Novel antitumor cisplatin and transplatin derivatives containing 1-methyl-7-azaindole: Synthesis, characterization, and cellular responses

Article abstract of DOI:10.1021/jm501420k

The current work investigates the effect of new bifunctional and mononuclear Pt(II) compounds, the cis- and trans-isomers of [PtCl₂(NH₃)(L)] (L = 1-methyl-7-azaindole, compounds 1 and 2, respectively), on growth and viability of huma

Full text of DOI:10.1021/jm501420k

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Article
Novel Antitumor Cisplatin and Transplatin Derivatives Containing 1
Methyl-7-Azaindole: Synthesis, Characterization and Cellular Responses
Jitka Pracharova, Teresa Saltarella, Tereza Radosova-Muchova, Simone
Scintilla, Vojt#ch Novohradský, Olga Novakova, Francesco Paolo Intini,
Concetta Pacifico, Giovanni Natile, Petr Ilik, Viktor Brabec, and Jana Kasparkova
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501420k • Publication Date (Web): 11 Dec 2014
Downloaded from http://pubs.acs.org on December 23, 2014
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Novel Antitumor Cisplatin and Transplatin Derivatives
Containing 1-Methyl-7-Azaindole: Synthesis, Characterization
and Cellular Responses
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†
‡
†
Jitka Pracharova, Teresa Saltarella, Tereza RadosovaꢀMuchova, Simone
‡
§,II
§
‡
Scintilla, Vojtech Novohradsky, Olga Novakova, Francesco P. Intini,
‡
‡
†
II
Concetta Pacifico, Giovanni Natile, Petr Ilik, Viktor Brabec, and Jana
,
§
Kasparkova*
†
Department of Biophysics, Centre of the Region Hana for Biotechnological and
Agricultural Research, Palacky University, Slechtitelu 11, 783 41 Olomouc, Czech
Republic
‡
Department of Chemistry, University of Bari "Aldo Moro", 70125 Bari, Italy
§
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i.,
Kralovopolska 135, CZꢀ61265 Brno, Czech Republic
II
Department of Biophysics, Faculty of Science, Palacky University in Olomouc,
Slechtitelu 11, 78371 Olomouc, Czech Republic
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ABSTRACT: The current work investigates the effect of new bifunctional and mononuclear
Pt(II) compounds, the cisꢀ and transꢀisomers of [PtCl (NH )(L)] (L = 1ꢀmethylꢀ7ꢀazaindole,
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compounds 1 and 2, respectively), on growth and viability of human carcinoma cells as well as
their putative mechanism(s) of cytotoxicity. The results show that substitution of 1ꢀmethylꢀ7ꢀ
azaindole for ammine in cisplatin or transplatin results in an increase of the toxic efficiency,
selectivity for tumor cells in cisplatinꢀresistant cancer cells and activation of the trans geometry.
The differences in the cytotoxic activities of 1 and 2 were suggested to be due to their different
DNA binding mode, different capability to induce cell cycle perturbations and fundamentally
different role of transcription factor p53 in their mechanism of action. Interestingly, both isomers
make it possible to detect their cellular uptake and distribution in living cells by confocal
microscopy without their modification with an optically active tag.
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INTRODUCTION
Cisplatin [cisꢀdiamminedichloridoplatinum(II)] and other platinumꢀbased anticancer agents are
well known to be effective in the treatment of many human malignancies. However, the ultimate
success of these agents is often limited by the natural or acquired drug resistance and side effects.
These drawbacks have fostered the search for new antitumor platinumꢀbased drugs with fewer
side effects and more effective outcomes. New mononuclear bifunctional platinum drugs have
been derived from cisplatin or ineffective transplatin [transꢀdiamminedichloridoplatinum(II)] by
replacing one or both ammine groups with other nonleaving Nꢀdonor ligands. The design of such
derivatives has been inspired by the observation that the carrier ligands can deeply affect the
pharmacological behavior of the drug including cell accumulation and distribution, activation,
DNA binding, and cellular responses to the DNA damage. In other words, nonleaving ligands of
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ꢀ6
cisplatin or transplatin derivatives can modulate their biological properties.
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, 8
Some of us have shown in recent papers that replacement of the ammine ligands of cisplatin
by 7ꢀazaindole derivatives results in considerable enhancement of cytotoxicity against several
human cancer cell lines. Thus, the rational chemical design can be applied also to this class of
compounds since key substituents at the 7ꢀazaindole ring system can play a major role in
controlling the chemical and biological properties, such as cellular accumulation and distribution,
effects at the level of cell cycle regulation, binding to DNA, propensity for DNA adduct repair,
and reactivity toward sulfurꢀcontaining nucleophiles. Hence, substituted 7ꢀazaindoles can offer a
good opportunity for the synthesis of novel therapeutic agents.
The rational design of novel platinum anticancer drugs can also benefit from early
information on the trafficking and localization of platinum drugs in tumor cells. Therefore,
confocal microscopy has been successfully applied to investigate the cellular distribution of Pt
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complexes. However, this relatively simple detection technique, which enables the visualization
of compounds in living cells, requires the use of drugs which, after illumination, emit radiation
detectable with sufficiently sensitive detectors. Cisplatin and most Pt anticancer complexes do
not possess this property and need to be modified with an optically active tag. These alterations
can affect chemical properties of the complexes, creating artifacts such as altered specificity and
other biological effects. Hence, in order to obtain significant information about the in vivo
trafficking and localization of a platinum drug in living tumor cells, the modified platinum
complex must be as similar as possible to the real drug.
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Several attempts have been made to obtain highꢀresolution images of the cellular accumulation
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and subcellular distribution of fluoresceinꢀtagged Pt(II) complexes (see Refs. as examples).
Interestingly, derivatives of cisplatin and transplatin with one ammine and one aromatic Nꢀdonor
heterocycle (L) (cisꢀ and transꢀ[PtCl (NH )L]) have proved to be endowed with good antitumor
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activity and one of these compounds, picoplatin [cisꢀamminedichlorido(2ꢀ
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methylpyridine)platinum(II)], is under advanced clinical trials with changing fortunes.
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opened the possibility to use a Nꢀdonor heterocycle which is intrinsically fluorescent, such as 7ꢀ
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azaindole, for monitoring the cell permeation and distribution of the platinum drugs. However,
coordination to platinum results in almost complete quenching of the fluorescence. In addition, 7ꢀ
azaindole exhibits a pHꢀdependent fluorescence. Hence, since the pH is different in different
cellular compartments, the use of a probe, whose fluorescence is dependent on pH, appears
disadvantageous. The latter drawback can be circumvented by derivatization of 7ꢀazaindole with
a methyl group.
In the present work we investigate the cytotoxic potential and molecular and cellular
pharmacology of a new cisplatin derivative bearing a nonleaving 1ꢀmethylꢀ7ꢀazaindole ligand
(hereafter called 1M7AI) and its trans isomer. We report the synthesis and characterization of
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both the cisꢀ and transꢀisomers of [PtCl (NH )(1M7AI)] (1 and 2, respectively, Figure 1), the
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crystal structure of 1, and several biological properties of 1 and 2, such as cytotoxicity, cellular
accumulation, impact on cell cycle regulation, triggering of apoptosis and involvement of p53
pathway in the mechanism of action. Moreover, the optical properties were utilized to follow the
cellular internalization and distribution of the complexes in tumor cells.
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RESULTS AND DISCUSSION
Synthesis and Characterization of the Platinum Complexes. The Pt(II) complexes under
investigation have one ammine and one Nꢀdonor aromatic heterocycle (1M7AI). For comparison,
both the cisꢀ and transꢀ[PtCl (NH )(1M7AI)] isomers were synthesized and fully characterized.
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The cis complex, cisꢀ[PtCl (NH )(1M7AI)] (1), was prepared according to a previously
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reported procedure for the synthesis of analogous compounds,
with some modifications.
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Starting from K[PtCl NH ], the reaction with 1M7AI leads to substitution of one of the two
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trans chlorides (both cis to the ammine) which are mutually transꢀlabilized. The reaction is quite
slow at room temperature (complete in 4 days) and affords the desired product in moderate yield,
the main sideꢀproduct being the cationic species [PtCl(NH )(1M7AI) ]Cl that can be easily
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removed (Scheme S1).
The synthesis of the trans complex, transꢀ[PtCl (NH )(1M7AI)] (2), requires a different
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2+
procedure in which cisꢀ[PtI (NH ) ] is first converted to cisꢀ[Pt(H O) (NH ) ] (reaction with
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3 2
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3 2
2
+
AgNO ) and then to cisꢀ[Pt(NH ) (1M7AI) ] (reaction with 1M7AI. 1:2 ratio). Treatment with
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3 2
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excess KI – a ligand with strong transꢀlabilizing effect – leads to substitution of two trans ligands
one ammine and one 1M7AI) and formation of transꢀ[PtI (NH )(1M7AI)]. The diiodido species
(
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can be converted to the dichlorido species (2) by reaction with AgNO (1:2 molar ratio) in water
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solution followed by addition of excess KCl (Scheme S2).
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Both 1 and 2 were characterized by elemental analysis, ESIꢀMS, and H and Pt NMR
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spectroscopy. The Hꢀspectra of both compounds show five signals between 9 and 6.5 ppm
belonging to aromatic protons, one singlet close to 5.2 ppm belonging to the methyl group, and
one broad signal close to 4.0 ppm belonging to the amminic protons. In general, protons are more
shielded in the trans than in the cis isomer. It is noteworthy the remarkable downfield shift (ꢁδ >
1.30 ppm) of the methyl protons upon coordination of 1M7AI to platinum which witnesses the
closeness of the methyl group to the deshielding cone originated by the platinum quadrupole in
the direction perpendicular to the coordination plane. The assignment of individual aromatic
protons was accomplished by 2D COSY experiments (Figure S1, see Schemes S1 and S2 for the
numbering of protons).
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The coordination of 1M7AI to platinum via N7 causes a JH6,Pt coupling which is greater for 1
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(1M7AI trans to Cl, J
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= 32 Hz) indicating
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H6,Pt
ꢀ
that the ligand trans to 1M7AI has a better σꢀdonor ability in 2 (NH ) than in 1 (Cl ). These
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results are in full agreement with literature data, and fully support the assignment of the isomer
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configurations (cis or trans). The Pt chemical shifts were nearly coincident for 1 and 2 (–1,970
and –1,967 ppm, respectively) and fall in the range found for analogous compounds with N Cl2
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set of donor atoms. It is interesting to note that also the methyl protons of 1M7AI are coupled
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with platinum ( J = 10 Hz) (Figure S2). A similar through space coupling has been observed in
H,Pt
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few other cases.
That this is a clear case of throughꢀspace coupling is also witnessed by the
absence of detectable coupling between Pt and H which are separated by four bonds, one less
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than the 1ꢀMe protons.
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X-ray Structure. An ORTEP drawing of the Xꢀray structure of complex 1 is shown in Figure
S3, while the association of two molecular units in a dimeric aggregate is shown in Figure 2.
Selected bond lengths and angles are given in Table 1.
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The Pt atom has the expected square planar coordination geometry, and the donor atoms are
two chlorides and two nitrogens in cis positions. The 1M7AI ligand is planar, and his plane is
nearly perpendicular to that of coordination [dihedral angle of 82.2(1)°] so to minimize steric
interactions with cis ligands.
The platinumꢀligand distances (2.045[7] Å for Pt−N and 2.300[2] Å for Pt−Cl) are in good
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agreement with values found in structurally related complexes.
The C−N and C−C distances
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in the heteroaromatic ligand are in accord with an extensive πꢀbond delocalization. The
complex displays a very short platinumꢀmethyl distance [Pt⋯C(10) distance of 3.203(8) Å];
moreover one methyl proton is directed toward the platinum atom indicating a Hꢀbond type
interaction [Pt⋯H(C) distance of 2.78(9) Å with a Pt⋯H–C angle of 105°], as confirmed by the
downfield shift of the methyl protons with respect to the uncoordinated ligand. In previous
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reports, similar M⋯H–C interactions for d square–planar complexes have been described as
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anagostic interactions. The same methyl forms a hydrogen bond with the chloride of another
complex unit related by an inversion center (Figure 2). Within the dimeric aggregate, two methyl
groups are placed at very short distance [C8···C8(ꢀx + 1, ꢀy, ꢀz+1) distance of 3.11(1) Å)], much
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shorter than the sum of the van der Waals radii (3.40 Å). Other C···C distances shorter than the
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sum of van der Waals radii have been found for methyl groups in the IsoStar database. However
in those cases (three) the C···C distances were longer (between 3.2 and 3.3 Å) than in the present
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The H atoms of the methyl group (found in the difference Fourier map) exhibit N−C−H angles
smaller than 109° (N1−C10−H10A, N1−C10−H10B, and N1−C10−H10C angles of 104°, 108°,
and 104°, respectively), arguably connected to Hꢀbond formation (HA and HC) and short
C10···C10 distance. Intermolecular interactions between amminic groups and chlorido ligands of
different molecular units (the chlorido not involved in interaction with the Me of 1M7AI) support
the growth of the supramolecular assembly of complex molecules [N2···Cl1(x + ½, ꢀ y + ½, ꢀ z +
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) 3.388(7) Å, (N2)H22···Cl1(x + ½, ꢀ y + ½, ꢀ z + 1) 2.82(9) Å, N2−H22···Cl1(x + ½, ꢀ y + ½, ꢀ
z + 1) 143(9)°; N2···Cl1(ꢀ x + ½+1, y ꢀ ½, z) 3.408(7) Å, (N2)H23···Cl1(ꢀx + ½+1, y ꢀ ½, z)
.69(9) Å, N2−H23···Cl1 (ꢀx + ½ +1, y ꢀ ½, z) 139(9)°]. In Figure S4 a centrosymmetric
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tetrameric aggregate of four subunits is shown. In each tetrameric aggregate there are two nonꢀ
classical C−H···π−C2−C3 contacts [distance between (C)H and π−C2−C3 centroid of 2.65 Å].
Cytotoxicity. The cytotoxic activity of 1 and 2 was determined against human ovarian
carcinoma cell lines A2780 (cisplatin sensitive) and A2780cisR (with acquired resistance to
cisplatin) and human breast adenocarcinoma cells MCFꢀ7 (inherently resistant to cisplatin)
commonly used to test cytotoxic activity of cisplatin derivatives and other antitumor
metallodrugs. The tumor cell lines were incubated with the platinum compounds and cell survival
in cultures was evaluated as described in the "Experimental section". The IC values
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(concentration of compound which causes death in 50 % of cells) for 72 h of treatment are
reported in Table 2; both compounds showed promising activity.
The activity of 1 and 2 was comparable to that of cisplatin in cisplatinꢀsensitive A2780 cells,
but, importantly, both compounds were markedly more toxic than cisplatin in cisplatinꢀresistant
A2780cisR cells and also moderately more toxic in MCFꢀ7 (inherently resistant to cisplatin).
Furthermore, the resistance factor [defined as the ratio of IC values in resistant (A2780cisR)
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and cisplatinꢀsensitive parent cells (A2780)] was 1.0 and 0.8 for 1 and 2, respectively, whereas it
was markedly higher for cisplatin (6.5). This suggests that the mechanism underlying the
biological action of 1 and 2 is somewhat different from that of cisplatin allowing both compounds
to overcome successfully the resistance mechanisms operating in the case of cisplatin.
Apart from the ability of compounds 1 and 2 to kill tumor cells resistant to clinically used
cisplatin, the low cytotoxicity of 1 in noncancerous human primary skin fibroblasts demands
special attention. The IC of 1 in noncancerous cells is more than one order of magnitude greater
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than that found for the sensitive cancer cell line A2780. Thus, 1 shows selectivity for tumor cells
relative to the nontumorigenic, normal, cells. Whereas the selectivity of 1, in comparison with
that of cisplatin, is less in A2780 cells (sensitive to cisplatin), it is higher in A2780cisR cells
(resistant to cisplatin). The trans derivative 2 is somewhat less selective than 1. Nevertheless its
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design and synthesis represents a broadening of the database of Pt ꢀdichlorido compounds in
which the trans geometry can be effectively activated by the replacement of one NH ligand in
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transplatin by a planar Nꢀdonor heterocycle.
Cellular Accumulation. One of the key factors affecting the biological activity of metalꢀ
based drugs is the intracellular accumulation. To examine the ability of 1 and 2 to enter and
accumulate in tumor cells, A2780, A2780cisR and MCFꢀ7 cells were incubated with the platinum
complexes for 5 and 24 h exposure times (A2780 cells were incubated with the platinum
complexes also for 3 and 12 h) and the platinum content measured. The amount of platinum
associated with the cells after only 10 s of treatment was also determined and assumed to
represent the level of platinum adsorption on the cells surface. These values were subtracted from
those found after 3, 5, 12 or 24 h exposure time. The platinum adsorption on the cell surface was
approximately the same for 1 and 2 and very similar to that of cisplatin (not shown).
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The intracellular accumulation of platinum from 1 was somewhat lower than that from
cisplatin in cisplatin sensitive A2780 cells while it was very similar to that from cisplatin in
resistant A2780cisR cells. Therefore the enhanced cytotoxic activity of 1 toward A2780cisR cells
is not due to enhanced accumulation of the drug. Unlike A2780 and A2780cisR cells, the
accumulation of platinum from 1 was greater than that from cisplatin in MCFꢀ7 cells (2ꢀ3ꢀfold
increase), in agreement with the greater cytotoxicity of 1 toward this cell line.
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Unlike 1, the cellular uptake of platinum from 2 was markedly greater (3 ꢀ 9ꢀfold) than that
from cisplatin in all cell lines tested in this work (Table 3, S1 and Figure S5). This is in line with
the lower polarity of trans compounds as compared to cis ones. A scale of hydrophobicity can be
deduced from log P values for octanol/water partition. Log P values for cisplatin, 1 and 2 were ꢀ
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1.92, ꢀ0.66 and 0.83, respectively, confirming that 2 is the most hydrophobic.
Notwithstanding the greater accumulation, the toxicity of 2 in these cancer cells is comparable
to that of 1 (Table 2). For compounds 1 and 2, the cellular uptake of platinum from these
complexes remains constant for sensitive and resistant ovarian cancer cells as does their
cytotoxicity. In contrast, the platinum uptake from cisplatin decreases by 1.5 ꢀ 2ꢀfold which
results in an even greater decrease of cytotoxicity (6.5ꢀfold). The greater toxicity of 1 and 2
toward MCFꢀ7 cells, which are intrinsically resistant to cisplatin, correlates with a greater degree
of platinum uptake from these drugs.
DNA Platination in Cells Exposed to Platinum Complexes. The cytostatic action of
conventional platinum anticancer drugs is connected with their binding to nuclear DNA. The
degree of DNA platination was determined by inductively coupled plasma mass spectrometry
(ICPꢀMS) for the three cell lines after their exposure to 1, 2 or cisplatin for 5 or 24 h (Table 4).
The amount of platinum associated with DNA, expressed as percentage of total platinum
accumulated in the cell, are reported in Table 5.
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After both 5 h and 24 h exposure times, the degree of DNA platination was comparable for 1
and cisplatin. Notably, while for cisplatin the degree of DNA platination was considerably lower
in cisplatinꢀresistant A2780cisR cells than in sensitive A2780 cells, this was not the case for
complex 1. These accumulation studies also suggest that in the case of cisplatin the reduced
toxicity toward A2780cisR cells is only in part due to reduced drug uptake, while there will be
also a contribution from a higher rate of DNA repair, as clearly shown by the results at 24 h
incubation time. Overall, DNA platination appears to correlate with toxicity of 1 and cisplatin in
the tumor cell lines tested in this work. This finding is in good agreement with the view that,
similarly to cisplatin, DNA is the biological target also for 1.
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Notably, for all cell lines and for all exposure times the amount of platinum bound to DNA
was significantly greater (1.5 ꢀ 9ꢀfold) in the case of 2 than in the case of 1. However, 2 had a
cytotoxicity comparable to that of 1 (Table 3). Assuming that DNA is the pharmacological target
also for Pt complexes with 1M7AI, these results suggest that 2 modifies DNA differently from 1
and that DNA adducts of 2 are inherently less effective than those of 1.
From the data reported in Tables 3 and 4 it is possible to estimate the percentage of total
platinum in the cells that binds to DNA. In most cases the percentage is lower for 2 than for 1 or
cisplatin (Table 5).
DNA and Protein Binding in Cell-free Media. The experiments described above reveal that
for 1 and 2 there is no direct correlation between the intracellular accumulation and the degree of
DNA platination. To clarify this result, the DNA binding properties of 1 and 2 were studied in
cell free media and compared to that of cisplatin. The platinum complexes were incubated with
calf thymus (CT) DNA at r (r is defined as the molar ratio of free platinum complex to
i
i
nucleotide phosphates at the onset of incubation with DNA) of 0.05 in NaClO (10 mM) at 310 K
4
in the dark. Aliquots were withdrawn at various time intervals, the free (unbound) platinum
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compound was removed by exhaustive dialysis, and the content of platinum and the
concentration of DNA were determined by flameless atomic absorption spectrometry (FAAS)
and absorption spectrophotometry, respectively. The results, shown in Figure 3A, indicate that
after 24 h of incubation the platinum bound to DNA is 69 ± 4% and 95 ± 3 % for 1 and 2,
respectively (cisplatin binds to DNA quantitatively). These DNA binding experiments indicate
that irreversible binding to DNA is somewhat slower for 1 than for 2 and cisplatin. Therefore, the
lower portion of platinum bound to nuclear DNA, with respect to the total platinum taken up by
the cell after 24 h, in the case of 2 cannot be a consequence of its lower DNA binding affinity. In
other words, other factors, such as greater trapping of more lipophilic 2 into the cell membrane or
greater reactivity of the trans compound toward intracellular nucleophiles other than DNA (such
as sulfurꢀcontaining species) may play a role.
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The possibility that the intracellular pool of 1 and 2 that can reach and bind DNA can be
reduced differently for these platinum complexes by interaction with intracellular nucleophiles
was tested by investigating the interaction of 1 and 2 with HSA. Therefore, 1 or 2 were incubated
with HSA at molar ratio 1:1 in Tris.HCl (10 mM, pH 7.4) and in the presence of NaCl (8 mM) at
310 K in the dark. Aliquots were withdrawn at various time intervals, the free (unbound)
platinum compound was removed by exhaustive dialysis, and the content of platinum and the
concentration of HSA were determined by FAAS and absorption spectrophotometry,
respectively. The reaction of 2 with HSA was noticeably faster than that of 1 (Figure 3B). Based
on this outcome, we can hypothesize that a significantly higher fraction of 2 is trapped in the
cytoplasm by reaction with intracellular proteins, peptides and/or other intracellular thiols and
cannot access DNA. The higher reactivity of 2 towards the biomolecules present in the cytoplasm
can thus be responsible for its lower efficiency to platinated DNA despite its higher accumulation
in the cell.
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Transcription Mapping. Samples of DNA (the NdeI/HpaI fragment of pSP73KB plasmid)
modified by 1 or 2 for analysis by transcription mapping were prepared in NaClO (10 mM) at
4
310 K. After 24 h incubation with the complex, the DNA sample was precipitated with ethanol,
then redissolved in the medium necessary for the transcription mapping analysis, while the rb
value (the number of molecules of platinum complex bound per nucleotide residue) in these
samples was determined by platinum FAAS and by measurement of absorbance at 260 nm. By
adopting this procedure, the transcription mapping analysis (described below) is performed in the
absence of unbound (free) Pt complex.
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Cutting of pSP73KB DNA by NdeI and HpaI restriction endonucleases yields a 212ꢀbase pairs
(
bp) fragment (a substantial part of its nucleotide sequence is shown in Figure 4B). This fragment
contains a T7 RNA polymerase promotor. This DNA template was modified by 1 or 2 at r = 0.01
b
and its ability to terminate the in vitro RNA synthesis by T7 RNA polymerase at the level or in
the proximity of platinumꢀDNA adducts was evaluated (Figure 4A). The major stop sites
observed for 1 correspond to the sites for cisplatin, indicating similar DNA binding modes for
both cis compounds. On the other hand, the pattern of stop sites obtained for compound 2 is
similar to that found for transplatin (e.g. much less regular with respect to those of the cis
compounds, Figure 4B).
Localization in Tumor Cells. A2780 cells were first exposed to 1 or 2 (30 µM) for 5 h and
then visualized by confocal microscopy (Figure 5). After the treatment, cells were carefully
washed with platinumꢀfree medium so that no residual free platinum complex was present in the
growing medium. No signal was yielded by the untreated cells (Figure 5C) or the cells treated
with free 1M7AI (Figure 5F) or cisplatin (not shown), which suggests that the signal yielded by
the cells treated with 1 or 2 comes mainly from the platinum complex containing 1M7AI ligand.
The resulting images show that the subcellular localization of the light is largely nonꢀnuclear.
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This is in good correlation with previously described data showing that only about 1% of total
cellꢀassociated platinum is localized on nuclear DNA. The signal appears to be localized mostly
in cell organelles (for example in lysosomes or late endosomes) rather than on cytoplasmic
membranes (Figures 5A,B). Data shown in Figures 5D,E are consistent with the thesis that the
intracellular accumulation of 1 and 2 increases with the time of treatment and that the
intracellular accumulation of 2 is significantly higher than that of 1.
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Cell Cycle Analysis. To further characterize the cytotoxic effect of 1 and 2, an analysis of the
cell cycle perturbations was performed in A2780 cells exposed to 1, 2 or to the reference
compound cisplatin. Activation of cell cycle checkpoints is a general cellular response to
cytotoxic agents. Previous studies have indicated that cisplatin and other platinum agents
3
1, 32
predominantly inhibit cell cycle progression at the S and/or G phase.
Cells were exposed to
2
1
, 2, or cisplatin at their IC concentrations (Table 2) for 24 or 72 h. Significant differences in
50
cell cycle modulation between 1 and 2 were observed already after 24 h of treatment and the
differences became more pronounced as the treatment time increased (Figure 6).
Exposure of A2780 cells to 1, 2, or cisplatin led to the appearance of a population in the subꢀ
G region (more evident after 72 h exposure), where apoptotic and necrotic cells are found.
1
Therefore, the appearance of a subꢀG peak is consistent with the onset of internucleosomal DNA
1
32
cleavage in late apoptosis. Importantly, treatment with 2 affected the G /G populations less
0
1
than treatment with 1 or cisplatin. Unlike 2, 1 and cisplatin caused significant accumulation of
cells in the Sꢀphase of the cell cycle at short exposure time (24 h) and cell cycle block in G2ꢀ
phase at long exposure time (72 h). These results are in agreement with data previously reported
3
2, 33
for cisplatin
and highlight the similarity between cell cycle perturbations induced by 1 and
cisplatin whereas 2, having trans geometry, behaves differently.
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Detection of Apoptosis and Necrosis. The levels of apoptosis and necrosis induced in A2780
cells by 1, 2 or cisplatin at various concentrations over 24 and 72 h exposure time was quantified
by the cell death detection ELISA colorimetric kit. The assay is based on the quantitative
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“
sandwich enzyme immunoassay” principle using mouse monoclonal antibodies directed against
DNA and histones. This allows the specific determination of monoꢀ and oligonucleosomes
histoneꢀassociated DNA fragments) in the cytoplasmic fraction of the cell lysates. The relative
(
amounts of cytoplasmic histoneꢀassociated DNA fragments are measured after the induction of
apoptosis or when these fragments are released from necrotic cells. Figure 7 shows DNA
fragmentation induced by 1, 2, or cisplatin in A2780 cells.
The results demonstrate that treatment with all tested antitumor agents leads to concentration
dependent apoptotic events in the A2780 cell line (Figure 7A). Importantly, 1 induces a lower
level of DNA fragmentation, due to apoptosis, in comparison with cisplatin over the whole range
of concentrations used. In contrast, 2 was less effective in inducing DNA fragmentation at low
concentration (IC ) compared to cisplatin, whereas at higher concentrations (corresponding to
2
0
IC and IC ) it was more effective than cisplatin. A similar ELISA assay was also used to detect
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the extent of necrosis induced by 1, 2, or cisplatin. Importantly, the level of necrosis was
significantly lower compared to that of apoptosis triggered by 1, 2, or cisplatin (Figure 7B). At
long treatment times, apoptosis predominates over necrosis, with 2 being less efficient in
inducing apoptosis compared to the cisꢀcompounds 1 and cisplatin (Figure 7B). The data are
consistent with the results of flow cytometric measurements (Figure 6) where 2 was the most
effective in inducing sub G population of cells after 24 h of incubation, but the least efficient
1
after 72 h.
To further explore this finding, we determined the cytotoxicities of 1, 2 and cisplatin also after
a short time of treatment. After 24 h of incubation of A2780 cells with the Pt(II) complexes, the
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IC values assessed by the MTT test [MTT = 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
5
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diphenyltetrazolium bromide] were 47 ± 2, 9.4 ± 0.8, and 14.9 ± 0.9 µM for 1, 2, and cisplatin,
respectively. These data also confirm that 2 is more effective in the early phases of the treatment,
whereas the cytotoxic effect of 1 is manifested mostly after longer period of incubation (Figure
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The Involvement of p53 Pathway in the Mechanism of Action. The promotion of apoptosis
by the platinum complexes may be connected with the induction of tumor suppressor p53, which
plays a proꢀapoptotic role.[Amaral, 2010 #6433] To investigate the possible involvement of p53
pathway in the mechanism of action of 1 and 2, the effect of these platinum complexes on the
+
/+
viability of human colon carcinoma cells expressing p53 (HCT116 p53 ) or non expressing p53
ꢀ/ꢀ
(HCT116 p53 ) was investigated. The cells were treated with 1, 2 or (for comparative purposes)
cisplatin for 72 h as described in the Experimental section. The IC values and DNA content in
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3
4
the treated cells were determined as described in a previously published article. The data
showed that cisplatin and 1 significantly reduced the number of cells in a doseꢀdependent
manner, being more effective in p53ꢀproficient cells compared to the p53ꢀknockꢀout cells (Table
2
and Figures 9A,B). These results suggest that transcription factor p53 plays an important role in
the mechanism of action of 1. In this regard, the mechanism of action of 1 appears to be similar to
that of cisplatin, which was shown to be more effective in p53ꢀwild type over the p53ꢀmutant
3
5
+/+
ꢀ/ꢀ
tumor cells. In contrast, the effectivity of 2 in killing p53 and p53 cells was not too different
(Table 2 and Figure 9C) indicating that 2 operates via a pathway that is p53ꢀindependent. That
our new transplatin derivative 2 operates via a p53ꢀindependent pathway is in a fundamental
contrast to what has been reported for other antitumorꢀactive derivatives of transplatin, namely
36
transplatinum complexes containing a planar, Nꢀdonor, heterocycle. Thus, this is to the best of
our knowledge the first demonstration that p53 does not play a role in the toxic effects induced in
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tumor cells by the transꢀplatinum drugs. A plausible implication of this finding is that 2 might be
active in vivo in contrast to other activated transplatinum complexes hitherto tested in vivo. The
work is in progress to test this challenging hypothesis.
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CONCLUSIONS
Two new bifunctional and mononuclear Pt(II) compounds, having cisꢀ and transꢀ geometry
and containing the 1ꢀmethylꢀ7ꢀazaindole ligand, were synthesized, fully characterized, screened
for their toxicity against cancer cell lines, evaluated for their efficiency to enter the cells, damage
DNA, affect cell cycle, and induce apoptosis. When compared with cisplatin, substitution of 1ꢀ
methylꢀ7ꢀazaindole for ammine results in an increase of the toxic efficiency and selectivity for
tumor cells in cisplatinꢀresistant cancer cells. On the other hand, such a substitution activates the
trans geometry so that the cytotoxicity of the trans isomer, compared to that of cisplatin, is nearly
equivalent in cisplatin sensitive tumor cells and markedly better in cisplatin resistant tumor cells.
The ability to overcome resistance to cisplatin appears to be associated with an increased level of
cellular DNA platination by these compounds as compared to cisplatin. Although the trans
isomer enters cells more easily due to its higher lipophilicity, it is also more easily trapped in the
cytoplasm by faster reactions with intracellular nucleophiles when compared with its cis
congener, with consequent reduction of binding to cellular DNA. For both isomers the cytotoxic
effect is accompanied by triggering of apoptosis and cell cycle arrest in S and G /M phases.
2
Interestingly, whilst the trans isomer demonstrates its biological action in the early phases of the
treatment, the cytotoxic effect of the cis isomer is more pronounced after longer incubation times.
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The differences in the biological (cytotoxic) activities of 1 and 2 may be associated with their
different DNA binding mode, different capability to induce cell cycle perturbations and
fundamentally different role of transcription factor p53 in their mechanism of action.
Notably, both isomers make it possible to detect their cellular uptake and distribution in living
cells by confocal microscopy without their modification with an optically active tag. Moreover,
our current work not only reinforces the potential utility of the transꢀplatinum compound to act as
antitumor drugs, but also indicates that transplatin derivatives containing a 7ꢀazaindole ligand can
be subjected to different chemical modifications resulting in different biological properties,
therefore representing an ideal starting point for further developments.
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EXPERIMENTAL SECTION
Commercial reagent grade chemicals and solvents were used as received without further
purification. Cisplatin, DMF, HSA, and propidium iodide were from SigmaꢀAldrich (Prague,
Czech Republic). 1ꢀMethylꢀ7ꢀazaindole was from Ark Pharm, Inc. (Libertyville, USA). CT DNA
7
(
42% G+C, mean molecular mass ca. 2x10 ) was prepared and characterized as described
3
7, 38
previously.
Plasmid pSP73KB (2455 bp) was isolated according to standard procedures.
Restriction endonucleases were purchased from New England Biolabs. Acrylamide and
bis(acrylamide) were obtained from Merck KgaA (Darmstadt, Germany). MTT was from
3
2
Calbiochem (Darmstadt, Germany). [αꢀ P]CTP was obtained from MP Biomedicals, LLC
(
Irvine, CA). RNase A was from Qiagen (USA). DNazol (genomic DNA isolation reagent) was
obtained from MRC (Cincinnati, OH). RPMI 1640 medium, fetal bovine serum (FBS),
trypsin/EDTA, and Dulbecco's modified Eagle's medium (DMEM) were from PAA (Pasching,
Austria). Penicilin, gentamycin and streptomycin were from Serva (Heidelberg, Germany).
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Riboprobe Gemini System II for transcription mapping containing T7 RNA polymerase was
purchased from Promega (Madison, WI). Cell death detection ELISA plus kit was from Roche
Molecular Biochemicals (Mannheim, Germany).
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Synthesis. K[PtCl (NH )] and cisꢀ[PtI (NH ) ] were prepared according to already reported
3
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2
3 2
1
5, 39
procedures.
The elemental analysis and the spectroscopic features were fully consistent with
the data reported in the literature.
Cisꢀ[PtCl (NH )(1M7AI)] (1). K[PtCl (NH )] (0.26 g, 0.71 mmol) was suspended in CH OH
2
3
3
3
3
(40 mL) and treated with a slight excess of 1M7AI (0.115 g, 0.84 mmol). The mixture was kept
under stirring at room temperature for 4 days and then the suspension was filtered through Celite
and the pale yellow solution was evaporated to dryness under reduced pressure. The solid residue
was triturated with CH Cl (8 mL) in order to remove the excess of ligand. The obtained
2
2
suspension was filtered off and the resulting yellow solid was dried under vacuum, washed with
warm water (328 K) to remove KCl, dried again under vacuum and analyzed by TLC using
dichloromethane/acetone 1:1 as eluent. The chromatogram showed one single spot,
corresponding to 1, in addition to some nonꢀeluting side products. Therefore, the raw material
was subjected to silica gel chromatography, using dichloromethane/acetone 1:1 as eluent. Were
obtained 0.14 g (0.34 mmol, 48% yield) of 1. Anal. Calculated for cisꢀ
[
PtCl (NH )(1M7AI)]·CH OH (C H N Cl Pt·CH OH): C, 24.16; H, 3.38; N, 9.40%. Found: C,
2
3
3
8
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3
2
3
1
23.83; H, 2.99; N, 9.65%. HꢀNMR (acetoneꢀd , numbering scheme of 1M7AI as in Schemes S1
6
and S2) δ: 8.89 (m, 1 H, H ), 8.06 (m, 1 H, H ), 7.55 (m, 1 H, H ), 7.10 (m, 1 H, H ), 6.63 (d, 1
6
4
2
5
195
H, H ), 5.24 (s, 3 H, CH ), 4.10 (b, 3 H, NH ) ppm. PtꢀNMR (acetoneꢀd ) δ: −1,970 ppm. ESIꢀ
3
3
3
6
+
MS: calculated for [C H N Cl PtNa] 437.98. Found: m/z (% relative to the base peak) 437.9
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2
+
(
100), [M + Na] .
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Transꢀ[PtCl (NH )(1M7AI)] (2). Compound 2 was prepared by a twoꢀstep process which
2
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involved the preparation of the intermediate species transꢀ[PtI (NH )(1M7AI)].
2
3
Transꢀ[PtI (NH )(1M7AI)] was prepared from cisꢀ[PtI (NH ) ] and 1M7AI. cisꢀ[PtI (NH ) ]
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1.01 g, 2.09 mmol) was reacted with AgNO (0.71 g, 4.19 mmol) in H O (50 mL) and kept at
3 2
3
28 K in the dark for 20 min. The AgI precipitate was separated by filtration and the clear
solution treated with a twofold excess of 1M7AI (0.55 g, 4.19 mmol). The mixture was heated at
43 K for 3 h; then the suspension was filtered through Celite and the yellow mother solution
3
2+
containing cisꢀ[Pt(NH ) (1M7AI) ] was treated with an aqueous solution of KI (1.74 g, 1.05
3
2
2
mmol), added dropwise over a period of 5 min. The solution was stirred at 343 K for 2 h,
meanwhile a brown precipitate formed. The brown solid was collected by filtration of the mother
liquor and left to dry under vacuum. The solid residue was triturated with a mixture of
acetone/dichloromethane (1:1, v/v; 100 mL), the solution was separated by filtration, the solvent
was evaporated under reduced pressure, and the solid residue analyzed by TLC using
dichloromethane/acetone 9:1 as eluent. The chromatogram showed two spots, corresponding (in
order of increasing R ) to transꢀ[PtI (NH )(1M7AI)] and transꢀ[PtI (1M7AI) ]. Therefore, the
f
2
3
2
2
raw material was subjected to silica gel chromatography, using dichloromethane/acetone 9:1 as
eluent. Were obtained 0.250 g (0.42 mmol, 20% yield) of transꢀ[PtI (NH )(1M7AI)]. Anal.
2
3
Calculated for transꢀ[PtI (NH )(1M7AI)] (C H N I Pt): C, 16.05; H, 1.84; N, 7.02%. Found: C,
2
3
8
11 3 2
1
16.65; H, 1.91; N, 6.89%. HꢀNMR (acetoneꢀd , numbering scheme of 1M7AI as in Schemes S1
6
and S2) δ: 8.72 (m, 1 H, H ), 7.97 (m, 1 H, H ), 7.50 (m, 1 H, H ), 7.03 (m, 1 H, H ), 6.59 (d, 1
6
4
2
5
195
H, H ), 5.17 (s, 3 H, CH ), 3.88 (b, 3 H, NH ) ppm. PtꢀNMR (acetoneꢀd ) δ: −3,240 ppm. ESIꢀ
3
3
3
6
+
MS: calculated for [C H N I PtNa] 621.07. Found: m/z (% relative to the base peak)
8
11 3 2
+
6
21.05(100), [M + Na] .
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The dichlorido complex 2 was prepared from transꢀ[PtI (NH )(1M7AI)] (0.11 g, 0.18 mmol)
2
3
dissolved in H O (50 mL) and treated with AgNO (0.060 g, 0.37 mmol). After 3 h stirring at 323
2
3
K and in the dark, the solution was filtered (to remove the AgI precipitate), treated with an excess
of KCl (0.13 g, 1.8 mmol) and kept at 328 K for 1 h, during which time a brown solid
precipitated from the solution. The suspension was concentrated to a minimum volume under
reduced pressure, while keeping the reaction vessel at 328 K, and then cooled to 277 K in order
to promote further precipitation of the desired product. The solid residue was transferred on a
glass filter, washed with a minimum amount of cold water, dried under vacuum and analyzed by
TLC using dichloromethane/acetone 9:1 as eluent. The chromatogram showed one single spot,
corresponding to 2, in addition to some nonꢀeluting side products. Therefore, the raw material
was subjected to silica gel chromatography, using dichloromethane/acetone 9:1 as eluent. Were
obtained 0.037 g (0.089 mmol, 50% yield) of 2. Anal. Calculated for transꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[
PtCl (NH )(1M7AI)]·¼CH COCH (C H N Cl Pt·¼CH COCH ): C, 24.44; H, 2.91; N, 9.78%.
2 3 3 3 8 11 3 2 3 3
1
Found: C, 24.70; H, 2.92; N, 10.09%. HꢀNMR (acetoneꢀd , numbering scheme of 1M7AI as in
6
Schemes S1 and S2) δ: 8.71 (m, 1 H, H ), 8.03 (m, 1 H, H ), 7.51 (m, 1 H, H ), 7.09 (m, 1 H, H ),
6
4
2
5
1
95
6
.61 (d, 1 H, H ), 5.17 (s, 3 H, CH ), 3.85 (b, 3 H, NH ) ppm. PtꢀNMR (acetoneꢀd ) δ: −1,967
3 3 3 6
ppm. ESIꢀMS: calculated for [C H N Cl PtNa] 438.18. Found: m/z (% relative to the base peak)
8
11
3
2
+
4
37.9(100), [M + Na] .
X-ray crystallography. Crystals suitable for Xꢀray investigation were obtained from
water/acetone (1:2 ratio). Reflections were collected with MoꢀKα radiation by using a Bruker
AXS X8 APEX CCD System. All reflections were indexed, integrated, and corrected for Lorentz,
polarization, and absorption effects using the program SADABS [Sheldrick G. M., SADABS
University of Göttingen Germany (1996)]. Data collection, data reduction, and unit cell
2
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2
2
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2
2
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6
refinement were carried out with the SAINTꢀIRIX package [Bruker: SAINTꢀIRIX
BrukeRAXSInc Madison Wisconsin USA (2003)].
The model was refined by fullꢀmatrix leastꢀsquare methods. All nonꢀhydrogen atoms were
refined anisotropically. The H atoms of the amino and methyl groups were found in a difference
Fourier map and were refined with Uiso(H) = 1.5 Ueq(parent). Hydrogen atoms of aromatic rings
were placed at calculated positions and refined given isotropic parameters equivalent to 1.2 times
those of the atom to which they are attached. All calculations and molecular graphics were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0
41
42, 43
44
carried out using SIR2004, SHELXL97, PARST97,
WinGX, and ORTEPꢀ3 for
4
5
Windows packages. Details of the crystal data are listed in Table 6.
Crystallographic data (without structure factors) have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no. CCDC 1024413. Copies of the
data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK;
tel: (+44) 1223ꢀ336ꢀ408; fax: (+44) 1223ꢀ336ꢀ003; eꢀmail: deposit@ccdc.cam.ac.uk; website
http://www.ccdc.cam.ac.uk/data_request/).
Cell Lines and Culture. The human ovarian carcinomas A2780 (parent cisplatinꢀsensitive)
and A2780cisR (cisplatin resistant variant of A2780) and the human breast adenocarcinoma
MCFꢀ7 (cisplatin inꢀborn resistance) cell lines were supplied by Professor B. Keppler, University
+/+
of Vienna (Austria). The human colon carcinoma HCT116 (parental, p53 ) and its p53ꢀnull
4
6
derivative (p53ꢀ/ꢀ) cells were a kind gift of Dr. M. Brazdova, Institute of Biophysics, Brno
Czech Republic). The A2780 and A2780cisR were grown in RPMI 1640 medium supplemented
(
−
1
−1
with streptomycin (100 µg mL ), penicilin (100 U mL ) and heat inactivated FBS (10%). The
acquired resistance of A2780cisR cells was maintained by supplementing the medium with
+/+
ꢀ/ꢀ
cisplatin (1 ꢂM) every second subculture. The MCFꢀ7, HCT116 p53 and HCT116 p53 cells
−
1
−1
were grown in DMEM supplemented with streptomycin (100 µg mL ), penicilin (100 U mL )
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and heat inactivated FBS (10%). The cells were cultured in a humidified incubator at 310 K in an
atmosphere of 5% CO and subcultured 2−3 times per week with an appropriate plating density.
2
Human skin fibroblasts (primary cell culture) was a kind gift from Professor T. Adam,
Laboratory of Inherited Metabolic Disorders, Department of Clinical Chemistry, Palacky
University and Hospital, Olomouc, Czech Republic. These cells were grown in DMEM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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42
43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
−
1
−1
supplemented with streptomycin (100 µg mL ), penicilin (100 U mL ) and 10% heat
inactivated FBS.
In Vitro Growth Inhibition Assay. Influence of platinum complexes on cell viability was
tested using the colorimetric MTT assay. Cells were seeded on 96ꢀwell tissue culture plates at a
4
density of 10 cells/well in 100 ꢂL of growth medium and incubated at 310 K in a humidified 5%
CO atmosphere for 16 h (overnight). After the incubation period the cells were treated with the
2
compounds under investigations and kept in the incubator for additional 72 h. The stock solutions
of platinum compounds were always freshly prepared in DMF before use. The final concentration
of DMF in cell culture medium did not exceed 0.1% (v/v), which was shown not to affect cell
growth. The final concentrations of tested compounds were in the range of 0 to 80 ꢂM in a
−
1
volume of 200 ꢂL/well. Subsequently, 10 ꢂL of MTT solution (5 mg mL ) was added to each
well, and plates were incubated for 4 h. At the end of the incubation time the medium was
removed and the formazan product was dissolved in 100 ꢂL of DMSO per well. Cell viability
was evaluated by measuring the absorbance at 570 nm (reference wavelength at 630 nm) using an
Absorbance Reader Sunrise Tecan Schoeller. IC values were calculated from curves
5
0
constructed by plotting cell survival (%) versus drug concentration (ꢂM). All experiments were
done in triplicate. The reading values were converted to the percentage of control (% cell
survival). Cytotoxic effects were expressed as IC .
5
0
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6
Cellular Accumulation. Cellular uptake of 1, 2, and cisplatin was measured in A2780,
A2780cisR and MCFꢀ7 cell lines. The cells were seeded on 100 mm tissue culture dishes (3x10
cells/dish in 10 mL of growth medium). After 48 h of incubation, the cells were treated with
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
2
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1
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9
0
tested compounds (at their IC values) for 10 s, 3, 5, 12 or 24 h (the concentrations were verified
5
0
by measuring the platinum content in the growing medium by FAAS). The attached cells were
harvested by trypsinization and the cell pellets washed twice with cold PBS (277 K). The pellets
were digested by using microwave acid (HCl, 11 M) digestion system (MARS5, CEM) to give a
fully homogenized solution. Final platinum content in the samples was determined by ICPꢀMS.
4
7
The results of cellular platinum uptake were corrected for adsorption effects.
Determination of Partition Coefficients of Platinum Complexes. Partition coefficients of 1
and 2 were determined in an octanol/saline system. Briefly, saline solution (100 mM NaCl) and
ꢀ3
nꢀoctanol were preꢀsaturated with nꢀoctanol and saline, respectively; then 1 or 2 (10 g, 2.41 x
ꢀ3
1
0 mmol) was dissolved in saline (3 mL and 10 mL for 1 and 2, respectively) at 310 K and the
complex concentration was determined by FAAS. Equal volumes of the drug solution and nꢀ
octanol were placed in a glass tube and the mixture shaken mechanically for 1 h at 310 K. Then
the mixture was centrifuged (1500 rpm, 5 min) and the bottom aqueous layer was removed and
subjected to platinum analysis by FAAS. The complex concentration in the nꢀoctanol phase was
calculated by the difference between the complex concentration in saline before and after nꢀ
octanol extraction. Each value reported in the text represents a mean of triplicate determination;
differences between the mean and individual values were <5%.
DNA Platination in Cells Exposed to Platinum Complexes. A2780, A2780cisR or MCFꢀ7
cells grown to near confluence were treated with 1, 2 or cisplatin at IC values and incubated for
5
0
5
or 24 h. After incubation, the cells were trypsinized and washed twice in iceꢀcold PBS. Cells
were lysed in DNAzol (DNAzol genomic DNA isolation reagent) supplemented with RNase A
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(
100 ꢂg mL ). The genomic DNA was precipitated from the lysate with ethanol, dried, and
resuspended in water. The DNA content in each sample was determined by UV
spectrophotometry. To avoid the effect of high DNA concentration on detection of platinum in
the samples, the DNA samples were digested in the presence of HCl (11 M) using a high pressure
microwave mineralization system. Experiments were performed in triplicate, and the values are
the means ± SD.
4
5
6
7
8
9
1
1
1
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1
1
1
1
1
1
2
2
2
2
2
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2
2
2
2
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9
0
Platination Reactions in Cell-free Media. CT or plasmid DNAs were incubated with the
platinum complexes in NaClO (10 mM) at 310 K in the dark. After 24 h, the samples were
4
exhaustively dialyzed against the medium required for subsequent biochemical or biophysical
analysis. An aliquot of these samples was used to determine r values by FAAS. Similarly, HSA
b
was incubated with the platinum complexes (molar ratio of 1:1) in Tris.HCl (10 mM, pH 7.4)
plus NaCl (8 mM) at 310 K in the dark. Aliquots were withdrawn at various time intervals,
quickly cooled to 193 K and exhaustively dialyzed against water. The platinum content in these
samples was determined by FAAS, while the concentration of HSA was determined by
absorption spectrophotometry.
DNA Transcription by RNA Polymerase In Vitro. Transcription of the pSP73KB DNA with
T7 RNA polymerase and electrophoretic analysis of the transcripts were performed as previously
48
ꢀ5
described. The concentration of DNA used in this assay was 3.9x10 M (relative to the
monomeric nucleotide content).
Cell Cycle Analysis. A2780 cells were seeded on 60 mm tissue culture dishes at a density of
6
2
x10 cells/dish in 6 mL of growth medium and incubated at 310 K in a humidified 5% CO
2
atmosphere overnight. After incubation, the cells were treated with the compounds at IC values
5
0
and stored at culture conditions. After 24 h, floating cells were collected, and attached cells were
harvested by trypsinization (trypsin/EDTA in PBS). Total cells (floating + attached) were washed
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2
2
2
2
2
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3
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5
6
twice in PBS (277 K), fixed in 70% ethanol, and stored at 253 K. Cell pellets were subsequently
rinsed with PBS buffer and the sediment was stained with solution of propidium iodide (50 µg
ꢀ1
ꢀ1
mL ) supplemented with RNase A (10 µg mL ) for 30 min at room temperature in the dark. The
DNA content of the cells was analyzed using flow cytometry (Cell lab quanta TM SCꢀMPL,
Beckman Coulter). The percentages of cells in individual cell cycle phases were analyzed using
Multicycle AV for Windows (Phoenix Flow Systems, USA).
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
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9
0
Detection of Apoptosis and Necrosis. The cell death detection ELISA plus kit (Roche
Molecular Biochemicals, Mannheim, Germany) was used as an indicator of apoptosis and
4
9
necrosis. In this assay, internucleosomal DNA fragmentation was quantitatively assayed by
antibodyꢀmediated capture and detection of cytoplasmic mononucleosomeꢀ and
oligonucleosomeꢀassociated histoneꢀDNA complexes. The cells were seeded on 96ꢀwell tissue
4
culture plates at a density of 10 cells/well. The treated A2780 cells were centrifuged (200 g, 10
min, room temperature), 20 ꢂL of the supernatant (necrotic fraction) was used in the ELISA for
detection of necrosis. A2780 cells were resuspended in 200 ꢂL of the lysis buffer supplied by the
manufacturer and incubated for 30 min at room temperature. After pelleting of the nuclei (200 g,
10 min, room temperature), 20 ꢂL of the supernatant (cytoplasmic fraction) was used in the
ELISA for detection of apoptosis following the manufacturer's standard protocol. Following
incubation with peroxidase substrate for 20 min, absorbance was determined at 405 nm
(reference wavelength was 490 nm) with a microplate reader (absorbance reader Tecan
INFINITE M200, Schoeller). Signals from wells containing the substrate only were subtracted as
background. Other details of this assay and data analysis were performed according to the
manufacturer's instructions.
Confocal Microscopy. A2780 cells were seeded on glass bottom dishes (P50Gꢀ0ꢀ30ꢀF,
6
MatTek Co., Ashland, USA) at the density 1.5x10 cells per dish and incubated at 310 K in a
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humidified 5% CO atmosphere overnight. The cells were then treated with 1, or 2, or 1M7AI (30
2
µM concentration) for 1 or 5 h. After this period, the cells were washed twice with PBS and
supplemented with fresh medium (without compound). The distribution of tested compounds in
tumor cells was analyzed by confocal microscopy. Samples were visualized with confocal
microscope Leica TSC SPꢀ5 X, sequentially scanned with the objective HCX PL APO lambda
blue 63.0x1.20 water UV, corrected with an appropriate beam path. Resolution of captured
images was 512·512 pix scanned with 100 Hz frequency. Samples were illuminated with UV
laser at 355 nm and the light returning from the samples was detected with highly sensitive
hybrid detectors at wavelengths 710ꢀ760 nm (for other details, see Supporting Information,
Section Determination of the Wavelength Used to Illuminate Cells Treated with the Platinum
Complexes in Confocal Microscopy Experiments). Signal intensity associated with single cells
was analyzed in the center of captured image (256·256pix) to omit potential objective
aberrations. The experiment was repeated twice with three replicates of each sample. Settings of
the imaging software (laser strength and gain) were kept constant within each experiment to
allow for comparison of the light intensities of samples.
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45
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60
Involvement of p53 Pathway in the Mechanism of Action. The effect of p53 status on the
3
4
cytotoxic activity of 1, 2 and cisplatin was tested as already described using two isogenic
+/+
human colon cancer cells, wild type p53ꢀcontaining HCT116 cells [parental cells (p53 ) and its
ꢀ/ꢀ 46
+/+
ꢀ/ꢀ
p53ꢀnull derivative (p53 )]. Briefly, HCT116 p53 and HCT116 p53 cells were seeded in
6well black plates (Corning) at the density 4000 cells/well in 100 µL of growth medium
DMEM) and incubated overnight (16 h). Then the cells were treated with 1, 2 or cisplatin for 6
9
(
h, washed twice with PBS and cultured in drugꢀfree medium for additional 66 h at 310 K in 5%
CO humidified atmosphere. DMFꢀsupplemented DMEM was introduced to the untreated
2
controls. After the incubation period, the medium was replaced with 0.1% triton Xꢀ100 in MQ
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6
water. The cells were lysed under continuous shaking on ice for 30 min. Subsequently, the cells
were freeze thawed for five 30 min cycles. An equal volume of 2x SYBR Green I was added to
100 µL of the lysate. DNA contents were measured with fluorescence reader Infinite 200
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
2
3
4
5
6
7
8
9
0
(TECAN) at excitation/emission wavelength 485/535 nm. Experiments were done in triplicate.
The data were normalized to the DNA content in control, untreated cells.
Physical Measurements. NMR spectra were collected at 295 K on a Bruker AVANCE DPX
1
1
3
00 MHz nominal H Larmor frequency. H chemical shifts were referenced to TMS by using the
residual protic peak of the solvent (acetoneꢀd , or D O) as internal reference. Oneꢀdimensional
6
2
1
95
1
195
Pt spectra were acquired using H decoupling sequences. Pt chemical shifts were referenced
to K PtCl (1 M in water, δ = −1,614 ppm). Elemental analyses were carried out on a CHN
2
4
Eurovector EA 3011 equipment. ESIꢀMS analyses were performed on an Agilent 1100 series LCꢀ
MSD Trap system VL. Absorption spectra were measured with a Beckmann DUꢀ7400
spectrophotometer. FAAS measurements were carried out with a Varian AA240Z Zeeman atomic
absorption spectrometer equipped with a GTA 120 graphite tube atomizer. The analysis with the
aid of ICPꢀMS was performed using Agilent 7500 (Agilent, Japan). The gels were dried and
visualized using a FUJIFILM BAS 2500 bioimaging analyzer.
ASSOCIATED CONTENT
Supporting Information
"Supporting Information Available: Product characterization including NMR and fluorescence data, data
on intracellular accumulation. This material is available free of charge via the Internet at
http://pubs.acs.org."
AUTHOR INFORMATION
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Corresponding Author
Phone: +420ꢀ541517174. Fax: +420ꢀ541240499.
*
Eꢀmail: jana@ibp.cz.
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60
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The authors acknowledge support from the National Program of Sustainability I (LO1204),
Operational Program Education for Competitiveness ꢀ European Social Fund
(CZ 1.07/2.3.00/20.0057) of the Ministry of Education, Youth and Sports of the Czech Republic
and the University of Bari “Aldo Moro”, the Consorzio Interuniversitario di Ricerca in Chimica
dei Metalli nei Sistemi Biologici (CIRCMSB), the Italian Ministero dell’Università e della
Ricerca (PON 01078 and FIRB RINAME RBAP114AMK). This research was also supported by
Palacky University in Olomouc (IGAPrF2014029). J.K.'s research was supported by the Ministry
of Education, Youth and Sports of the Czech Republic (Grant LD14019). The authors
acknowledge that their participation in the EU COST Action CM1105 enabled them to exchange
regularly the most recent ideas in the field of metallodrugs with several European colleagues.
ABBREVIATIONS USED
1
, cisꢀ[PtCl (NH )(1M7AI)]; 2, transꢀ[PtCl (NH )(1M7AI)]; 1M7AI, 1ꢀmethylꢀ7ꢀazaindole; bp,
2 3 2 3
base pair; cisplatin, cisꢀdiamminedichloridoplatinum(II); CT, calf thymus; DMEM, Dulbecco's
modified Eagle's medium; FAAS, flameless atomic absorption spectrometry; FBS, fetal bovine
serum; IC , concentration of compound which causes death in 50 % of cells; ICPꢀMS,
5
0
inductively coupled plasma mass spectrometry; MTT, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
29
ACS Paragon Plus Environment