Antiproliferative activity of Pt(IV) conjugates containing the non-steroidal anti-inflammatory drugs (NSAIDs) Ketoprofen and Naproxen

Article abstract of DOI:10.3390/ijms20123074

Cisplatin and several non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to act synergistically or at least additively on several tumor cell lines. Dual-action cisplatin-based Pt(IV) combos containing ketoprofen and naproxen offer good antipr

Full text of DOI:10.3390/ijms20123074

International Journal of
Molecular Sciences
Article
Antiproliferative Activity of Pt(IV) Conjugates
Containing the Non-Steroidal Anti-Inflammatory
Drugs (NSAIDs) Ketoprofen and Naproxen †
Mauro Ravera ‡ , Ilaria Zanellato ‡ , Elisabetta Gabano , Elena Perin, Beatrice Rangone,
Marco Coppola and Domenico Osella *
Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Viale T. Michel 11,
15121 Alessandria, Italy; mauro.ravera@uniupo.it (M.R.); ilaria.zanellato@uniupo.it (I.Z.);
elisabetta.gabano@uniupo.it (E.G.); elena.perin@uniupo.it (E.P.); beatrice.rangone@uniupo.it (B.R.);
marco.coppola@uniupo.it (M.C.)
* Correspondence: domenico.osella@uniupo.it
† Partially presented at the XVIII Workshop on PharmaBioMetallics, Arezzo, Italy, 22–24 March 2019.
‡ These authors contribute equally to the work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 May 2019; Accepted: 22 June 2019; Published: 24 June 2019
Abstract: Cisplatin and several non-steroidal anti-inflammatory drugs (NSAIDs) have been proven
to act synergistically or at least additively on several tumor cell lines. Dual-action cisplatin-based
Pt(IV) combos containing ketoprofen and naproxen offer good antiproliferative performance on a
panel of human tumor cell lines, including a malignant pleural mesothelioma (MPM) one, a very
chemoresistant tumor. The main reason of the increased activity relies on the enhanced lipophilicity of
these Pt(IV) conjugates that in turn promotes increased cellular accumulation. A quick Pt(IV)
→
Pt(II)
reduction generates the active cisplatin metabolite. The NSAID adjuvant action seems to be almost
independent from cyclooxygenase-2 (COX-2) expression in the tumor cells under investigation (lung
A-549, colon HT-29, HCT 116, SW480, ovarian A2780, and biphasic MPM MSTO-211H), but it seems
to rely (at least in part) on the activation of the NSAID activated gene, NAG-1 (a member of the
transforming growth factor beta, TGF-β, superfamily), which has been suggested to be involved in
NSAID antiproliferative activity.
Keywords: multifunctional Pt(IV) prodrugs; antiproliferative activity; NSAID; COX-2; NAG-1;
mesothelioma
1. Introduction
There are two main problems when a single drug is used in clinics: (i) There is no pharmacological
agent with a “perfect” selectivity (and this causes systemic toxicity); (ii) even the most successful
therapies may lose potency with time because of the development of acquired resistance. Combination
therapy is aimed to overcome these limitations: Employing two or more synergistic drugs together
reduces their individual dose and therefore their toxicity. At the same time, when two or more drugs
which have different biochemical mechanisms of action are combined, the probability of selecting
tumor cells resistant to all the drugs is reduced.
These considerations apply to Pt-based anticancer compounds, the prototype of which is
cisplatin ((SP-4-2)-diamminedichloridoplatinum(II) and cis-[PtCl2(NH3)2]) [1]. The development
of a cisplatin-based combination chemotherapy such as the PVB (cisplatin, vinblastine and bleomycin)
protocol in the late 1970s resulted in a dramatic improvement in the prognosis of patients with
metastatic germ cell tumors, and overall cure rates exceeded 80%. Today, the bleomycin, etoposide,
Int. J. Mol. Sci. 2019, 20, 3074; doi:10.3390/ijms20123074
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cisplatin (BEP) combination is considered to be the standard treatment of patients with testicular cancer,
with survival rates of 90% [2].
However, the administration of two or more drugs can suffer from drawbacks such as different
bioavailabilities, the pharmacokinetics and metabolism of the drugs, and possible drug–drug
interactions. The preparation of a single molecule containing different chemically bonded drugs
(combo) may represent a more effective strategy to address this task [3].
In this framework, octahedral Pt(IV) complexes play a role because of their peculiar chemical
features [4–7]. The octahedral, coordinatively saturated geometry of Pt(IV) is generally characterized by
kinetic inertness that can minimize off-target reactions and allow for oral administration. Additionally,
Pt(IV) complexes are selectively (or at least preferentially) reduced in the hypoxic tumor environment
to the corresponding cytotoxic Pt(II) metabolites with the loss of their axial ligands (activation by
reduction) [6,8–10]. Recently, Pt(IV) chemistry has been used to design multi-functional conjugates.
One or two adjuvant or synergistic agents are conjugated to the octahedral Pt(IV) core in axial position
so that they and the parent Pt(II) drug will be released simultaneously and will act in a synergistic or at
least in an additive way [4,7,11–14] (Figure 1).
Figure 1. Reduction reaction of a cisplatin-based Pt(IV) prodrug; D = second drug.
Various agents with different cellular targets have been coordinated to Pt(IV) complexes to obtain
such combos, and a number of them contain the inhibitors of cyclooxygenase (COX) enzymes. There are
two main isoforms of COX, namely COX-1 and COX-2, responsible for the formation of biological
mediators of inflammation, including prostaglandins (as prostaglandin E2, PGE2), prostacyclin,
and thromboxane. COX-1 is constitutively expressed in most tissues, whereas the COX-2 level is
generally very low unless induced in response to inflammatory and other physiological stimuli.
Indeed, an enhanced COX-2 expression is found in several tumors, and it appears to play a role
in carcinogenesis [15]. For this reason, COX inhibitors, including the large class of non-steroidal
anti-inflammatory drugs (NSAIDs), as well as the COX-2 selective inhibitors (e.g., celecoxib), are
suggested for cancer prevention, especially as colon adenocarcinoma is concerned [16]. NSAIDs
are commonly used for their analgesic and antipyretic effects by blocking the generation of PGE2
via the inhibition of COX activity [17–19]. Regrettably, NSAIDs showed a modest tumor growth
inhibition when employed as a single agent; however, they have been tested as adjuvant agents in
some chemotherapeutic protocols [20]. The overall mechanisms of their action are so far not fully
understood, but both COX-dependent and -independent pathways seem to play a role [17,18].
Conventional NSAIDs, such as aspirin, ibuprofen, naproxen, indomethacin, and several others,
inhibit both COX-1 and COX-2. Unfortunately, NSAIDs seem to exercise their anti-cancer adjuvant
effects only through the inhibition of the inducible COX-2, whereas unwanted side effects, such as
gastrointestinal problems, are thought to arise from the inhibition of the constitutive COX-1 [21–23].
The first example of a Pt(IV) complex containing acetylsalicylic acid (2-acetoxybenzoic acid,
aspirin) as a ligand is asplatin or platin-A (1, Figure 2). Asplatin exhibited an antiproliferative activity

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comparable or slightly better to that of cisplatin alone or of an equimolar mixture of cisplatin and
aspirin [24 26]. Interestingly, an in vivo assay has demonstrated that asplatin possesses a higher
antitumor efficacy with lower toxicity in comparison with cisplatin [25].
–
Figure 2. Sketch of the compounds under investigation.
Following this research, several cisplatin-, oxaliplatin-, or kiteplatin-based Pt(IV) complexes
containing NSAIDs (i.e., indomethacin, ibuprofen, flurbiprofen, naproxen, and wogonin) as axial
ligands have been reported in literature [27–35].
In the present work, two complexes containing axial ketoprofen (2-(3-benzoylphenyl)propanoic
acid) or naproxen (2-(6-methoxynaphthalen-2-yl)propanoic acid) (Figure 2) were synthesized and
tested for their biological features against a panel of human cancer cell lines. Ketoprofen and naproxen
are two classic NSAID propionic acid derivatives, acting as inhibitors of both COX-1 and COX-2 [23,36].
The presence of a carboxylic functionality in their structure allows an easy coordination to the Pt(IV)
core through an “esterification” reaction of the hydroxido synthon [37].
2. Results and Discussion
2.1. Synthesis and Characterization of the Pt(IV) Complexes
The synthesis of complexes
(OC-6-44)-acetatodiamminedichloridohydroxidoplatinum(IV) (
2
and
3
began with the preparation of the intermediate
, Figure 3) from cisplatin and hydrogen
A
peroxide in acetic acid according to published procedures [38]. The following step required the
conversion of the carboxyl group of NSAIDs into its more reactive acyl chloride using oxalyl chloride
and N,N-dimethylformamide (DMF) as a catalyst. The acyl chlorides of the NSAIDs were then
coordinated to the Pt core by a microwave-assisted reaction with
a small amount of pyridine (able to neutralize the hydrochloric acid released during the reaction)
to obtain and . The microwave heating was applied because there is neat advantage in terms of
A in anhydrous acetonitrile with
2
3
reaction speed, yield, and the purity of products, with respect to the traditional thermal syntheses.
This improvement is well-known in organic chemistry, but it has been seldom reported in the syntheses
of Pt(II) and Pt(IV) complexes [39–42]. The synthesis of complex 3 has been already reported in the
literature, but this was done with a different procedure [35].

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Figure 3. Scheme of the syntheses used in this work: (i) 50% w/w H2O2 in CH3COOH (room
temperature, 4 h), (ii) acyl chloride of non-steroidal anti-inflammatory drugs (NSAIDs) in acetonitrile,
under microwave heating (55 ◦C, 1 h).
Finally, as a reference compound, 1 was prepared, according to the procedure reported by Dhar
et al., by reacting (OC-6-44)-diamminedichloridodihydroxidoplatinum(IV) with the anhydride of
acetylsalicylic acid [24].
All the complexes were characterized with reversed-phase high performance liquid
chromatography (RP-HPLC) coupled with electrospray ionization-mass spectrometry (ESI-MS)
and multinuclear nuclear magnetic resonance (NMR; see Figures S1–S13, Supplementary Material).
Their lipophilicity was evaluated by RP-HPLC measurements [43]. Indeed, the chromatographic
retention was due to partitioning between C18 chains of the stationary phase (a model of the cell
membrane) and the aqueous eluent (a model of the water inside and outside cells). The logarithm of
the RP-HPLC capacity factor k0 (k0 = (tR
−
t0)/t0—where tR = retention time of the compound under
investigation and t0 = column dead time)—usually correlates with the logarithm of the octanol/water
partition coefficient, log Po/w (typically employed to represent lipophilicity). The log k0 values for the
complexes under investigation were measured on a C18 HPLC column by using a 70% methanol/30%
aqueous 15 mM formic acid eluent. In these conditions (high methanol content), the tR, and consequently
the log k’, are compressed, but maintain the correct trend. Therefore, the aspirin-containing
tended to be the most hydrophilic of the series, while naproxen-containing was slightly more
lipophilic than ketoprofen-containing , as expected from the log Po/w values of the free NSAIDs (log
o/w = 1.13 for aspirin, 2.77 for ketoprofen, and 3.06 for naproxen) [44].
Despite the “traditional” claim that Pt(IV) complexes are inert, several members of this family have
shown changes in the coordination sphere when challenged in a solution [ ]. Therefore, compounds
and were dissolved in a 2 mM HEPES (HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
1 results
3
2
P
6
2
3
pH = 7.5) buffer with 5% v/v ethanol to improve the solubility, and the HPLC traces were recorded up
to 7 days. All compounds showed a very low decomposition during the experiment (from 24 h to
7 days:
2 94.5% to 79.8%; 3 98.3% to 75.9% vs. the original peak). In both cases, the ESI-MS spectra
showed that the main by-product derived from the chloride/water exchange in the equatorial position
of the original octahedral assembly.
The redox properties of the Pt(IV) complexes could be evaluated through their reduction potential.
However, a suitable (high) potential may not guarantee their in vivo reduction, as the kinetics plays an
important role [
complexes [
45], such reduction was studied via NMR using 15NH3-labeled complexes. Usually, the
[1H, 15N] the HSQC spectra of 15N-cisplatin-based Pt(IV) complexes when reduced in cytosol show
only the presence of cisplatin (15NH3 δ = –66.7 ppm and 1H
= 4.0 ppm, with satellite peaks possibly
visible at 63.7 ppm and 46 47]. The production of
69.6 ppm, 1JPt-N = 331 Hz and 2JPt-H = 63 Hz) [41
and15N-
(synthesized according to the actual procedures from
48] after 2 h of treatment with cytosol extracted from A2780 ovarian cancer cells (data
9]. Since the high molecular weight cytosolic bioreductants mainly reduce the Pt(IV)
9,
δ
−
−
,
,
cisplatin was verified for both 15N-
2
3
15N-cisplatin) [38
not shown).
,

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2.2. Antiproliferative Activity
The half-maximal inhibitory concentrations, IC50, were determined for
2
and
3
, along with the
clinically-employed reference complexes cisplatin and oxaliplatin, as well as asplatin 1, free ketoprofen,
and naproxen for comparison purposes (Table 1). Six human cancer cell lines were challenged with
the compounds under investigation: Lung carcinoma A-549, colon adenocarcinoma HT-29, HCT
116, SW480, ovarian endometroid adenocarcinoma A2780, and malignant pleural mesothelioma
(MPM) MSTO-211H (a mixed or biphasic phenotype consisting of both epithelioid and sarcomatoid
components). The MPM cell line was added to the panel, since promising preclinical results have
been reported on the activity of celecoxib on MPM, albeit apparently independent of COX-2 protein
levels [49].
In these cell lines, the level of COX-2 expression was evaluated just before the viability tests by a
quantitative reverse transcription polymerase chain reaction (RT-qPCR) [50,51], resulting observably
high in A-549 and HT-29, medium in HCT 116, low in MSTO-211H, and very low in A2780 and SW480
(Figure 4). A significant amount of amplicon was found in all samples (i.e., the cycle quantification
value Cq within the dynamic linear range of the standard curve. See Section 4).
Figure 4. Cyclooxygenase (COX)-2 cDNA level of expression (
∆Cq) in A-549, HT-29, HCT 116,
MSTO-211H, A2780, and SW480 cells. Data are means SEM (standard error of mean) of at least
±
three independent replicates and were compared by means of a one-way ANOVA analysis of the
variance-Tukey test (no indication = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. SW480).
Table 1. Lipophilicity (log k’) and antiproliferative (IC50) data for complexes
oxaliplatin, aspirin, ketoprofen, and naproxen. IC50 data ( M) were obtained after 72 h of treatment of
the following cancer cell lines: Lung A-549, colon HT-29, HCT 116, SW480, ovarian A2780 cancer cell
lines, and malignant pleural mesothelioma MSTO-211H. Data are means standard deviation (SD) of
1–3, together with cisplatin,
µ
±
at least three independent replicates. Numbers in parenthesis are the ratios R = IC50 (cisplatin)/IC50
(Pt(IV) conjugate) reported with two significant figures.
IC50 (µM)
Compounds
log k’
A-549
HT-29
HCT 116
MSTO-211H
SW480
A2780
cisplatin
oxaliplatin
aspirin
ketoprofen
naproxen
−0.50
−0.28
−0.08
0.40
3.60 ± 0.90
0.74 ± 0.25
1672 ± 152
725 ± 69
701±28
2.72 ± 0.39
0.92 ± 0.08
2835 ± 885
828 ± 229
764 ± 158
4.42 ± 0.21
(0.62)
3.05 ±0.28
1.16 ± 0.09
2578 ± 772
984 ± 179
640 ± 91
1.50 ± 0.083
(2.0)
1.33 ± 0.35
1.01 ± 0.55
639 ± 374
518 ± 296
427 ± 159
1.74 ± 0.21
(0.75)
2.27 ± 0.12
0.48 ± 0.02
1607 ± 386
830 ± 173
927 ± 257
0.217 ± 0.07
(1.1)
0.460 ± 0.110
0.171 ± 0.008
1597 ± 455
676 ± 36
0.50
353 ± 64
6.4 ± 2.7
0.552 ± 0.123
asplatin, 1
−0.32
0.14
(0.56)
0.825 ± 0.388
(4.4)
(0.83)
0.486 ± 0.235
0.184 ± 0.088
0.198 ± 0.035
0.0948 ± 0.023
0.063 ± 0.033
(7.3)
2
3
(5.6)
(17)
(6.7)
(24)
0.486 ± 0.075
0.313 ± 0.186
(8.7)
0.149 ± 0.076
(20)
0.161 ± 0.040
(8.3)
0.0844 ± 0.0287
(27)
0.045 ± 0.016
0.18
(7.4)
(10)

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At the first glance, the Pt(IV)-NSAID complexes
2
and
3
appear to have been more active than
cisplatin, oxaliplatin, and 1 on all cell lines tested ranging from 2 to 24 times. Moreover, naproxen-based
2 was (almost always) slightly more active than ketoprofen-based 3.
To extract quantitative trends from the bulk of the IC50 data, the ratio R of the activity of
cisplatin-based Pt(IV) derivatives 1, 2, and 3 vs. the activity of parent cisplatin against each cancer
cell was calculated and inserted in Table 1. Since the antiproliferative activity is the inverse of
the corresponding IC50 value, it follows that: R = IC50 (cisplatin)/IC50 (Pt(IV) conjugate). What is
noteworthy is that the trend of the R values does not parallel the expression of COX-2. In particular,
within the colon cancer family, the HCT 116 and SW480 cells, which have lower level of COX-2 with
respect to HT-29, resulted more sensitive to 2–3 treatments. From this scenario, the antiproliferative
activity seems to be more related to the lipophilicity of the complexes (log k’) than to the COX-2
expression in these tumor cell lines. In addition, the IC50 values of the free NSAIDs ketoprofen
and naproxen resulted to be quite similar (in the low mM range) and seemed to be unrelated to the
COX-2 expression.
Moreover, the combination index (CI) values [52–54] between free cisplatin and the two NSAIDs
were calculated for HT-29 and HCT 116, showing a high and medium COX-2 expression, respectively.
In all cases, the CI pointed out an almost additive combination (CI around 1), once again independently
from COX-2 expression (Figure 5 and Figures S14–S17, Supplementary Material).
Figure 5. (a) Residual HCT 116 viability data and concentration response curves (four parameters
logistic function) after 72 h of treatment of cisplatin (red circles), ketoprofen (black squares), and a
mixture of cisplatin and ketoprofen (final cisplatin:ketoprofen ratio = 1:500, dark yellow triangles).
Data are means
±
SEM of at least three replicates. (b) Residual viability data for the 1:500 mix were used
to obtain the Combination Index (CI) value by using the equation of Chou and Talalay for non-mutually
exclusive drugs (see Materials and Methods).
The intracellular Pt accumulation of 1–3 and cisplatin was assessed to verify the relationship
existing with their lipophilicity (log k’). This parameter, which is the final balance between cellular influx
and efflux [55], is here expressed as the accumulation ratio (AR), the ratio between the intracellular
[Pt] and the extracellular [Pt] (in the culture medium) [56]. For this purpose, A2780 cells were treated
for 4 h with 10 µM concentrations of all Pt complexes, and the AR values were calculated from [Pt]
experimentally measured by inductively coupled plasma-mass spectrometry (ICP-MS) (Figure 6).
The AR trend paralleled the differences in antiproliferative activity (1/IC50) between the group formed
by 1 and cisplatin and that formed by 2 and 3.

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Figure 6. Accumulation ratio (AR) of cisplatin,
1
,
2
, and
3
, measured on A2780 ovarian cancer cells
SEM of at least
SEM of at least three independent replicates and
treated for 4 h with 10
µM concentrations of the Pt complexes. Data are means
±
three independent replicates. Data are means
were compared by means of a one-way ANOVA analysis of a variance-Tukey test (no indication = not
significant; *** p < 0.001 vs. cisplatin).
±
In dicarboxylato Pt(IV) complexes, the combination of (hydrophilic) cisplatin and two (amphiphilic)
carboxylate anions causes the neutralization of their negative charges while the lipophilic chains
protrude toward the exterior, thus generating very lipophilic conjugates. These combo Pt(IV) prodrugs
enter cells via passive diffusion (the main if not the only mechanism of their cellular uptake) more
efficiently than the single components (synergistic cellular accumulation) [57].
2.3. Gene Modulation and RT-qPCR
The apparent absence of relationship between COX-2 expression and sensitivity towards free
ketoprofen and naproxen or
2 and 3 suggests that these drugs might inhibit cell growth through a
COX-independent mechanism.
To shed more light on the underlying biochemical mechanism, a RT-qPCR analysis was carried
out. Preliminarily, to perform equitoxic treatments, the IC50 values after 24 h of continuous treatment
(the time interval of the corresponding RT-qPCR experiment) were obtained. In this experiment, the
variation of the expression of five genes (i.e., BAD, BAX, BCL-2, COX-2, and NAG-1) was analyzed.
Two cell lines, namely A-549 (high COX-2 expression) and HCT 116 (low COX-2 expression), were
chosen for this experiment (Figure 7).
Apoptosis, the phenomenon of programmed cell death, is triggered through two major routes:
The extrinsic and the intrinsic (mitochondrial) pathways. The second mechanism is regulated by the B
cell leukemia-2 gene product (BCL-2) family. This family contains both pro-apoptotic (e.g., BAX, BAK,
and BAD) and anti-apoptotic (e.g., BCL-2 and BCL-XL) members [58]. Agents able to increase the levels
of pro-apoptotic proteins or decrease that of anti-apoptotic proteins are good anticancer candidates.
As already reported in the introduction, albeit with the acknowledgment that the anti-tumorigenic
mechanism of NSAIDs is not fully understood, both COX-dependent and -independent pathways
seem to play a role. Recent studies have revealed the existence of the NSAID activated gene
(NAG-1), a member of the transforming growth factor beta, TGF-
antiproliferative activity [61 62]. Importantly, NAG-1 is induced by NSAIDs in cells also devoid of
any COX activity [63 64] The induced expression of NAG-1 can thus be correlated with the growth
inhibition of cancer cells that follows a COX-independent mechanism [18,65].
β, superfamily [59,60], involved in
,
,

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Figure 7. Relative gene expression of BAD (pro-apoptotic), BAX (pro-apoptotic), BCL-2 (anti-apoptotic),
COX-2 (cyclooxygenase-2), and NAG-1 (NSAID-activated gene 1) in ( ) HCT 116 (lower COX-2
expression) and ( ) A-549 (higher COX-2 expression) cells following equitoxic 24 h treatments (IC50
with cisplatin, , ketoprofen, and naproxen. From the left to right of each sequence: Control, (light
yellow), (light green), ketoprofen (dark yellow), naproxen (dark green), and cisplatin (light blue). The
a
b
)
2,
3
2
3
RT-qPCR experiment was performed in triplicate, and the results were normalized to references genes
in untreated control. The arrows indicate the upregulated (arrow up) or downregulated (arrow down)
gene expression with respect to internal threshold value as determined by the CFX Manager software.
Cisplatin
2
and
3
showed similar effects on both anti-apoptotic and pro-apoptotic genes in both
Pt(II) reduction plays the paramount role.
cell lines, indicating that cisplatin generated by Pt(IV)
→
Their major effect was the downregulation of anti-apoptotic BCL-2, more intensely on A-549 than HCT
116, and the up-regulations of pro-apoptotic BAD and BAX. On the contrary, these contrasting but
negligible effects (i.e., no changes compared to regulation threshold) were caused by free ketoprofen
and naproxen. Pt(IV) conjugates and free NDAIDs did not significantly change the COX-2 expression
on A-549 cells, whereas it smoothly increased it on HCT 116 cells with a substantial contribution of
cisplatin to increase the expression of COX-2.

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In order to confirm the RT-qPCR data (i.e., the COX-2 expression remained almost unchanged
on A-549 cell lines or even slightly increased on HCT 116), a semi-quantitative immunochemical
analysis was carried out using a primary antibody against COX-2. Indeed, the fluorescence microscopy
indicated a similar amount of COX-2 protein in the nuclear region after 24 h treatment with equitoxic
concentrations of 2 and 3. Only a slight decrease in cytosolic staining was observed in treated A-549
cells, and this was attributed to the inactive form of the enzyme (see Figures S18 and S19, Supplementary
Material) [66,67].
Interestingly, 2 and 3 and, to a lesser extent, free ketoprofen and naproxen increased the expression
of the above-discussed NAG-1. In addition, cisplatin increased NAG-1 expression, thus augmenting
the overall NAG-1 stimulation.
3. Materials and Methods
3.1. General Procedures
All the chemicals (Sigma Aldrich-Merck KGaA, Darmstadt, Germany or Alfa Aesar–Thermo
Fisher GmbH, Kandel, Germany) were used without further purification. The complex (OC-6-44)-
acetatodiamminedichloridohydroxidoplatinum(IV) (
A
) was prepared according to previously
published procedures [38
,
68]. The reactions under microwave heating were performed by using a
CEM Discover® SP System equipped with a focused single mode, a self-tuning cavity, an air-cooling
system, and an automated power control based on temperature feedback that supplied power in 1 W
increments from 0 to 300 W.
The purity of all the synthesized compounds was assessed by analytical RP-HPLC.
Chromatographic analyses were carried out using a C18 Phenomenex Phenosphere-NEXT (5-µm,
250 × 4.6 mm internal diameter) column on a Waters HPLC-MS instrument (equipped with
Alliance 2695 separations module, 2487 dual lambda absorbance detector, and 3100 mass detector).
The UV-visible detector was set at 210 nm, the eluent was a 30/70 mixture of 15 mM formic acid/CH3OH,
and the flow rate was 0.5 mL
·
min−1. Mass spectra were recorded using source and desolvation
temperatures set to 150 ◦C and 250 ◦C, respectively, with nitrogen used as both drying and nebulizing
gas. The cone and the capillary voltages were usually +30 V (positive ion mode) or 30 V (negative ion
−
mode) and 2.70 kV, respectively. The quasi-molecular ion peak [M + H]+ of the synthesized complexes
was assigned on the basis of the m/z values and of the simulated isotope distribution patterns. The NMR
spectra were measured on a NMR-Bruker Avance III spectrometer operating at 500 MHz (1H), 125.7
1
MHz (13C), and 107.2 MHz (195Pt). H and 13C NMR chemical shifts were reported in parts per million
(ppm) and referenced to solvent resonances. 195Pt NMR spectra were recorded using a solution of
K2[PtCl4] in saturated aqueous KCl as the external reference. The shift for K2[PtCl4] was adjusted to
−1628 ppm from Na2PtCl6 (δ = 0 ppm).
3.2. Synthesis of (RS)-2-[3-(benzoyl)phenyl]propanoyl Chloride (Acyl Chloride of Ketoprofen)
(RS)-2-[3-(benzoyl)phenyl]propanoic acid (0.034 g, 0.134 mmol) was placed into a 10 mL flask
and dissolved in 2 mL of dichloromethane; then, 0.115 mL of oxalyl chloride (1.34 mmol) were added
together with a drop of dimethylformamide. After the end of gas development, the transparent and
colorless mixture was allowed to react at room temperature overnight. The yellow solution was dried
1
by a rotary evaporator at 60 ◦C obtaining a yellow–green oil. H-NMR (CDCl3): 1.52 (3H, d, J = 7.2 Hz,
H3), 3.80 (1H, q, J = 7.2 Hz, H2), 7.40–7.47 (3H, m, H5, H6, H9), 7.56 (2H, t, J = 7.7 Hz, H13, H15), 7.64
(1H, d, J = 7.7 Hz, H7), 7.78 (3H, m, H12, H14, H16) ppm; 13C-NMR (CDCl3): 18.7 (C3), 57.2 (C2), 128.5
(C13, C15), 129.1 (C6), 129.6 (C7), 129.9 (C12, C16), 130.1 (C9), 131.8 (C14), 132.8 (C5), 137.3 (C4), 137.9
(C11), 138.4 (C8), 175.3 (C1), 196.1 (C10) ppm.

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3.3. Synthesis of 2-(6-metoxynaphtalen-2-yl)propanoyl Chloride (Acyl Chloride of Naproxen)
2-(6-metoxynaphtalen-2-yl)propanoic acid (0.030 g, 0.132 mmol) was placed into a 10 mL flask
and dissolved in 2 mL of dichloromethane; then, 0.114 mL of oxalyl chloride (1.32 mmol) were added
together with a drop of dimethylformamide. After the end of gas development, the transparent and
colorless mixture was allowed to react at room temperature overnight. The yellow solution was dried
1
by a rotary evaporator at 60 ◦C obtaining a yellow–green oil. H-NMR (CDCl3): 1.62 (3H, d, J = 7.0 Hz,
H3), 3.87 (3H, s, H14), 4.22 (1H, q, J = 7.0Hz, H2), 7.10 (1H, s, H9), 7.13 (1H, m, H5), 7.30 (1H, d, J = 8.4
Hz, H11), 7.64 (1H, s, H13), 7.69 (2H, m, H6, H10) ppm; 13C-NMR (CDCl3): 18.7 (C3), 55.4 (C14), 57.4
(C2), 105.7 (C13), 119.4 (C11), 125.9 (C6), 127.0 (C9), 127.7 (C5), 128.9 (C8), 129.4 (C10), 132.5 (C7), 134.2
(C4), 158.1 (C12), 175.7 (C1) ppm.
3.4. Synthesis of (OC-6-44)-acetatodiamminedichlorido(RS)-2-[3-(benzoyl)phenyl]propanoatoplatinum(IV) (
2)
(OC-6-44)-acetatodiamminedichloridohydroxoplatinum(IV) (0.050 g, 0.134 mmol) was
A
suspended in 2 mL of anhydrous acetonitrile in a microwave tube. (RS)-2-[3-(benzoyl)phenyl]propanoyl
chloride was then dissolved in 1 mL of acetonitrile which was added to the suspension together with
30
with a power of 80 W. The resulting yellow–green solution was filtered (polytetrafluoroethylene filter,
porosity: 0.45 m) to remove unreacted reagents, and then it was dried with a rotary evaporator.
µ
L of pyridine. The mixture was allowed to react in a microwave reactor for one hour, at 55 ◦C,
µ
The resulting residue was dissolved in 2 mL of dichloromethane, and then 10 mL of diethyl ether were
added to get the precipitation of a yellow solid. The precipitate was separated by centrifugation and
washed several times with diethyl ether, water and, finally, diethyl ether again, drying the residue
under nitrogen flow before any change of solvent and at the end. Yield: 0.043 g, 52%. ESI-MS: found
613 m/z; calcd for C18H23Cl2N2O5Pt, [M + H]+ 613 m/z; 1H-NMR (DMSO-d6): 1.36 (3H, d, J = 7.2 Hz,
H5), 1.91 (3H, s, H2), 3.80 (1H, q, J = 7.2 Hz, H4), 6.50 (6H, m, NH3), 7.47 (1H, t, J = 7.6 Hz, H8),
7.56–7.70 (6H, m, H7, H9, H11, H15, H16, H17), 7.75 (2H, d, J = 7.6 Hz, H14, H18) ppm; 13C-NMR
(DMSO-d6): 19.8 (C5), 22.8 (C2), 46.2 (C4), 127.8 (C8), 128.3 (C9), 128.6 (C15, C17), 128.8 (C11), 129.7
(C14, C18), 132.1 (C16), 132.6 (C7), 136.8 (C13), 137.0 (C10), 142.6 (C6), 178.1 (C1), 181.4 (C3), 195.7 (C12)
ppm; 195Pt-NMR (DMSO-d6): 1214 ppm.
3.5. Synthesis of (OC-6-44)-acetatodiamminedichlorido2-(6-metoxynaphtalen-2-yl)propanoatoplatinum(IV) (3)
(OC-6-44)-acetatodiamminedichloridohydroxidoplatinum(IV) (0.050 g, 0.133 mmol) was
suspended in 2 mL of anhydrous acetonitrile in a microwave tube. 2-(6-metoxynaphtalen-2-yl)
propanoyl chloride was dissolved in 1 mL of acetonitrile and added to the suspension together with
30
with a power of 80 W. The resulting yellow solution was filtered with a polytetrafluoroethylene filter
(porosity: 0.45 m) to remove unreacted reagents, and it was subsequently dried by a rotary evaporator
µ
L of pyridine. The mixture was allowed to react in a microwave reactor for one hour, at 55 ◦C,
µ
obtaining a light-yellow residue. The complex was precipitated by adding 2 mL of dichloromethane
and then 10 mL of diethyl ether, causing the formation of a sticky yellow solid. The solid residue was
tritured with diethyl ether to get a yellow powder and then dried under nitrogen flow. The residue
was further washed with water, dried under nitrogen flow, and kept in desiccator. Yield: 0.052 g, 65%.
1
ESI-MS: found 589 m/z; calcd for C16H23Cl2N2O5Pt, [M + H]+ 589 m/z. H-NMR (DMSO-d6): 1.40 (3H,
d, J = 7.2 Hz, H5), 1.91 (3H, s, H2), 3.81 (1H, q, J = 7.2 Hz, H4), 3.86 (3H, s, H16), 6.53 (6H, m, NH3),
7.13 (1H, m, H7), 7.27 (1H, m, H13), 7.47 (1H, m, H15), 7.73 (3H, m, H8, H11, H12) ppm; 13C-NMR
(DMSO-d6): 19.8 (C5), 22.8 (C2), 46.4 (C4), 55.1 (C16), 105.7 (C15), 118.3 (C13), 125.4 (C8), 126.3 (C11),
126.9 (C12), 128.3 (C10), 129.1 (C7), 133.0 (C9), 137.4 (C6), 156.9 (C14), 178.2 (C1), 181.9 (C3) ppm;
195Pt-NMR (DMSO-d6): 1217 ppm.

Int. J. Mol. Sci. 2019, 20, 3074
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3.6. Lipophilicity and Stability in Water
A chromatogram for each complex (0.25 mM) at a fixed eluant composition (70% methanol/30%
aqueous 15 mM formic acid) was performed on a C18 column (Phenomenex Phenosphere-NEXT,
5
µ
m, 250
×
4.6 mm internal diameter). The corresponding retention time tR was used to calculate
t0)/t0, where t0 is the column dead time (i.e., the tR of KCl used as an internal
log k0:k0 = (tR
−
reference) [46,69,70].
The stability in water of the compounds was checked by dissolving the Pt complexes ([Pt] = 0.5 mM)
in a 2 mM HEPES buffer (HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH = 7.5)
containing 5% v/v ethanol as a cosolvent. The HPLC traces (see above for the instrumental conditions)
were recorded along one week.
3.7. Antiproliferative Activity
The compounds under investigation were tested on ovarian endometrioid adenocarcinoma A2780
(ICLC HTL98008 from Interlab Cell Line Collection, ICLC, Genova, Italy), lung adenocarcinoma A-549
(ICLC HTL03001), biphasic malignant pleural mesothelioma MSTO-211H (ICLC HL01018), colon
carcinoma HCT 116 (ECACC 91091005 from European Collection of Cell Cultures, ECACC, Salisbury,
UK), and both HT-29 and SW480 (a kind gift from Prof. Claudia Cantoni, University of Genova,
Italy). An RPMI 1640 medium as used for the A2780, A-549, and SW480 cells, McCoy’s 5A medium
was used for HCT 116 and HT-29 cells, and Dulbecco’s Modified Eagle Medium (DMEM) was used
for MSTO-211H. 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. Cisplatin was
dissolved in a 0.9% w/v NaCl aqueous solution brought to pH 3 with HCl (final stock concentration
1 mM). All Pt(IV) complexes were dissolved in absolute ethanol (final stock concentration 1–5 mM)
◦
and stored at
−
20 C. The mother solutions were diluted in a 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). The concentration was confirmed by means
of inductively coupled plasma-optical emission spectroscopy (ICP-OES). Ketoprofen and naproxen
were freshly dissolved in 0.1 M NaHCO3 (final stock concentration 5 and 2.5 mM, respectively). Cells
were treated with the compounds under investigation for 72 h. To assess the growth inhibition of
the compounds under investigation, a cell viability test (i.e., the resazurin reduction assay) was used.
Briefly, 2
end of the treatment, viability was assayed by 100
Scientific, Geel, Belgium) in a fresh medium for 1 h at 37 ◦C, and the amount of the reduced product,
resorufin, was measured by means of fluorescence (excitation = 535 nm, emission = 595 nm) with
×
103 cells per well were seeded in black sterile tissue-culture treated 96-well plates. At the
mL−1 resazurin (Acros Chemicals-Thermo Fisher
µg
·
λ
λ
a Tecan Infinite F200Pro plate reader (Tecan, Grödig, Austria) [71]. 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. The half-maximal inhibitory 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 NSAIDs and cisplatin, HCT 116 and HT-29 cells were treated with increasing
concentrations of cisplatin, ketoprofen or naproxen, and a mixture cisplatin:NSAIDs in a fixed 1:500
ratio, according to their respective IC50 values. The experiment was repeated three times. According to
the method of Chou and Talalay [52
–54], the interaction between cisplatin and NSAIDs was computed
in terms of combination index (CI) for non-mutually exclusive drugs by using the following equation:
C1m
C1a
C2m
C2a
C1mC2m
C1aC2a
CI =
+
+
(1)

Int. J. Mol. Sci. 2019, 20, 3074
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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. The interpretation of CI
values is defined such that CI = 1 indicates an additive effect, and CI < 1 and CI > 1 indicate synergism
and antagonism, respectively.
3.8. Cellular Uptake
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
medium. At time zero, 100 L 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 1
+ 2% EDTA (HyCloneTM-, Thermo
µM) in a complete
µ
×
Fisher Scientific, Waltham, MA, USA) and harvested in a fresh complete medium. The cell pellet was
lysed in 1% w/v sodium dodecyl sulphate and the protein content was determined by means of the
bicinchoninic acid method (BCA assay Kit, PierceTM, Thermo Fisher, Waltham, MA, USA). The exact
volume of the sample was determined by weight. The cell number was computed by a standard curve
with known amount of the same cells. Control and cisplatin-treated cells showed the same values
as those previously reported. Before the ICP-MS measurement, the HNO3 was diluted to a final 1%
concentration. To obtain the Pt cellular concentration, the total cellular volume of each sample was
obtained considering the mean cell diameter and cell number estimated by means of an automatic
cell counting device (Countess®, Life Technologies-Thermo Fisher, Waltham, MA, USA). The ratio
between the internal and the external cell Pt concentration, namely, the accumulation ratio (AR) was
computed as previously reported [72].
3.9. Quantitative Reverse Transcription PCR (RT-qPCR)
HCT 116 and A-549 cells (2
The treatment was performed with equitoxic concentrations (i.e., HCT 116: 3
and , 1 mM for both ketoprofen and naproxen, and 30 M for cisplatin; A-549: 5
complexes and , 1 mM for both ketoprofen and naproxen, and 50 M for cisplatin). After 24 h,
×
106) were seeded on T25 flasks and allowed to attach for 24 h.
M for both complexes
M for both
µ
2
3
µ
µ
2
3
µ
RNA was extracted and purified by DNAse treatment with a commercial kit (RNASPIN MINI, GE
Healthcare, Pittsburgh, PA, USA); then it was quantified and checked for purity by means of absorbance
at
Waltham, MA, USA) with the above-mentioned microplate reader. For each sample, 1
λ
= 260, 280, and 340 nm in UV-Star® half-area UV transparent plate (Fisherbrand-Thermo Fisher,
µg of RNA was
retrotranscribed (RT) to cDNA with the Revertaid cDNA First strand kit (Thermo Fisher, Waltham,
MA, USA) using random hexamer primers at 45 ◦C, following the manufacturer’s instructions. qPCR
was performed in triplicate on each sample (10 ng) to detect the expression levels of BAD, BAX, COX-2,
NAG-1, and the reference genes RNA18S, HPRT1, and GAPDH. Primer sequences were designed using
the NCBI tool and checked for target specificity, including splice variants (see Supplementary Material,
Table S1). Reactions were based on the (PowerUp SYBRTM, Thermo-Fisher) in the presence of 0.4
primer pairs except for RNA18S (0.2 M), according to the manufacturer’s instructions, in a reaction
volume of 10 L. In order to compute reaction efficiency, a standard curve was performed for each
µM
µ
µ
master mix. qPCR was performed in triplicate using and the CFX368 thermal cycler (Bio-Rad, Hercules,
CA, USA). The reaction conditions were 95 ◦C for 1 min, followed by 45 cycles at 98 ◦C for 5 s, and the
annealing–extension step for 5 s at 60 ◦C, with data collection. At the end of these cycles, a melting
curve (65–95 ◦C, with the plate read every 0.5 ◦C) was performed in order to assess the specificity of the
amplification product by single peak melting temperature verification. Results were normalized on the
reference genes and on the control according to the
∆Cq and ∆∆Cq method, respectively [50]. All data
analyses were performed with the built-in software (CFX Manager, Bio-Rad, Hercules, CA, USA).

Int. J. Mol. Sci. 2019, 20, 3074
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4. Conclusions
The Pt(IV)-NSAID complexes
2
and
3
showed higher antiproliferative activity on the cancer cell
lines under investigation when compared to cisplatin, 1, and the corresponding free NSAIDs, ketoprofen
and naproxen. The trend of activity can be explained in terms of lipophilicity of the compounds
(evaluated here by means of their log k’), that affects their cellular accumulation. This is often the main
reason of the increased activity of most Pt(IV) conjugates, defined as synergistic cellular accumulation.
On the contrary, the trend of activity seems to be scarcely dependent on COX-2 gene expression,
suggesting that a COX-2-independent mechanism may play a role (Figure 8). This mechanism has been
tentatively identified in the activation of NAG-1, a protein that has antitumorigenic and pro-apoptotic
propensity, linked, at least in part, to the chemoprevention activity of NSAIDs [59]. However, analyzing
the variation of pro-apoptotic BAD and BAX and anti-apoptotic BCL-2 genes, it appears clear that
cisplatin, released after Pt(IV)
→
Pt(II) reduction, is the component of the combos mainly responsible
for the overall antiproliferative activity—also because of its own NAG-1 activation. In conclusion, most
of the biological effects are related to the cisplatin metabolite. The NSAID axial ligands increase the
overall lipophilicity and, hence, the cellular accumulation of these combos, offering a minor (additive)
contribution in terms of NAG-1 activation.
Figure 8. Proposed mechanism of action of the Pt(IV)-NSAID complexes.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/12/
3074/s1.
Author Contributions: Data curation: M.R., I.Z., E.G., B.R., M.C., E.P, and D.O. Investigation: M.R., I.Z., E.G.,
B.R., M.C., and E.P. Supervision: M.R., I.Z., and D.O. Writing — original draft: M.R., I.Z., E.G., and D.O.
Funding: This research has the financial support of the Azienda Sanitaria Ospedaliera “S.S. Antonio e Biagio e
Cesare Arrigo” (ASO) of Alessandria (Italy), within the HERMES project.
Acknowledgments: We were indebted to the Inter-University Consortium for Research on the Chemistry of
Metals in Biological Systems (CIRCMSB, Bari, Italy) for providing opportunities for stimulating discussions during
the annual meetings. We thank Maria Grazia Bottone and Beatrice Ferrari (University of Pavia, Italy) for the
support in immunochemical analyses.

Int. J. Mol. Sci. 2019, 20, 3074
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Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
A2780
A-549
AR
ovarian endometroid adenocarcinoma cell line
lung carcinoma cell line
accumulation ratio
COX
cyclooxygenase
Cq
cycle quantification value
DMEM
DMF
Dulbecco’s Modified Eagle Medium
N,N-dimethylformamide
DNA
EDTA
ESI-MS
FBS
deoxyribonucleic acid
2,20,200,2000-(ethane-1,2-diyldinitrilo)tetraacetic acid or ethylenediaminetetraacetic acid
electrospray ionization - mass spectrometry
fetal bovine serum
HCT 116
HEPES
HT-29
IC50
ICP-MS
ICP-OES
k0
colorectal carcinoma cell line
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
colon adenocarcinoma cell line
half-maximal inhibitory concentrations
inductively coupled plasma - mass spectrometry
inductively coupled plasma – optical emission spectroscopy
RP-HPLC capacity factor
MPM
malignant pleural mesothelioma
MSTO-211H biphasic malignant pleural mesothelioma cell line
NAG-1
NMR
NSAIDs
PBS
PTFE
RNA
NSAID activated gene 1
nuclear magnetic resonance
non-steroidal anti-inflammatory drugs
phosphate buffered saline
polytetrafluoroethylene
ribonucleic acid
RP-HPLC
RPMI
RT-qPCR
SD
reversed-phase high performance liquid chromatography
Roswell Park Memorial Institute
quantitative reverse transcription polymerase chain reaction
standard deviation
SEM
standard error means
SW480
t0
colorectal adenocarcinoma
column dead time
tR
retention time
UV
ultraviolet
∆Cq/∆∆Cq
cDNA level of expression
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