The cisplatin-based Pt(iv)-diclorofibrato multi-action anticancer prodrug exhibits excellent performances also under hypoxic conditions

Article abstract of DOI:10.1039/c7dt04614f

Multi-action cisplatin-based mono- (1) and di-clofibric acid (2) Pt(iv) "combo" derivatives were synthesized via both traditional and microwave assisted procedures. The two complexes offered very good performances (IC₅₀ values in a nanomolar ra

Full text of DOI:10.1039/c7dt04614f

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Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
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The cisplatin-based Pt(IV)-diclorofibrato multi-action anticancer
prodrug exhibits excellent performances also in hypoxic
conditions.§
Received 00th January 20xx,
Accepted 00th January 20xx
Elisabetta Gabano,a* Mauro Ravera,a Francesca Trivero,a Stefano Tinello,a Andrea Gallina,a Ilaria
Zanellato,a Marzia B. Gariboldi,b Elena Monti,b and Domenico Osellaa
DOI: 10.1039/x0xx00000x
www.rsc.org/
Multi-action cisplatin-based mono- (1) and di-clofibric acid (2) Pt(IV) “combo” derivatives were synthesized via both
traditional and microwave assisted procedures. The two complexes offered very good performances (IC50 values in
nanomolar range) on a panel of human tumor cell lines, including the highly chemoresistant malignant pleural
mesothelioma ones. Moreover, both 1 and 2 bypass the cisplatin resistance. Indeed, cisplatin and clofibric acid, the
metabolites of the Pt(IV)→Pt(II) intracellular reduction, proved to act synergistically. The adjuvant action of clofibric acid
relies on the activation of peroxisome proliferator-activated receptor α (PPARα) that, in turn, decreases the level of
Hypoxia-Inducible Factor-1α. Both compounds induced extensive apoptosis in tumor cells, also via oxidative stress. Finally,
2 exhibited excellent performances also under the hypoxic conditions typical of solid tumors, where cisplatin is less
effective.
In this context, the well-established Pt(IV) chemistry
permits to design dual- or even multi-functional drug
candidates that act as combination-therapy single-molecule,
Introduction
Although targeted- and immuno- therapies are nowadays
considered among the most promising developments in
fighting cancer, conventional cytotoxic chemotherapy, in
particular the Pt-based one, still represents the first-line
armory against very aggressive cancers.
called “combo”.2-6 One or two adjuvant/synergistic agents
(generally containing carboxylic functions) can be conjugated
to the octahedral Pt(IV) assembly (generally based on the
cisplatin square-plane scaffold) in axial positions. These Pt(IV)
derivatives are considered prodrugs, because they can reach
tumor cells in their intact form and then can be reduced in the
hypoxic (reducing) intracellular milieu to cytotoxic cisplatin,
with the simultaneous loss of the two axial ligands (activation
by reduction).6-9 Importantly, Pt(IV) complexes are generally
much more lipophilic than their progenitors, i.e. hydrophilic
cisplatin and amphiphilic carboxylate anions (at physiological
pH). This means that their assembly permits a more efficient
cellular uptake (via passive diffusion) than that of the separate
components (synergistic cellular accumulation).10
In principle, there are many opportunities to target and kill
cancer cells, because they use multiple and redundant
pathways to survive. The simplest approach to a simultaneous
attack on different molecular targets/mechanisms is the
combination chemotherapy, where two or more drugs act in
additive or (better) synergistic way. To improve patient
compliance, different drugs are incorporated into the same
formulation; however, combining multiple medicaments in a
single preparation can be challenging, because of
pharmacokinetic differences between the individual
components. Alternatively, a single chemical entity, containing
highly integrated different pharmacophores, can be designed.1
The auxiliary drug selected for the present work is (2-(p-
chlorophenoxy)-2-methylpropionic acid (clofibric acid, CA).
Fibrates are widely used in the treatment of dyslipidemia. The
CA mechanism of action relies on activation of peroxisome
proliferator-activated receptor α (PPARα), a member of the
PPAR nuclear receptor superfamily. PPARα controls the fatty
acid metabolism. Interestingly, PPARα agonists have been
reported to activate apoptotic cell death in different cancer
cell lines through different and sometimes contrasting
biochemical mechanisms.11 In particular, a brief report claimed
(data not shown in such a paper) that combination treatment
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in vivo with CA and cisplatin produces efficient antitumor spheroid (3D) cultures obtained from non-small cell lung
effects on ovarian cancer cells.12
cancer cells the cytotoxicity of cisplatin-based Pt(IV) complexes
Furthermore, the activation of PPARα by CA is able to was unaffected (but not increased) under hypoxic conditions.18
promote the degradation of Hypoxia-Inducible Factor-1α (HIF- More recently, Keppler et al. showed that the antiproliferative
1α),13 the oxygen-dependent subunit of the transcription potency is almost identical in normoxic and hypoxic conditions
factor HIF-1, that acts as the master regulator of cellular for most of the Pt(IV) derivatives and in most of the 2D and 3D
adaptation to hypoxia.14 HIF-1 activation has been implicated cell lines investigated.19, 20
in solid tumor progression,15 as well as in poor response to
Noteworthy, renal dysfunction, that is an important
several chemotherapeutic protocols, including those adverse effect of cisplatin, was attenuated by co-treatment
employing cisplatin.16 Very recently, some Pt(IV) complexes with fibrates, albeit in doses much higher than that released
with indazole-based axial ligands, specifically targeting HIF-1
α
,
from the combo derivatives
In this context, one clofibrate and one acetate (compound
) or two clofibrates (compound ) were coordinated as axial
1 and 2
(Scheme 1).21, 22
exhibited moderately improved anticancer activity in hypoxic
with respect to normoxic conditions.17 The finding that
1
2
clofibrates induce PPARα activation and thus promote HIF-1α ligands in the Pt(IV) octahedral scaffold, completed with two
degradation suggests that combo Pt(IV)-CA mono- and di- ammonia molecules and two chlorides (cisplatin-based Pt(IV)
derivatives could offer interesting performances under hypoxic derivatives) (Scheme 1). These compounds were tested in vitro
conditions.
on several human tumor cell lines having different chemo-
In principle, the activity of all the Pt(IV) complexes should sensitivity (ovarian, testis, lung, colon, and breast cancer cells).
be favored by the hypoxic microenvironment encountered in Additionally, several malignant pleural mesothelioma (MPM)
solid tumors. Indeed, the reduction Pt(IV)
→Pt(II) should be cell lines were also tested. Moreover, the occurrence of
promoted under hypoxic (reducing) conditions, even if the synergy between cisplatin and CA was verified in vitro by
activity of the generated Pt(II) metabolite will be subsequently means of the combination index (CI) procedure. Finally,
hindered in the same milieu.12 The relationship between additional experiments were carried out using the most active
hypoxia and cytotoxicity of Pt(IV) prodrugs has not been compound 2 on two selected cell lines (namely A2780 and HCT
extensively investigated so far. However, in a seminal paper 116) under normoxic and hypoxic conditions.
Hambley et al. reported that in monolayer (2D) and tumor
Scheme 1. Synthetic pathways for compounds 1 and 2: i) H2O2 in CH3COOH (RT, 3-4 h); ii) H2O2 in H2O (microwave heating, 70 °C, 20 min); iii) clofibroyl chloride (in acetone +
pyridine, reflux overnight or microwave heating 55 °C, 1 h). The numbering scheme for the identification of the NMR signals is reported.
(cisplatin or CDDP), prepared according to the Dhara’s
24
Cisplatin was oxidized to the mono- and di-
method.23,
Results and Discussion
hydroxido intermediates 0A and 0B, using hydrogen peroxide
in acetic acid or in water, respectively.25, 26
Synthesis and characterization of Pt(IV) complexes
The following step of the syntheses involves the
substitution reaction of the axial hydroxido/s with the
carboxylic acid/s. However, to perform the substitution
All the complexes under investigation were synthesized
starting
from
(SP-4-2)-diamminedichloridoplatinum(II)
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reaction, a more reactive form of CA is necessary and, min of incubation, the [1H, 15N] HSQC spectra showed the
therefore, this acid was turned into its corresponding clofibroyl absence of the original signals of Pt(IV) complex (15N-1
δ = -
1
chloride using oxalyl dichloride in the presence of a catalytic 40.3 ppm and H
1
δ
= 6.4 ppm; 15N-2
δ
= -39.8 ppm and H
= -
amount of dimethylformamide, DMF. The reaction between
δ
= 6.4 ppm) and the presence of that of cisplatin (15NH3
δ
0B and clofibroyl chloride was more complicated than that 67.0 ppm with satellite peaks at -63.7 ppm and -70.2 ppm, 1JPt-
employing 0A, being the solubility of final complex and N = 330 Hz and 2JPt-H = 64 Hz; 1H
δ = 4.0 ppm).
unreacted CA very similar. This problem required slight
Antiproliferative activity and synergism
modification of the usual synthetic procedure (see
Experimental section).
The half-maximal inhibitory concentrations, IC50, were
Furthermore, in the present work, the microwave heating determined on a panel of human cancer tumor cells. Ovarian
was applied to the synthesis of 0B , and with neat A2780, testis NT2/D1, lung A549, colon HCT 116, breast MCF7
,
1
2
advantage in terms of speed, maintaining or even improving cancer cells, together with five malignant pleural
yield and purity of the traditional syntheses (see Experimental mesothelioma cells (BR95, MG06, MSTO-211H, and MM98, the
section). In the field of Pt compounds, the microwave-assisted latter both as wild type and cisplatin-resistant subline MM98R)
heating is rather unexplored. The successful application in this were challenged with
1 and 2, along with cisplatin and CA for
work is in tune with the few literature data reporting that comparison purpose (Table 1).
microwaves can speed up and/or make possible reactions that
Table 1. Antiproliferative data (IC50) of cisplatin, CA, and Pt(IV) complexes 1-2 (72 h
continuous treatment, CT, normoxic conditions: 21% O2). The panel of cancer cell lines
includes: ovarian carcinoma A2780, testicular cancer metastatic cell tumor NT2/D1,
lung carcinoma A549, colorectal cancer HCT 116, breast adenocarcinoma MCF7, and
five malignant pleural mesotheliomas (epithelioid phenotypes BR95 and MG06,
biphasic MSTO-211H, sarcomatoid MM98, and its cisplatin-resistant subline MM98R).
Data are reported as means ± standard deviation of at least three independent
replicates.
hardly occur when thermally driven.23, 27-30
Complexes and
were also obtained starting from 15NH3-
1
2
labeled cisplatin.31 These 15N-containing complexes are
extremely useful to follow the reduction reactions of the Pt(IV)
prodrugs challenged with cytosolic extract.8, 26, 32 According to
Gibson et al., this test seems to be more realistic than the
common kinetic study with models of bio-reductants such as
glutathione or ascorbic acid, since the in vivo reduction of
Pt(IV) complexes seems to be mainly mediated by cytosolic
macromolecules.8, 32, 33
IC50 [µM]
Cell lines
Cisplatin
0.5 ± 0.1
0.11 ± 0.05
3.6 ± 0.9
2.3 ± 0.3
6.5 ± 0.9
6.2 ± 0.9
4.1 ± 1.5
1.4 ± 0.3
3.2 ± 1.0
19.4 ± 2.8
6.1
CA
1
2
A2780
NT2/D1
A549
(1.0 ± 0.2) × 103
(2.8 ± 0.6) × 103
(1.4 ± 0.3) × 103
(1.2 ± 0.2) × 103
(1.4 ± 0.2) × 103
(0.95 ± 0.03) × 103
(1.6 ± 0.6) × 103
(1.0 ± 0.4) × 103
(4.6 ± 0.9) × 103
(3.6 ± 0.3) × 103
0.8
0.081 ± 0.006
0.031 ± 0.002
0.86 ± 0.03
0.31 ± 0.08
1.1 ± 0.1
0.028 ± 0.006
0.022 ± 0.008
0.12± 0.04
0.042 ± 0.009
0.26 ± 0.02
0.44 ± 0.06
0.5 ± 0.1
Finally,
1 and 2 were characterized by RP-HPLC-ESI-MS and
multinuclear (1H, 13C, 195Pt and 15N) NMR spectroscopy using
both mono- and bi-dimensional techniques, such as
heteronuclear single quantum coherence spectroscopy
(HSQC), to assign the whole set of signals (ESI contains the
most relevant mono- and bi-dimensional NMR spectra). In
general, the 1H and 13C NMR spectra of the Pt complexes show
a high-frequency shift of the signals of the atoms nearer to the
metal center with respect to the free ligands, pointing out the
occurred coordination.
HCT 116
MCF7
BR95
1.66 ± 0.06
2.6 ± 0.2
MG06
MSTO-211H
MM98
MM98R
RFa
0.4 ± 0.2
0.041 ± 0.007
0.23 ± 0.05
0.24 ± 0.02
1.0
1.49 ± 0.09
1.7 ± 0.5
1.2
a resistance factor RF = IC50(MM98R) / IC50(MM98)
195Pt and 15N NMR spectroscopy provides information on
the oxidation state of the metal and on the nature of the
coordinated ligands.34-38 Accordingly, the 195Pt NMR signals of
1
CA showed a modest antiproliferative activity when
employed as a single agent, with millimolar IC50 values on all
the cell lines tested. On the contrary, cisplatin exhibited IC50
and
2 exhibit chemical shifts around 1050-1250 ppm well-
suited with the presence of two chlorides, two carboxylates
and two amines coordinated to Pt(IV) in an octahedral
geometry.26, 39-41 The 15N NMR signals of 15N-labeled complexes
values in the low micromolar range, whereas
one-two orders of magnitude more active than cisplatin,
depending on the chemosensitivity of the cell type. Both and
were able to bypass cisplatin resistance with resistance
factors (RF = IC50(MM98R) / IC50(MM98) near to 1. While the
antiproliferative potency of is similar to that of the mixture
of free drugs, the antiproliferative potency of is much higher
1 and 2 were
1
1
and 2 exhibit chemical shifts around -40 ppm compatible
with the presence of chlorides trans to 15NH3 in Pt(IV)
2
complexes.38,36
1
Solution behavior and reduction
2
(Figure 1a), in tune with a higher cellular accumulation (see
later).
Complexes
HEPES
1
and
2
proved to be stable towards hydrolysis in
(HEPES 4-(2-hydroxyethyl)-1-
buffer
=
The evaluation of the pharmacological interaction between
the two metabolites produced by reduction into the cells,
cisplatin and CA, was performed by using the combination
piperazineethanesulfonic acid, pH = 7.5) with methanol as a
co-solvent for one week at 37 °C, as verified by means of HPLC-
MS (see Experimental for details).
45
index (CI).44, This method evaluates the effect of two (or
The reduction of 15N-labeled complexes
1 and 2 was
followed by 15N NMR spectroscopy using cell extracts from
A2780 ovarian cancer cells (see ESI, Figures S16-S19). After 30
more) drugs and quantifies synergism or antagonism by
determining how the combination effect differs from the
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simple additive effect. The CI equation, which considers both Therefore, the high concentration of such a drug is essential to
the potency and the shapes of the dose-effect curves, is used guarantee its active intracellular level. On the contrary, when
to precisely understand drug combinations: CI ≅ 1 indicates an the two drugs, i.e. hydrophilic cisplatin and amphiphilic
additive effect, CI < 1 and a CI > 1 indicate synergism and clofibrate, are ideally assembled in a lipophilic Pt(IV) scaffold
antagonism, respectively (see Experimental section).
(Scheme 2), synergistic accumulation into the cells can be
43
For this reason, complexes 1 and 2 have
exploited.42,
behaviour similar to or even better than the mixture of free
cisplatin and CA (Figure 1a).
Scheme 2. The lipophilic complex 2 may be regarded as the combination of hydrophilic
and amphiphilic moieties.
Apoptosis induction
It is well recognized that the failure of many chemotherapeutic
treatments arises from the inability to induce apoptosis in
cancer cells. The adducts formed by DNA-platination produce
the sequential activation of several signal transduction
47
The
pathways that culminate in apoptotic cell death.46,
induction of apoptosis (both via intrinsic and extrinsic
pathway) is generally associated with the activation of
caspases 3 and 7. Therefore, the measurement of their activity
is a convenient way to assess whether the cells are undergoing
apoptosis concomitant to proliferation arrest. To elucidate if
1
and 2 induced apoptosis in A2780 cells, the caspase 3/7
activity was measured with a DEVD-based fluorogenic assay
(DEVD = Asp-Glu-Val-Asp). Figure 2 shows the activity of
Figure 1. a) Concentration–response curves of A2780 cells challenged for 72 h with CA
(black squares), cisplatin (red dots), mixture 1:1000 cisplatin/CA, (magenta upward
triangles), 1 (green diamonds), 2 (blue downward triangles). Data are means ± standard
deviation of a representative experiment. Residual viability was assessed by means of
caspase 3/7 when the A2780 cells are challenged with
1, 2,
cisplatin, and CA at increasing concentrations. The ability of
the resazurin reduction assay and data were fitted with a four-parameter sigmoidal activation of caspase 3/7 was in the order:
function (black, red, magenta, green and blue lines, respectively). b) Combination index
2 > 1 > cisplatin >>
CA. Thus, the di-CA complex 2 was able to trigger apoptotic cell
(CI) plot of the mixture 1:1000 cisplatin/CA. Residual viability data were compared to
those obtained for cisplatin and CA to obtain the value. (CI < 1: synergism; CI ≅ 1
additive effect; CI > 1 antagonism).
death more effectively than the mono-CA complex
cisplatin.
1 and
Figure 1b clearly indicates that cisplatin and CA have a
synergistic action in the whole range of concentrations
employed, so that the two metabolites released within the
cells by the Pt(IV) complexes increase each other's
effectiveness.
The employed mixture of the free drugs is in 1:1000
cisplatin/CA ratio according to their IC50 values (Table 1 and
Figure 1). However, it has been previously shown that
bioactive carboxylic acids, such as valproic acid (VPA) present
as amphiphilic carboxylate at physiological pH, barely enter
cells. Indeed the intracellular concentration is about 3 orders
of magnitude lower than that originally administered.42
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the retention is due to partitioning between the C18 chains of
the stationary phase (representing the cellular membrane) and
the aqueous eluent (representing the water inside and outside
cells).40,
50
The retention times (tR) of the complexes were
determined on a C18 column by using a 30% aqueous formic
acid (15 mM) / 70% MeOH mixture as an eluent and the data
were expressed as log k’ (k’ = (tR - t0) / t0, where t0 is the
column dead-time). Recently, some models for the prediction
of the log Po/w values for both Pt(II) and Pt(IV) complexes have
been developed and made publicly available. The final and
recommended
model
(***ASNN5
model,
http://ochem.eu/article/76903) was here applied to the
studied Pt complexes.51 Finally, the Pt(IV) complexes were
tested for their solubility in water. Data are reported in Table
2.
Figure 2. Caspase-3/7 activity (fold increase) of A2780 cells treated with CA
(squares), cisplatin (dots), 1 (diamonds), 2 (triangles). Data, normalized to untreated
control and residual viability, are means ± standard deviations of three experiments
performed in triplicate.
Table 2. Solubility and lipophilicity data.
Complex Solubility [mM] log k’
Calculated log Po/w
cisplatin
7.7a
-0.50
-2.0
(experimental value -2.13)b
Apoptosis is characterized by chromatin condensation in
early phases and later by nuclear fragmentation.48 Accordingly,
an increasing number of A2780 nuclei characterized by
Hoechst 33342 (a DNA intercalating dye) fluorescence was
1
2
0.44
0.01
0.19
0.88
0.49
1.97
apparent water solubility;52 b mean experimental value of shake-flask literature
a
data.53-57
observed after 24 h in the order 2 > 1 > CA (10 µM 1 and 2, and
1 mM CA; Figure 3). In addition, a relevant fraction of nuclear
fragmentation (nuclei with the typical punctuate morphology)
As expected, solubility and lipophilicity are inversely
correlated and in tune with the chemical structures of the
complexes.
was observed only as a consequence of treatment with
tune with caspase activity (Figure 2).
2, in
The intracellular Pt accumulation of
1 and 2 was measured
as a key parameter in understanding the mechanism of action
of the complexes. This parameter, that is the final balance
between cellular influx and efflux,58 is here expressed as
accumulation ratio (AR), the ratio between the intracellular Pt
concentration and the extracellular (in the culture medium) Pt
concentration at a given time.59 The AR values were measured
on A2780 cells after 4 h of continuous treatment (CT) with 10
µM concentrations of the Pt compounds (Figure 4). The AR
trend is in agreement with the lipophilicity data.
Finally, the platination of genomic DNA, expressed as pg Pt
µg-1 DNA, is reported in Figure 4. By comparing Figure 4a and
Figure 4b it is clear that the DNA platination, and hence the
antiproliferative potency, is roughly proportional to the
cellular accumulation.
Figure 3. Chromatin condensation and fragmentation of A2780 cell nuclei following
24 h (1 mM CA or 1 or 2), along with the untreated control (NT). Pictures were taken by
a fluorescent microscope (standard DAPI filters, 200X magnification) on living cells
immediately after Hoechst 33342 staining.
Lipophilicity, cell uptake and DNA platination
It has been reported that Pt(IV) complexes enter cells mainly
(if not exclusively) by passive diffusion.49 For this reason,
lipophilicity is a key feature for such a process. In a first
attempt to determine the lipophilicity of the complexes under
study, the traditional octanol-water shake-flask method was
employed, but the rather low water solubility of the complexes
prevented the achievement of reliable and reproducible data.
Therefore, this method was substituted using both a HPLC
procedure and in silico calculations. RP-HPLC techniques are
often used to measure the lipophilicity of a compound, since
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oxidase 1 (ACOX1) and carnitine palmitoyltransferase 1A
(CPT1A), the key players of fatty acid oxidation.61 Figure 5
shows that all the Pt complexes induced a striking increase
(p < 0.001 vs. control) of ACOX1 and CPT1A. For ACOX1, the
results seem to indicate the primary role of CA, as far as gene
upregulation increases from 12 for cisplatin to about 27 for
1
and 2. In addition, the modulation of genes ruling the
antiproliferative and cytotoxic activity (cyclins (A, E, and D1),
p53, and p21) was also investigated. A strong upregulation of
cyclins was observed following treatment with cisplatin (Figure
5). This effect was not balanced by the (expected, but
limited)62 downregulation induced by CA in
1 and 2. Thus, both
Pt(IV) compounds showed a cyclin upregulation similar to that
of cisplatin. The strong upregulation of p53 confirms this
observation.
Figure 4. a) Accumulation ratio, AR, and b) DNA platination measured on A2780 ovarian
cancer cells after treatment with 10 ꢀM concentrations of cisplatin (CDDP) and
complexes 1 and 2 for 4 h of continuous treatment. Data are means ± standard
deviations of three experiments performed in triplicate and were compared to cisplatin
by means of a two-tailed t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).
On the contrary, p21 was upregulated in the order
2 > 1 > cisplatin > CA, i.e. the same trend observed for
apoptosis induction. In fact, the expression of p21 increases in
response to DNA damage, that induces cell cycle arrest.63
Another gene expected to be upregulated by CA treatment
is heme oxygenase (HMOX1), albeit in a PPARα-independent
way.60 Figure 5 shows that HMOX1 was strikingly upregulated
Gene modulation
In order to shed more light on the biochemical mechanisms
operating for
1 and 2, a quantitative polymerase chain
reaction, by reverse transcription quantitative PCR (RT-qPCR)
analysis, was carried out. With the aim to perform equitoxic
treatments, the IC50 values were calculated from the
also by cisplatin, but, to a larger extent, by the combos
. Since this gene is usually increased by oxidative stress,
reactive oxygen species (ROS) were evaluated in the same time
interval (24 h).
1 and
2
corresponding dose-response curves (24 h CT) of
with free CA and cisplatin.
1, 2, along
It was shown that CA activates PPARα in A2780 cells.60 This
transcription factor upregulates target genes, such as acyl-CoA
Figure 5. Relative gene expression of ACOX1, CPT1A, Cyclin D1, E, A, p21, p53, HMOX1 in A2780 cells following equitoxic 24 h treatments with CA (5 mM, black bars), cisplatin (30
µM, red bars), 1 (6 µM green bars), 2 (2 µM blue bars). The RT-qPCR experiment was performed in triplicate, and results were normalized to references genes and untreated
control (white bars). All data are means ± standard deviations and were compared to untreated control by means of a one-way ANOVA test (at least p < 0.05, except ACOX1 and
CPT1A after CA treatment).
ROS induction
negligible increase of ROS after CA treatment. On the contrary,
treatment with Pt complexes increased ROS in the order
The levels of ROS were measured by means of the
dichlorofluorescein diacetate (DCF) assay. Figure 6 shows a
6 | Dalton Trans., 2012, 00, 1-3
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64
1
>
2
≅
expression.
cisplatin,
in tune with the trend of HMOX1 As mentioned above, tumor hypoxia leads to chemoresistance
in many, but not in all tumor cell lines.13, 19, 65-73 This can be
related to an increased expression and activity of hypoxia-
induced HIF-1α, a well-known regulator of tumor growth,
angiogenesis, and metastasis. Treatment with CA and other
fibrates
is
able
to
diminish
HIF-1α,
restoring
chemosensibility.13
The influence of hypoxia was evaluated by assessing the
viability of A2780 and HCT 116 cells following exposure to the
more active compound
2 and to the reference parent
compounds cisplatin and CA, under normoxic (21% O2) or
hypoxic (1% O2) conditions. Preliminary control experiments
indicated that the maximum period of hypoxia well tolerated
by these cells is 48 h. Accordingly, the IC50 values were
calculated from the corresponding dose-response curves (48 h
CT) and reported in Table 3. Obviously, compared to the
standard treatment time (72 h CT), the potency is lower
(higher IC50), as expected for genotoxic drugs that require
some population doubling times for working at the best. A 2-3
time increase of IC50 was observed on passing from normoxic
Figure 6. Fold ROS induction compared to untreated control (NT). A2780 cells were
treated with 1 mM CA, 1 µM cisplatin (CDDP), 1, and 2 for 24 h. H2O2 (+) was used as
positive control of the DCF assay. Data are means ± standard deviations (n = 5) and
were compared to control by means of a one-way ANOVA test with Dunnett’s post test
(* p < 0.5; ** p < 0.01; *** p < 0.001).
to hypoxic conditions for cisplatin. On the contrary,
2 exhibits
moderately better performances on both cell lines in hypoxic
when compared to normoxic conditions.
Evaluation of the percentage of apoptotic cells was carried
out on the same cell lines after 48 h CT with cisplatin and
flow cytometric analysis (Figure 7).
2 by
Hypoxia vs. normoxia
Being
2 the most potent of the two newly synthesized
compounds, the following biological tests were limited to it.
Table 3. Antiproliferative effect of CA, cisplatin and 2 on A2780 and HCT 116 cells following 48-h exposure under normoxic or hypoxic conditions. IC50 values are the
means ± standard error mean of 3-5 independent experiments.
IC50 [ꢀM], 48 h
Cell lines
CA
hypoxic
Cisplatin
2
normoxic
ratio normoxic hypoxic ratio
Normoxic
hypoxic
ratio
0.6
A2780
(5.3 ± 0.6)
×
×
103 (8.7 ± 0.2)
103 (5.1 ± 0.1)
×
×
103
103
1.6
0.8
1.7 ± 0.6
12 ± 5
5.4 ± 0.9
22 ± 5
3.2
1.9
0.05 ± 0.02 0.03 ± 0.02
0.4 ± 0.1 0.3 ± 0.1
HCT 116 (6.2 ± 0.7)
0.7
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Dalton Transactions
Figure 8. Effect of equitoxic treatments (48 h) with cisplatin, CA, and 2 on HIF-1α
protein level in A2780 (A) and HCT 116 (B) cells. C = control, N = normoxic conditions, H
= hypoxic conditions. β-tubulin served as the loading control.
Conclusions
Cisplatin and CA show a synergistic antiproliferative activity.
Their “combo” derivatives, namely the cisplatin-based Pt(IV)
mono-CA
1
and especially di-CA
2,
offer excellent
performances on a number of human tumor cell lines. This
result relies mainly on the synergistic cellular accumulation of
both drugs by virtue of the lipophilicity of the Pt(IV)
conjugates. This is confirmed by the analysis of the gene
expression in normoxia that is mainly influenced by the
cytotoxic action of the released cisplatin, albeit a contribution
Figure 7. Flow cytometric assessment of the percentage of apoptotic A2780 (top) and
HCT 116 (bottom) cells following 48-h CT under normoxic (white bars) and hypoxic
(grey bars) conditions with cisplatin and 2 at their respective normoxic IC50 values.
Values are the means ± standard error means of 2-4 independent experiments. a:
p < 0.05 vs. control; b: p < 0.01 vs. control; c: p < 0.01 vs. control and cisplatin.
of CA was observed as well. Both
apoptosis in a more efficient way than free cisplatin. The
contribution of CA to the antiproliferative propensity of , the
1 and 2 are able to trigger
In ovarian A2780 cells, cisplatin induced a significant
increase of apoptotic cells under normoxic, as compared to
hypoxic conditions. On the contrary, no significant differences
in apoptosis between normoxic and hypoxic conditions were
2
most potent combo, is more evident under hypoxia. In fact,
the released CA induces the HIF-1α degradation thus
observed for
2. The HCT 116 cell line exhibits a different
augmenting the ability of
2 to bypass the well-known hypoxia-
behavior: i) a smaller increase in apoptosis cells was observed
in response to cisplatin treatment, a well-known phenomenon
that can be attributed to a deficit in mismatch repair in this cell
line;74 ii) a significant increase in apoptotic cells was observed
related cisplatin resistance.
Experimental
when
2 was employed in hypoxic conditions.
Cell cycle analysis (Fig. S20, see ESI) indicates that
2
General procedures
significantly increased the percentage of cells in G2/M as
compared to control and cisplatin, with a corresponding
decrease of the G1 sub-population in both A2780 and HCT 116
cells under normoxic conditions. This behavior was maintained
in HCT 116 cells following hypoxic incubation. Except for this,
cell distribution throughout the different phases was
substantially unaffected by oxygen levels.
K2[PtCl4] and all other chemicals (Alfa Aesar and Sigma-Aldrich)
were used without further purification. Complex (OC-6-44)-
acetatodiamminedichloridohydroxidoplatinum(IV)
(0A) was
prepared according to an already published procedure.26 The
reactions under microwave irradiation were performed by
using a CEM Discover® SP System equipped with a focused
single mode and self-tuning cavity, an air cooling system, an
automated power control based on temperature feedback,
supplying power in 1 W increments from 0 to 300 W.
The ability of the released CA to decrease HIF-1α level was
verified by Western blot analysis after equitoxic treatments of
CA, cisplatin and
2 under hypoxia. Figure 8 indicates that 2
The purity of all the compounds was assessed by analytical
RP-HPLC (see below), elemental analysis and determination of
Pt content. Elemental analyses were carried out with an
EA3000 CHN Elemental Analyzer (EuroVector, Milano, Italy).
For all the compounds, the experimental values of the
elemental analyses correspond to calculated values within
induced a more marked degradation of HIF-1α, with respect to
the references, as expected for the synergistic contribution of
cisplatin and CA. This effect explains the ability of
2 to work
under hypoxic conditions, restoring the HIF-1α level observed
in normoxia.
8 | Dalton Trans., 2012, 00, 1-3
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±0.4%. Platinum content determination was performed by mixture was stirred overnight at room temperature. The
inductively coupled plasma-optical emission spectrometry solvent and excess chloride were removed under reduced
(ICP-OES Spectro Genesis equipped with a crossflow nebulizer, pressure (60 °C) to quantitatively yield a yellow/orange oil.
Spectro Analytical Instruments, Kleve, Germany) or inductively Elemental analysis: found C, 51.22; H, 4.45. Calc. for
1
coupled plasma-mass spectrometry (ICP-MS, Thermo Optek X C10H10Cl2O2 (233.09) C, 51.53; H, 4.32. H NMR (500.13 MHz,
1.64 (s, 6H, H5), 6.85 (d, 2H, H7, 3J = 8.95 Hz), 7.24
Series 2), accordingly to the platinum content of the sample DMSO-d6): δ
3
and the sensitivity of the chosen instrument. Mineralization of (d, 2H, H8, J = 8.95 Hz) ppm. 13C NMR (125.76 MHz, DMSO-
the samples was performed by the addition of 70% w/w HNO3 d6): 25.1 (C5), 85.6 (C4), 121.2 (C7), 128.6 (C9), 129.6 (C8),
δ
to each sample, followed by incubation for 1 h at 60 °C in an 153.1 (C6), 178.3 (C1) ppm.
ultrasonic bath. Before the ICP-OES or ICP-MS measurements,
Synthesis of complex 1
the HNO3 was diluted to a final 1% v/v concentration. In order
to measure the platinum concentration by ICP-OES, the The oily clofibroyl chloride previously obtained (1.33×10-3 mol,
299.797 nm line was selected. A Pt standard stock solution of 10 eq) was dissolved in anhydrous acetone (3 mL) and added
1000 mg L-1 was diluted in 1.0% v/v nitric acid to prepare to a suspension of complex 0A (0.05 g, 1.33×10-4 mol, 1 eq) in
calibration standards. Instrumental ICP-MS settings were anhydrous acetone (2 mL). Excess pyridine (50 μL, 4 eq) was
optimized in order to yield maximum sensitivity for platinum. added dropwise to the reaction mixture which was then stirred
For quantitative determination, the most abundant isotopes of and heated at reflux overnight. The bright yellow/orange
platinum and indium (used as internal standard) were solution was evaporated under reduced pressure and the
measured at m/z 195 and 115, respectively.
resulting oil was treated with hexane, diethyl ether and finally
The NMR spectra were measured on a NMR-Bruker water to yield 0.046 g (60%) of a yellowish powder for complex
Advance III operating at 500 MHz (1H), 125.7 MHz (13C) and
1. Elemental analysis: found C, 24.98; H, 3.37; N, 4.51; Pt,
107.2 MHz (195Pt with a spectral window of 2000 ppm), 34.22. Calc. for C12H19Cl3N2O5Pt (572.73) C, 25.17; H, 3.34; N,
1
respectively. H and 13C NMR chemical shifts were reported in 4.89; Pt, 34.06. 1H NMR (500.13 MHz, DMSO-d6):
δ 1.45 (s, 6H,
parts per million (ppm) referenced to solvent resonances. 195Pt H5), 1.93 (s, 3H, H1), 6.53 (m, 6H, NH3), 6.90 (d, 2H, H7, J =
NMR spectra were recorded using a solution of K2[PtCl4] in 8.90 Hz), 7.20 (d, 2H, H8, J = 8.95 Hz) ppm. 13C NMR (125.76
3
3
saturated aqueous KCl as the external reference. The shift for MHz, DMSO-d6):
K2[PtCl4] was adjusted to -1628 ppm from Na2PtCl6 ( = 0 ppm). 124.7 (C9), 128.6 (C8), 154.3 (C6), 177.9 (C2), 180.7 (C3) ppm.
15N NMR spectra were recorded using a solution of 15NH4Cl in 195Pt NMR (107.51 MHz, DMSO-d6):
1208 ppm. ESI-MS
δ 22.6 (C1), 25.7 (C5), 79.7 (C4), 120.7 (C7),
δ
δ
1 M HCl as the external reference. [1H, 15N] HSQC spectra (positive ion mode): 574.1 m/z. Calc. for [C12H20Cl3N2O5Pt]+
(Heteronuclear Single Quantum Correlation) were obtained 573.7 m/z [M+H]+.
with the standard Bruker sequence hsqcetgpsiz with 0.2 s
Microwave assisted synthesis of complexes 1
acquisition time, 8 scans, 1.3 s relaxation delay, and 128 F1
points. DEPT-45 (Distortionless Enhancement by Polarization A solution in anhydrous acetone of clofibroyl chloride
Transfer) spectra were recorded with 100 scans, 3.5 s (1.33×10-3 mol, 10 eq) was added in a microwave vessel to a
1
relaxation delay, 0.5 s acquisition time and 75 Hz for J(15N, suspension of complex 0A (0.05 g, 1.33×10-4 mol, 1 eq) in
1H).
anhydrous acetone to obtain a maximum volume of 5 mL.
Chromatographic analyses were carried out using a C18 Excess pyridine (50 μL, 4 eq) was added dropwise to the
Phenomenex Phenosphere-NEXT (5-ꢀm, 250×4.6 mm ID) stirring reaction mixture and the vessel was capped and
column on a Waters HPLC-MS instrument (equipped with introduced into the microwave cavity. The microwave unit was
Alliance 2695 separations module, 2487 dual lambda programmed to heat the vessel content to 55 °C over a 5-min
absorbance detector, and 3100 mass detector). The UV-visible ramp period and then hold at this temperature for 1 h; the
detector was set at 210 nm. Mass spectra were recorded using power was automatically set at 50 W. During this time, the
source and desolvation temperatures set to 150 and 250 °C, mixture was stirred with a magnetic bar. After heating, the
respectively, with nitrogen used both as a drying and as a vessel was allowed to cool to room temperature before
nebulizing gas. The cone and the capillary voltages were removing it from the cavity. The solution obtained was
usually +30 V (positive ion mode) or -30 V (negative ion mode) transferred in a round bottomed flask and dried under
and 2.70 kV, respectively. Quasi-molecular ion peaks [M+H]+, vacuum. The resulting oil was treated with hexane, diethyl
[M+Na]+, [M-H]- and/or [M+Cl]- were assigned on the basis of ether and then water to yield a yellowish powder. Yield: 0.052
the m/z values and of the simulated isotope distribution g, 68%.
patterns.
Microwave assisted synthesis of complex 0B
Cisplatin (0.083 g, 2.77×10-4 mol, 1 eq) was suspended in 4 mL
Synthesis of the clofibroyl chloride (2-(4-chlorophenoxy)-2-
methylpropanoyl chloride)
of milliQ water in a microwave vessel and a wide excess of
Clofibric acid (CA, 1 eq) was dissolved in dichloromethane and hydrogen peroxide 35% w/w (0.6 mL, 1.99×10-2 mol, 70 eq)
one drop of dimethylformamide (DMF), as catalyst, was added. was added dropwise. The vessel was capped and introduced
While the solution was stirring, oxalyl dichloride (5 eq) was into the microwave cavity. The microwave unit was
added dropwise and effervescence was observed. The reaction programmed to heat the vessel content to 70 °C over a 5-min
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ramp period and then hold at this temperature for 15 min; the The syntheses of complexes
Dalton Transactions
1
and
2
containing 15N ammonia
75
power was automatically set at 15 W. During this time, the were the same of those containing 14NH3,25,
but starting
mixture was stirred with a magnetic bar. After heating, the from cis-[PtCl2(15NH3)2].31 The relevant characterization data
vessel was allowed to cool to room temperature before are reported below.
removing it from the cavity. The solution obtained was 15N-1. Elemental analysis: found C, 25.32; H, 3.11; N, 5.34; Pt,
transferred in a round bottomed flask and dried under 33.67. Calc. for C12H19Cl315N2O5Pt (574.71) C, 25.08; H, 3.33; N,
vacuum. The resulting yellow oil was treated with ethanol first 5.22; Pt, 33.94. 15N NMR (50.70 MHz, H2O):
δ -40.3 with
and then diethyl ether to yield a bright yellow powder (yield: satellite peaks at -37.7 ppm and -42.9 ppm (1J15N-195Pt = 261.2
2
0.085 g, 92%). Elemental analysis: found H, 2.33; N, 8.45; Pt, Hz, J1H-195Pt = 55.0 Hz) ppm. ESI-MS (positive ion mode): 576.0
58.16. Calc. for Cl2H8N2O2Pt (334.06) H, 2.41; N, 8.39; Pt, 58.40. m/z [M+H]+. Calc. for [C12H20Cl315N2O5Pt]+ 575.7 m/z [M+H]+.
195Pt NMR (107.51 MHz, D2O): 860 ppm. ESI-MS (negative ion 15N-2. Elemental analysis: found C, 32.70; H, 3.43; N, 4.32; Pt,
δ
mode): 333.0 m/z [M-H]-. Calc. for [Cl2H7N2O2Pt]- 332.9 m/z 26.58. Calc. for C20H26Cl415N2O6Pt (729.31) C, 32.94; H, 3.59; N,
[M-H]-.
4.11; Pt, 26.75. 15N NMR (50.70 MHz, H2O):
δ -39.8 ppm. ESI-
MS (positive ion mode): 730.3 m/z [M+H]+. Calc. for
[C20H27Cl415N2O6Pt]+ 730.0 m/z [M+H]+.
Synthesis of complex 2
The oily acyl clofibroyl chloride (3.74×10-4 mol, 2.5 eq) was
dissolved in anhydrous acetone (3 mL) and added to a
suspension of 0.05 g (1.50×10-4 mol, 1 eq) of complex 0B in The solution behavior of the investigated complexes (0.5 mM
Solution behavior and reduction
anhydrous acetone (2 mL). Excess pyridine (50 μL, 2 eq) was for complex
1
, 0.3 mM for complex
2
) was studied in 2 mM
4-(2-hydroxyethyl)-1-
added dropwise to the reaction mixture, which was then HEPES buffer
(HEPES
=
stirred and heated at reflux overnight. The yellow/orange piperazineethanesulfonic acid, pH = 7.5) with a variable
suspension was dried under vacuum and the resulting oil was amount of methanol (from 10% to 80% depending on the
treated with hexane first, then with a solution 0.1 M of NaOH solubility of the complex) as a cosolvent along one week.
to eliminate the excess clofibric acid and finally water to yield
The reduction of the complexes (0.5 mM for complex
1 and
0.039 g (36%) of a yellowish powder. Elemental analysis: found ≤ 0.1 mM for complex
2
) with ascorbic acid (AA:Pt molar ratio
C, 32.80; H, 3.29; N, 3.72; Pt 26.52. Calc. for C20H26Cl4N2O6Pt = 10:1) was studied in HEPES (2 mM, pH 7.5)/MeOH 50/50 at
1
(727.32) C, 33.03; H, 3.60; N, 3.85; Pt, 26.82. H NMR (500.13 37 °C. All these reactions were followed by monitoring the
MHz, DMSO-d6): δ 1.46 (s, 12H, H5), 6.56 (m, 6H, NH3), 6.93 (d, decrease of the area of the chromatographic peaks of the Pt
3
3
4H, H7, J = 8.95 Hz), 7.21 (d, 4H, H8, J = 8.95 Hz) ppm. 13C complexes in HPLC-UV-MS. The mobile phase was a mixture of
NMR (125.76 MHz, DMSO-d6): 25.7 (C5), 79.7 (C4), 120.9 15 mM aqueous HCOOH and CH3OH in a ratio depending on
(C7), 124.8 (C9), 128.6 (C8), 154.3 (C6), 180.6 (C3) ppm. 195Pt the lipophilicity of the complex (aqueous formic acid/methanol
δ
NMR (107.51 MHz, DMSO-d6):
δ
1196 ppm. ESI-MS (positive 30/70 for
1
, 20/80 for
For 15N NMR measurements, 20 µl of a 150-mM solution of
or saturated solution of in DMF were diluted in 450 µl cell
2).
ion mode): 728.3 m/z. Calc. for [C20H27Cl4N2O6Pt]+ 728.3 m/z
[M+H]+.
1
2
extract and D2O (30 μL) was added (final D2O content = 6%,
final [Pt] ≤ 3 mM) at 25 °C. The complete cell extract was
Microwave assisted synthesis of complex 2
A solution of clofibroyl chloride (3.74×10-4 mol, 2.5 eq) in prepared according to literature procedures32, 76 by lysing 160
anhydrous acetone was added in a microwave vessel to a million A2780 ovarian cancer cells in 1.6 mL of deionized water
suspension of complex 0B (0.05 g, 1.50∙10-4 mol, 1 eq) in followed by centrifugation to remove the insoluble material
anhydrous acetone to obtain a maximum volume of 5 mL. and by reducing the volume to 225 μL.
Excess pyridine (50 μL, 2 eq) was added dropwise to the
Lipophilicity and water solubility
stirring reaction mixture and the vessel was capped and
introduced into the microwave cavity. The microwave unit was Chromatographic analysis was used to evaluate the capacity
50, 77
programmed to heat the vessel content to 55 °C over a 5-min factors of the compounds as reported elsewhere.40,
ramp period and then hold at this temperature for 1 h; the Briefly, a chromatogram for each complex (0.5 mM) with
power was automatically set at 50 W. During this time, the eluant composition (15 mM formic acid/CH3OH 30/70) was run
mixture was stirred with a magnetic bar. After heating, the on a C18 column Phenosphere-NEXT (5-ꢀm, 250×4.6 mm ID).
vessel was allowed to cool to room temperature before The corresponding retention time tR was used to calculate
removing it from the cavity. The solution obtained was log k’ (k’ = (tR - t0) / t0); KCl was the internal reference to
transferred in a round bottomed flask and dried under determine the column dead-time, t0.
vacuum. The resulting oil was washed with hexane, then with a
The water solubility of the Pt(IV) complexes was
solution 0.1 M of NaOH to eliminate the excess clofibric acid, determined preparing their saturated solution in ultrapure
and finally with water to yield a yellowish powder. Yield: 0.041 water, which were stirred in the dark at 25 °C. After 24 h the
g, 37%.
solid residue was filtered off (0.20 µm cellulose filters) and the
Pt content of the solutions was determined by means of ICP-
OES.
Synthesis of 15NH3-containing complexes 1 and 2
10 | Dalton Trans., 2012, 00, 1-3
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Antiproliferative activity
Half-inhibiting concentration (IC50), defined as the
concentration of the drug reducing cell viability by 50%, was
obtained from the dose-response sigmoid using Origin Pro
(version 8, Microcal Software Inc., Northampton, MA, USA).
In order to verify the synergy between CA and cisplatin,
A2780 cells were treated with increasing concentrations of
cisplatin, CA, and a mixture cisplatin:CA in a fixed 1:1000 ratio,
according to their respective IC50 values. The experiment was
repeated three times. According to the method of Chou and
Talalay,45, 80, 81 the interaction between cisplatin and CA was
computed in terms of combination index (CI) for non-mutually
exclusive drugs by using the following equation:
The compounds under investigation were tested on a panel of
commercial human cancer cell lines: ovarian endometrioid
adenocarcinoma A2780 (ICLC HTL98008), colon carcinoma HCT
116 (ECACC 91091005), breast invasive ductal carcinoma MCF7
(ECACC 86012803), embryonal carcinoma of the testis NTERA-
2 clone D1 (also known as NT2/D1, ICLC HTL97025), lung
adenocarcinoma A549 (ICLC HTL03001), and biphasic
malignant pleural mesothelioma (MPM) MSTO-211H (ICLC
HL01018). Moreover, four cell lines derived from pleural
effusions of untreated MPM patients were used: BR95 and
MG06 (epithelioid phenotypes), MM98 (sarcomatoid
phenotype) and its cisplatin-resistant subline MM98R derived
from wild type by exposure to sub-lethal concentrations of
cisplatin for several months.78 The non-MPM cells and MSTO-
211H were purchased from European Collection of Cell
Cultures (ECACC, UK) or Interlab Cell Line Collection (ICLC,
Genova, Italy), whereas the other MPM cell lines were
obtained from the Hospital of Alessandria (Pathology Unit).
BR95 and MG06 cells were growth in Ham’s F10 cell
medium (GIBCO, Invitrogen Life Science, San Giuliano
Milanese, Italy), DMEM (Sigma-Aldrich) was used for MSTO-
211H, MM98 and MM98R, whereas A2780 NT2/D1 and A549
were grown in RPMI 1640, MCF7 in DMEM supplemented with
non-essential aminoacids, and McCoy’s 5A was used for HCT
116 cells. All media contained L-glutamine (2 mM), and were
supplemented with penicillin (100 IU mL-1), streptomycin (100
mg L-1) and 10% heat inactivated fetal bovine serum (FBS). Cell
culture and treatment were carried out at 37 °C in a 5% CO2
humidified chamber. Cells were challenged with the
compounds under study for 72 h of continuous treatment.
Cisplatin was dissolved in 0.9% w/v NaCl aqueous solution
brought to pH = 3 with HCl (final stock concentration 1 mM).
The Pt(IV) complexes were dissolved in ethanol (final stock
concentration 5 mM) and stored at -18 °C. The stock
concentration was confirmed with ICP-OES measurements.
The mother solutions were diluted in complete medium to the
required concentration range and, where present, the total co-
solvent concentration never exceeded 0.2% (this
concentration was found to be non-toxic to the cells tested).
To assess the growth inhibition of the compounds under
investigation, a cell viability test, i.e. the resazurin reduction
assay, was used. Briefly, 2-5×103 cells per well (depending on
the cell line) were seeded in black sterile tissue-culture treated
96-well plates. At the end of the treatment, viability was
assayed by 100 µg mL-1 resazurin (Acros Chemicals, France) in
fresh medium for 1 h at 37 °C, and the amount of the reduced
product, resorufin, was measured by means of fluorescence
ꢀ1ꢂ ꢀ2ꢂ ꢀ1ꢂꢀ2ꢂ
ꢀꢁ =
+
+
ꢀ1ꢃ ꢀ2ꢃ
ꢀ1ꢃꢀ2ꢃ
where C1 and C2 are the drug concentrations used in the mix
(C1m and C2m) or alone (C1a and C2a) to obtain the same level
of residual viability. Based on the actual experimental data, the
CI values were calculated by solving the equation over an
entire range of residual viability (from 5 to 95%, obtained from
the sigmoidal regression). These data were then used to
generate residual viability vs. CI plots, which is an effect-
oriented means of presenting synergism or antagonism.
Interpretation of CI values is defined such that CI = 1 indicates
an additive effect, and CI < 1 and a CI > 1 indicate synergism
and antagonism, respectively.
Cellular accumulation and DNA platination
A2780 cells were seeded in T25 flasks and allowed to grow
until around 80% confluence. Then, the treatment was
performed for 4 h with the complexes under investigations (10
μM) in complete medium. At time zero, 100 μL of medium was
taken out from each sample to check the extracellular Pt
concentration. At the end of the exposure, cells were washed
three times with phosphate buffered saline (PBS), detached
from the Petri dishes using 0.05% Trypsin 1X + 2% EDTA
(HyClone, Thermo Fisher) and harvested in fresh complete
medium. An automatic cell counting device (Countess®, Life
Technologies), was used to measure the number and the mean
diameter from every cell count. For cellular accumulation,
about 5×
106 cells were transferred into a borosilicate glass
tube and centrifuged at 1100 rpm for 5 min at room
temperature. The supernatant was carefully removed by
aspiration, while about 200 μL of the supernatant was left to
limit the cellular loss. Cellular pellets were stored at -20 °C
until mineralization and determination of the Pt content by
ICP-MS.
The level of Pt found in cells after drug treatment and
normalized upon the cell number (cellular Pt accumulation)
was expressed as ng Pt per 106 cells. The mean cellular
volume, calculated from the actual mean cell diameter
measured for every sample, was used to obtain the cellular Pt
concentration. The ratio between the cellular and extracellular
(in the culture medium) concentrations is defined as
accumulation ratio, AR.82
(excitation
λ = 535 nm, emission λ = 595 nm) with a Tecan
Infinite F200Pro plate reader (Tecan, Austria).79 In each
experiment, cells were challenged with the drug candidates at
different concentrations and the final data were calculated
from at least three replicates of the same experiment
performed in triplicate. The fluorescence of 8 wells containing
medium without cells were used as a blank. Fluorescence data
were normalized to 100% cell viability for non-treated cells.
For DNA platination measurement,3 about 10 106 cells
×
were transferred into a plastic tube and centrifuged at 1100
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Dalton Transactions
rpm for 5 min at room temperature. The supernatant was observed using a standard DAPI filter set (excitation λ = 350
removed by aspiration and the cell pellet stored at -20 °C until nm, emission = 461 nm) of a fluorescence microscope (Zeiss
λ
the total genomic DNA extraction with a commercial kit Axiolab), equipped with a digital photo camera (Nikon digital
(GenUPTM gDNA kit, Biotechrabbit, Hennigsdorf, Germany). Sights, DS-U3). Pictures were taken at 20X magnification.
According to the manufacturer’s instructions, during cell lysis,
Quantitative Reverse Transcription PCR (RT-qPCR)
DNA was purified employing RNAse A and proteinase K, then
extracted on silica centrifugation columns. After washing, DNA A2780 cells (2×106) were seeded on T25 flasks and allowed to
was eluted in 300
the same amount of elution buffer (blank) were diluted in TE concentrations (i.e. 6 µM complex
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to 80 L, to fit 0.5 cisplatin, and 5 mM CA). After 24 h, RNA was extracted and
cm path length in UV-transparent microplates half-area wells purified by DNAse treatment with a commercial kit (RNASPIN
(UV-Star®, Greiner Bio-one). Absorbance at 260 nm (A260 MINI, GE Healthcare); then it was quantified and checked for
relative to nucleic acids) and 280 nm (A280, relative to proteins) purity by means of absorbance at = 260, 280, and 340 nm in
ꢀ
L of elution buffer. The sample (8
ꢀ
L) and attach for 24 h. The treatment was performed with equitoxic
1, 2 µM complex 2, 30 µM
ꢀ
,
λ
were recorded from triplicate wells with a Tecan Infinite UV-Star half area UV transparent plate (Brand) with the above-
F200Pro plate reader (Tecan, Austria). For each measurement, mentioned microplate reader. For each sample, 1 µg of RNA
A260 and A280 were corrected by subtraction of the background, was retrotranscribed (RT) to cDNA with the Revertaid cDNA
then the purity of the samples was verified by means of the First strand kit (Thermo-Fisher) using random hexamer primers
A260 to A280 ratio, that always resulted > 2. After the at 45 °C, following the manufacturer’s instructions. qPCR was
subtraction of mean A260 of blank wells, the DNA concentration performed in triplicate on each sample (10 ng) to detect the
was computed from the corrected A260 by means of a expression levels of ACOX1, CPT1A, p53, p21, Cyclin D1, E,
calibration curve based on calf thymus DNA: an absorbance of HMOX1, and the reference genes RNA18S, HPRT1, GAPDH.
1 unit at 260 nm corresponds to 100
ꢀ
remaining DNA elution buffer was transferred into
g of DNA per mL. The Primer sequences were designed using the NCBI tool and
checked for target specificity including splice variants; they are
a
borosilicate glass tube, weighted to compute the total amount reported in Table S1 (see ESI). Reactions were based on the
of DNA, and then stored at -20 °C until mineralization. SsoFast EvaGreen Supermix (Bio-Rad) in the presence of 0.4
Mineralization and subsequent Pt content determination were µM primer pairs except for RNA18S (0.2 µM), according to the
performed as above-mentioned for the whole cells. The manufacturer’s instructions, in a reaction volume of 10 µL. In
amount of Pt bound to DNA was expressed as pg of Pt per
of DNA experimentally found.
ꢀ
g
order to compute reaction efficiency, a standard curve was
performed for each master mix. qPCR was performed in
triplicate using and the CFX368 thermal cycler (Bio-Rad). The
reaction conditions were 95 °C for 1 min, followed by 45 cycles
Caspase-3/7 activity
The assay was performed on A2780 cells similarly to a previous 98 °C for 5 seconds and anneal–extend step for 5 seconds at
79
report, though with some modifications. Briefly, 2×105 cells 60 °C, with data collection. At the end of these cycles, a
were seeded in 96-well black tissue culture (TC) plates in melting curve (65 °C to 95 °C, with plate read every 0.5 °C) was
complete medium, the day before treatment with the performed in order to assess the specificity of the
complexes at different concentrations. After 24 h, cells were amplification product by single peak melting temperature
washed and lysed with 25 µL of lysis buffer (10 mM HEPES, 2 verification. Results were normalized on the reference genes
mM EDTA, 2 mM DTT, 0.1 % CHAPS, pH 7.4). The caspase 3/7 and on the control according to the ꢁꢁCq method. All data
inhibitor N-Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) 10 mM analyses were performed with the built-in software (CFX
(Sigma-Aldrich) was added to control wells (data not shown). Manager, Bio-Rad).
Then, 200 µL the caspase-3 fluorescent substrate, Ac-DEVD-
ROS assay
AFC (Sigma-Aldrich), 100 mM in lysis buffer, was added to all
the wells. The activity was followed for 30 min by means of The 2’,7’-dichlorofluorescein diacetate (H2DCF-DA) dye was
= 520 nm, used to detect reactive oxygen species (ROS) levels. 2x105
fluorescence at excitation λ = 390 nm, emission
λ
normalized vs. the blank. Data were normalized to residual A2780 cells were seeded in black sterile 96-well TC plates the
viability and final fold activity (with respect to control wells) day before treatment (8 wells/treatment), performed the
was calculated as the mean of at least three independent following day, to a final concentration of 1 mM CA, 1 µM
replicates performed in duplicate for each condition.
cisplatin, 1 and 2. After 24h, 3 wells of each treatment were
used to assess residual viability by means of the resazurin
reduction assay, while the remaining wells were loaded with
Hoechst 33342 staining
A2780 cells (2×105) were seeded on Nunc™ Lab-Tek™ 4- 10 µM H
chamber slides and allowed to attach for 24 h. The following Immediately before the end of loading H
day, the treatment was added to a final concentration of 1 mM concentration) was added as positive control, while a control
CA, 1 µM and . After 24 h, the medium was replaced with column received 10 mM N-acetylcysteine (NAC), a well-known
2
DCF-DA in EBSS at 37 °C for 30 min, in the dark.
5 mM (final
2
O2
1
2
the staining solution, consisting of 5 ng mL-1 Hoechst 33342 in anti-oxidant, as negative control of each condition (data not
Earle’s Balanced Salt solution (EBSS). Cells were incubated in shown). After 2 washes by EBSS, oxidized DCF fluorescence
the dark for 5 min, washed thrice with EBSS and immediately
12 | Dalton Trans., 2012, 00, 1-3
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was measured at excitation
λ
nm. Data were normalized on viability.
= 485 nm and emission
λ
= 535
1α antibody (BD Biosciences). Equal loading of the samples
was verified by re-probing the blots with a mouse monoclonal
anti-β-tubulin antibody (Sigma Aldrich). Protein bands were
Hypoxia vs. normoxia
visualized using
a
peroxidase-conjugated anti-mouse
Hypoxia was induced by placing the cells for 48 h into a secondary antibody and the Westar Supernova kit (Cyangen).
modular incubator chamber (Billups Rothenberg Inc., Del Mar,
CA, USA) flushed with a mixture of 1% O2, 5% CO2 and 94% N2
Conflicts of interest
at 37 °C. The antiproliferative effect was assessed using the (3-
(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide
There are no conflicts of interest to declare.
(MTT) assay.83 Briefly, 3×103 cells per well were seeded onto
96-wells plate and allow to grow for 24 h prior to the
treatment with the drugs and placed under normoxic and
hypoxic conditions for 48 h. At the end of treatment, MTT was
added to each well (final concentration 0.4 mg mL-1) and left
reacting for 3 h at 37 °C. The formazan crystals, formed
through MTT metabolism by viable cells, were dissolved in
Acknowledgements
We thank Compagnia di San Paolo (Torino) within the research
project BIPLANES. We are indebted to Inter-University
Consortium for Research on the Chemistry of Metals in
Biological Systems (CIRCMSB, Bari) for providing opportunities
of stimulating discussion.
DMSO and the optical densities were measured at λ = 570 nm
using Universal Microplate Reader EL800 (Bio-Tek
a
Instruments). IC50 values were obtained by nonlinear
regression analysis, using the GraphPad PRISM 3.03 software
(GraphPad Software Inc., San Diego CA, USA).
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The cisplatin/clofibrato combos are multi-action Pt(IV) complexes active on a panel of human tumor cell lines,
also in hypoxic conditions