless otherwise
indicated in figure legends) and then left for a ‘‘recovery’’
phase of 72 h in drug-free culture medium. Following
‘‘recovery’’, the number of viable cells was determined using
the MTT assay. Absorbance (l = 550 nm) was measured in a
SpectraMAX 250 plate reader (Molecular Devices). Cell viability
was plotted as a function of drug concentration and the
cytotoxic potency (IC50) estimated by non-linear regression of
the general dose–response curve [22]. The viable cell number
(cells remaining (% total)) was expressed as a percentage of the
number obtained in the absence of drug treatment for the
highest concentration of drug tested.
2.6.
Confocal fluorescence microscopy
Monolayers were grown on coverslips in 6-well tissue culture
plates to enable assessment of fluorescent drug and organelle
specific probe localisation by confocal microscopy. Drugs (Pt-
1C3 or 1C3) or probes were added from concentrated stocks
directly to fresh culture medium and incubated at 37 8C for
periods as indicated in the figure legends. The coverslips were
then briefly washed in PBS and overlaid with 20% (v/v) glycerol
Table 1 – Cytotoxicity of platinum and anthraquinone
containing compounds in the DLD-1 cell line
Drug
Exposure
time (h)
Cells
remaining
(% total)
Potency
(IC50) (mM)
Cisplatin
2
4
77 ꢀ 2
59 ꢀ 8
10 ꢀ 1
85 ꢀ 19
34 ꢀ 6
24
2.2 ꢀ 0.3
Pt-1C3
1C3
2
4
38 ꢀ 5
25 ꢀ 7
19 ꢀ 4
11 ꢀ 1
2
4
67 ꢀ 8
48 ꢀ 6
25 ꢀ 2
21 ꢀ 6
JM118
2
4
29 ꢀ 15
20 ꢀ 8
16 ꢀ 7
5.9 ꢀ 2.9
Fig. 2 – Drug induced cytotoxicity profiles. Dose-dependent
cytotoxicity produced in DLD-1 cells cultured as monolayers
exposed to the following: (a) cisplatin and JM118 for 4 h, (b)
doxorubicin for 4 h and cisplatin for 24 h and (c) 1C3 and Pt-
1C3, each for 4 h. The general dose–response equation was
fitted using non-linear least squares regression and the
data points represent mean W S.E.M. of at least three
independent observations. Cells remaining after the drug
exposure and subsequent 72-h recovery period were
determined using the MTT assay and the number in the
absence of added drug was assigned a value of 1.0.
Doxorubicin
2
4
18 ꢀ 9
16 ꢀ 9
0.30 ꢀ 0.08
0.17 ꢀ 0.04
DLD-1 cells were exposed to a range of drug concentrations over
various exposure times and then allowed to recover for a further
72-h period. The number of cells remaining was measured with an
MTT assay and plotted as a function of drug concentration. The
percentage of cells remaining was determined at the maximal
achievable drug concentration, which was 20 mM. The potency to
generate cytotoxicity was estimated by non-linear regression
analysis of the dose–response curve and all values represent the
mean ꢀ S.E.M. of at least three independent observations.

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Fig. 3 – Cellular localisation of Pt-1C3. DLD-1 cells were grown to 80% confluency on glass coverslips in 6-well tissue culture
plates and drugs were localised using confocal fluorescence microscopy. Panels (a–c) were obtained from cells incubated in
the presence of Pt-1C3 (12 mM) for 4–5 h at 37 8C in the presence of the cell permeant nuclear stain SYTO21 (5 mM, 2 h, 37 8C).
Panel (a) highlights localisation of the nuclear stain (lex = 488 nm, 10–15% laser strength, lem = 505–530 nm). Panel (b)
highlights localisation of Pt-1C3 (lex = 543 nm, 76% laser strength, lem > 560 nm). Panel (c) is the overlay of panels (a and b).
Panels (d–f) were obtained from cells incubated in the presence of Pt-1C3 (12 mM) for 4–5 h at 37 8C in the presence of the
mitochondrial stain MitoTracker Green (0.2 mM, 30 min, 37 8C). Panel (d) highlights localisation of the mitochondrial stain

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1141
in PBS. Inverted coverslips were then placed on microscope
slides and the edges sealed with clear nail varnish.
The fluorescent compounds were detected using a Zeiss
LSM510 confocal laser scanning microscope (Carl Zeiss,
Welwyn Garden City, UK). SYTO21 (nuclear stain), MitoTracker
(mitochondrial stain) and LysoTracker (lysosomal stain) were
all excited with an Argon laser at l = 488 nm and detected
using an emission filter set at l = 505–530 nm. Doxorubicin, Pt-
1C3 and 1C3 were all excited at l = 543 nm with an emission
filter set for l > 560 nm.
advanced colorectal cancer. Thus it was thought that
investigation of novel platinum compounds in colon cancer
cell lines was relevant. The DLD-1 cells express Bcl2, a mutated
version of p53 (Ser241Phe) and are considered MMR deficient.
These features can potentially decrease the efficacy of
platinum compounds, however, previously we have exten-
sively characterised this cell line and it was sensitive to
cisplatin (IC50 = 14 mM).
Fig. 2 shows examples of dose–response curves obtained
for the various platinum complexes and doxorubicin in the
treated DLD-1 cells. A 2-h exposure to cisplatin was associated
with a viable cell count of 77 ꢀ 2% at the highest dose used
(20 mM). Extrapolation of the curve estimated that cisplatin
displayed a potency of 85 ꢀ 19 mM. However, increasing the
exposure time to 24 h resulted in cytotoxicity in 90% of the cell
population at the same concentration (i.e. 20 mM) and a
significantly greater potency of 2.2 ꢀ 0.3 mM (Fig. 2b and
Table 1). In contrast, a 4-h exposure to the hydrophobic
cisplatin derivative, JM118, was sufficient to generate cyto-
toxicity in 80% of the DLD-1 cells at 20 mM. The cytotoxic
potency of a 4-h exposure of cells to JM118 was 5.9 ꢀ 2.9 mM
(Fig. 2a and Table 1). Presumably this difference in efficacy,
particularly in a temporal manner, relates to the greater extent
and rate of JM118 accumulation in cells, compared with the
more hydrophilic cisplatin.
2.7.
Statistical and regression analyses
A minimum data set was obtained from at least three
independent observations and values were expressed as
mean ꢀ S.E.M. Statistical comparisons of mean values were
achieved using one-way ANOVA with Bonferroni’s multiple
comparison test and a P value < 0.05 was considered sig-
nificant. All non-linear regression analyses were generated
using the GraphPad Prism3.0 program.
3.
Results
3.1.
Cytotoxicity of doxorubicin and the Pt-complexes
Doxorubicin is a widely used anthracycline class che-
motherapeutic agent that displays significant hydrophobicity.
The hydrophobicity is due to an anthraquinone moiety and
the cytotoxicity of doxorubicin is due to inhibition of
Topoisomerase II activity. A 4-h exposure to DLD-1 cells
produced significant cytotoxicity as evident by the viable cell
population of less than 20% (Fig. 2). The potency of doxorubicin
for a 4-h exposure was 0.17 ꢀ 0.04 mM and similar effects were
observed for a 2-h exposure (Table 1). Clearly this compound
displays a rapid and extensive intracellular accumulation in
the DLD-1 colon cancer cell line.
The compounds used in this study are shown in Fig. 1. JM118 is
considerably more hydrophobic than cisplatin based on the
previously reported log P values [23]. The platinum complex
containing an anthraquinone moiety is designated Pt-1C3 and
the hydrophobic organic group is attached to the platinum via
a short diaminoalkyl bridge. The anthraquinone molecule
containing the diaminoalkyl chain is designated 1C3. The
hydrophobic Pt-1C3 and 1C3 complexes are only sparingly
soluble in aqueous buffer and therefore, the solvent DMF was
used in the stock solutions. Initial examination of cytotoxicity
used a procedure described previously [24], which involved a
72-h exposure of cells to a series of slow-acting Pt(IV)-
complexes. However, DMF produced necrotic cell damage
during this prolonged exposure. This toxicity was time-
dependent since exposure of DLD-1 cells to DMF (0.05–2%, v/
v) for periods up to 12 h did not generate any cytotoxic effect
(data not shown). However, longer exposures produced a time-
dependent toxicity. As a consequence, the DMF concentra-
tions were maintained below 1% (v/v) and cells were exposed
for a maximum of 4 h. Short exposure times represent a more
realistic pharmacokinetic scenario. Cisplatin and JM118 were
sufficiently soluble in 100 mM KCl buffer to alleviate the need
for added solvent.
Could the incorporation of a hydrophobic anthraquinone
moiety into a platinum complex also facilitate uptake into
cells and achieve greater efficacy in eliciting cytotoxicity?
Fig. 2c shows the dose response curve for the cytotoxicity of
the Pt-1C3 complex and of the anthraquinone moiety, both
following a 4-h exposure to the DLD-1 cells. The 4-h exposure
to Pt-1C3 produced cytotoxicity in 75% of the cell population at
20 mM, the maximum achievable concentration, whilst a 2-h
exposure produced a marginally less pronounced reduction in
cell number (Table 1). The extrapolated IC50 of Pt-1C3 was
11 ꢀ 1 mM following a 4-h exposure and thus, the potency and
extent of cell kill induced was greater than observed for
cisplatin, but not significantly different to JM118 (P < 0.05,
Table 1). The potency and extent of cytotoxicity was
independent of exposure time and indicates that the com-
pound, unlike cisplatin, achieved a rapid intracellular accu-
mulation and distribution. However, the anthraquinone
The DLD-1 cell line was used for a variety of reasons, in
particular since the efficacy of a number of novel platinum
chemotherapeutic agents have been examined using it.
Furthermore, oxaliplatin is used as a first line treatment for
(lex = 488 nm, 10–15% laser strength, lem = 505–530 nm). Panel (e) highlights localisation of Pt-1C3 (lex = 543 nm, 76% laser
strength, lem > 560 nm). Panel (f) is the overlay of panels (d and e). Panels (g–i) were obtained from cells incubated in
the presence of Pt-1C3 (12 mM) for 4–5 h at 37 8C in the presence of the lysosomal stain LysoTracker Green (0.075 mM,
60 min, 37 8C). Panel (g) highlights localisation of the lysosomal stain (lex = 488 nm, 10–15% laser strength, lem = 505–
530 nm). Panel (h) highlights localisation of Pt-1C3 (lex = 543 nm, 76% laser strength, lem > 560 nm). Panel (i) is the overlay
of panels (g and h).

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b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 1 3 6 – 1 1 4 5
Fig. 4 – Cellular localisation of doxorubicin. DLD-1 cells were grown to 80% confluency on glass coverslips in 6-well tissue
culture plates and drugs were localised using confocal fluorescence microscopy. Panels (a–c) were obtained from cells
incubated in the presence of doxorubicin (12 mM) for 4–5 h at 37 8C in the presence of the cell permeant nuclear stain
SYTO21 (5 mM, 2 h, 37 8C). Panel (a) highlights localisation of the nuclear stain (lex = 488 nm, 10–15% laser strength,
lem = 505–530 nm). Panel (b) highlights localisation of doxorubicin (lex = 543 nm, 76% laser strength, lem > 560 nm). Panel (c)
is the overlay of panels (a and b). Panels (d–f) were obtained from cells incubated in the presence of doxorubicin (12 mM) for
4–5 h at 37 8C in the presence of the mitochondrial stain MitoTracker Green (0.2 mM, 1 h, 37 8C). Panel (d) highlights

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 1 3 6 – 1 1 4 5
1143
moiety on its own was also able to generate cytotoxicity in
DLD-1 cells following either a 4 or a 2-h exposure (Fig. 2c and
Table 1). At the highest possible concentration of 1C3 (i.e.
20 mM), the extent of cell kill was 52% and the extrapolated
potency was 21 ꢀ 6 mM for a 4-h exposure. Neither the potency
nor the extent of cytotoxicity was significantly different than
observed for the platinum containing anthraquinone. These
results suggest that the nature of the cytotoxic effect may not
be due to the platinum and is more likely to have been
generated by the anthraquinone moiety.
atomic absorption spectroscopy failed to detect any platinum
incorporated into DNA extracted from DLD-1 cells following
drug exposure up to 24 h. Mislocalisation of Pt-1C3 to the
lysosomes is therefore responsible for the lack of difference
in cytotoxicity produced by the anthraquinone complexed
platinum from that of the organic molecule per se. This
suggests that the Pt-1C3 complex remains largely intact in the
cells, within the lysosomes. Exactly the same sub-cellular
distribution profile was observed for the 1C3 parent compound
as for Pt1C3.
3.2.
Sub-cellular localisation of Pt-1C3 and 1C3
3.3.
Sub-cellular localisation of doxorubicin
The possible lack of any platinum-dependent cytotoxicity
could have resulted from failure of the complex to
The distribution of the established anthraquinone containing
anticancer agent, doxorubicin, was also examined in the DLD-
1 cells to ascertain whether the lack of nuclear localisation of
Pt-1C3 was a general feature of anthraquinones. Fig. 4a–c
indicates that doxorubicin did in fact localise within the
nuclear compartment of DLD-1 cells, which is in agreement
with the potent and extensive cytotoxicity of this compound.
There was no appreciable distribution of doxorubicin within
either the mitochondrial (Fig. 4d–f) or lysosomal (Fig. 4g–i)
compartments, the latter being in contrast to Pt-1C3.
a
accumulate within the nuclear compartment. The anthra-
quinone moiety displays fluorescent properties that enabled
investigation of drug distribution using confocal fluorescence
microscopy. The 1C3 moiety on its own showed no discernible
difference in intracellular distribution compared to Pt-1C3.
Thus only the intracellular distribution of Pt-1C3 is shown in
Fig. 3 in comparison with fluorescent probes specific to
defined organelles. For example, Fig. 3a shows that localisa-
tion of the probe SYTO21 was confined to the nuclear
compartment. In contrast, the distribution of Pt-1C3
(Fig. 3b) was punctate, and as demonstrated by the overlay
in Fig. 3c, distinct from the nuclear compartment. This
indicates that the Pt-1C3 compound, whilst rapidly taken up
by DLD-1 cells, did not enter the nucleus.
4.
Discussion
Cellular uptake of cisplatin has long been recognised as a
limiting factor in the efficacy of this important clinical
anticancer drug. The mechanism of uptake is not fully
understood, but is likely to involve sparing trans-membrane
diffusion [26] and a role for the copper import pump has also
been implicated [27,28]. Once inside the cell cisplatin reaches
the nuclear compartment and a great deal is known about the
molecular interaction with DNA and the nature of the intra-
strand cross-links formed [29]. However, the precise cellular
distribution and the process by which cisplatin reaches the
nucleus are yet to be elucidated. One of the factors precluding
our understanding is the lack of simple analytical or imaging
techniques for most platinum anticancer drugs. Fluorescently
labelled cisplatin derivatives provide valuable information on
cellular distribution [30,31], yet the similarity of these
derivatives to the parent compound must be viewed with
caution. Nonetheless, fluorescein [31], carboxyfluorescein [30]
and Alexa Flour 546 [32] derivatives of cisplatin demonstrate
the potential for sub-cellular sequestration of drugs in non-
nuclear compartments, predominantly Golgi vesicles and
lysosomes.
Two potential ‘‘non-nuclear’’ compartments in which the
Pt-1C3 complex may accumulate are the mitochondria and
lysosomes. Fig. 3d–f demonstrates the relative distributions of
Pt-1C3 and the mitochondrial marker, MitoTracker Green. In
particular, the overlay panel (Fig. 3f) indicates that the
distributions for these two compounds were mutually
exclusive. The sub-cellular distribution of the lysosomal
probe, LysoTracker Green, also shows a punctate pattern
(Fig. 3g). Moreover, inspection of the overlay in Fig. 3i indicates
that the fluorescent dye and Pt-1C3 are similar—the yellow
stain in the overlay is indicative of co-localisation. The
distribution patterns displayed in Fig.
3 were obtained
following 4-h incubation and there was no difference with a
1 or 2-h exposure to drug and fluorescent probe (data not
shown). Fluorescence quenching of intercalators in the
presence of DNA has been reported previously [25]. DNA
titration experiments displayed minimal quenching of 1C3
or Pt-1C3 fluorescence in the presence of calf thymus DNA,
with fluorescence intensity remaining above 60% of the initial
peak intensity (data not shown). In addition, preliminary
results indicate that the whole cell accumulation of Pt-1C3 is
approximately five times higher than that of cisplatin (data not
shown). The results suggest that the Pt-1C3 complex remains
largely intact in the cells, within the lysosomes. Furthermore,
In an effort to more effectively generate platinum–DNA
adducts, an alkyl–amino anthraquinone moiety was incorpo-
rated into a novel Pt(II)-complex. This strategy was to promote
adequate cellular accumulation and nuclear localisation of the
Pt(II)-complex by virtue of the hydrophobicity and DNA
localisation of the mitochondrial stain (lex = 488 nm, 10–15% laser strength, lem = 505–530 nm). Panel (e) highlights
localisation of doxorubicin (lex = 543 nm, 76% laser strength, lem > 560 nm). Panel (f) is the overlay of panels (d and e).
Panels (g–i) were obtained from cells incubated in the presence of doxorubicin (12 mM) for 4–5 h at 37 8C in the presence of
the lysosomal stain LysoTracker Green (0.075 mM, 30 min, 37 8C). Panel (g) highlights localisation of the lysosomal stain
(lex = 488 nm, 10–15% laser strength, lem = 505–530 nm). Panel (h) highlights localisation of doxorubicin (lex = 543 nm, 76%
laser strength, lem > 560 nm). Panel (i) is the overlay of panels (g and h).

1144
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 1 3 6 – 1 1 4 5
intercalating properties of the anthraquinone group. Anthra-
cyclines are well established to intercalate with DNA [33] and
many studies have shown that doxorubicin preferentially
accumulates in the nuclear compartment of cells [34,35], likely
due to the nuclear DNA sequestration of drug that comes into
contact with it. The presence of the anthraquinone in the Pt-
1C3 structure may generate also novel DNA adducts, thereby
evading the DNA repair processes that limit platinum based
chemotherapy. The cytotoxic potency of the Pt-1C3 compound
was significantly higher compared to cisplatin and was similar
to the 1C3 moiety per se. Further examination of the Pt-1C3
and 1C3 compounds indicated that both were rapidly and
efficiently accumulated within the lysosomal compartment of
the cells and that no platinum was observed in the nucleus.
Lysosomal trapping of basic compounds, such as Pt-1C3, will
contribute to reduced efficacy of the compound to produce
DNA damage and resultant apoptosis.
anthraquinone complex used in the present investigation
was associated with rapid intra-lysosomal sequestration in
DLD-1 colon carcinoma cells. This may partly explain the lack
of sensitivity exhibited by colon carcinoma to platinum based
chemotherapy and highlights the need for extensive screening
of potential new anticancer agents in numerous cell lines.
Future efforts to improve the intracellular fate of anthra-
quinone–platinum complexes will need to improve DNA
targeting efficacy and evade the intra-lysosomal sequestration
in order to generate cytotoxicity. Modification of the ‘‘linker’’
group and substitution within the anthracycline moiety may
prove useful in ensuring that the drug reaches the nucleus.
Acknowledgements
This research was funded by a Cancer Research UK Program
grant (SP1861/0401).
The results in the present investigation clearly demon-
strate that sequestration of the novel Pt-1C3 drug within
lysosomes may limit its effectiveness as an anticancer drug.
Both Pt-1C3 and 1C3 were localised to the lysosomes and
displayed similar cytotoxicity profiles. The mechanism of Pt-
1C3 cytotoxicity is unlikely to involve DNA damage as the
results indicate that little, if any, of the compound reaches the
cell nucleus. Thus, the toxicity was due to lysosomal
accumulation, most probably leading to disruption. This
phenomenon has previously been observed as a late devel-
opment in the cascade of toxicity produced by cisplatin in
renal cells [36] and neural ganglia [37]. Conversely, the
accumulation of platinum complexes within lysosomes and
other acidic compartments is a well-established detoxification
mechanism [38] and has been implicated in mediating
resistance to protonatable drugs such as daunorubicin
[39,40]. The sequestration of drug away from the nucleus
and thus preventing attainment of sufficient drug concentra-
tion at the target region is readily classified as a pharmaco-
kinetic means of conferring resistance. There are two possible
mechanisms to account for the accumulation of Pt-1C3 within
the lysosomes. Firstly, the presence of a protonatable primary
amine group will render the compound susceptible to pH-
dependent accumulation within the highly acidic internal
environment of lysosomes. Alternatively, the relative amphi-
philicity of Pt-1C3 will enable rapid partitioning of the
compound in the plasma membrane, but prevent diffusion
through the bilayer core. Subsequent membrane internalisa-
tion will traffic Pt-1C3 to the lysosomal compartment via early
and late endosomes. Regardless of the precise mechanism, the
ultimate consequence is sequestration of the compound away
from the nuclear compartment.
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