Platinum (IV) Derivatives with Cinnamate Axial Ligands as Potent Agents Against Both Differentiated and Tumorigenic Cancer Stem Rhabdomyosarcoma Cells

Article abstract of DOI:10.1002/anie.201913996

To design an anticancer drug capable of inhibiting not only the proliferation of the differentiated tumor cells but also reducing the tumorigenic capability of cancer stem cells (CSCs), the new Pt^IV prodrugs with axial cinnamate ligands were sy

Full text of DOI:10.1002/anie.201913996

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: Platinum (IV) Derivatives with Cinnamate Axial Ligands Are
Potent Agents Against Both Differentiated and Tumorigenic
Cancer Stem Rhabdomyosarcoma Cells
Authors: Juraj Zajac, Vojtech Novohradsky, Lenka Markova, Viktor
Brabec, and Jana Kasparkova
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201913996
Angew. Chem. 10.1002/ange.201913996
Link to VoR: http://dx.doi.org/10.1002/anie.201913996
http://dx.doi.org/10.1002/ange.201913996

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
Platinum (IV) Derivatives with Cinnamate Axial Ligands Are
Potent Agents Against Both Differentiated and Tumorigenic
Cancer Stem Rhabdomyosarcoma Cells
[a]‡
[a]‡
[a]
[a]
[a]
Juraj Zajac, Vojtech Novohradsky, Lenka Markova, Viktor Brabec and Jana Kasparkova*
Abstract: To design a new anticancer drug capable of inhibiting not
only the proliferation of the differentiated tumor cells but also
reducing the tumorigenic capability of cancer stem cells (CSCs), the
new Pt(IV) prodrugs with axial cinnamate ligands were synthesized.
We demonstrate their superior antiproliferative activity in monolayer
and 3D spheroid antiproliferative activity tests using panel of cancer
cell lines. Interestingly, an outstanding activity was found against
rhabdomyosarcoma cells, one of the most problematic and poorly
treatable pediatric tumors. The results also suggest that Pt(II) moiety
and cinnamic acid are released from the molecules of the
investigated Pt(IV) prodrugs in cancer cells and are reduced by
intracellular reductants. Whereas the released Pt(II) compound
inhibits antiproliferative activity of cancer cells by DNA-damage
mediated mechanism, the released cinnamic acid can trigger
processes leading to differentiation, making the CSCs more
sensitive to killing by the platinum part of the complex. To the best of
our knowledge, Pt(IV) complex with axial cinnamate ligands is the
first Pt(IV) prodrug capable of overcoming CSCs resistance and
induce death in both CSCs and bulk cancer.
cells but not CSCs. The presence of even a very small amount
of CSCs may lead to the development of therapeutic resistance,
metastases, and relapse, which fundamentally diminishes
therapeutic effectiveness of numerous anticancer drugs. Hence,
a possible intriguing scenario for the response to cellular
exposure to antitumor drugs with a limited efficiency to kill CSCs
is to combine these drugs with trans-CA which potentiates
differentiation of CSCs. Thus, the formation of these newly
differentiated cells may considerably increase sensitivity of
tumor cells to chemotherapeutics incapable to kill CSCs.
Nevertheless, the combination of drugs administered as a
mixture of single agents may not inevitably reach the cellular
targets simultaneously with the desired dose and ratio. As
regards anticancer platinum drugs, an efficient approach in this
respect can be to utilize the advantageous properties of
octahedral Pt(IV) complexes. The design of platinum(IV)
complexes enables conjugation of biologically active ligands in
axial positions; the ligands can be released simultaneously with
DNA damaging Pt(II) moiety after the complexes are
[5]
accumulated and reduced inside the cells, where they act by
different mechanisms on different cellular targets. Thus, these
conjugates are able to bring into the tumor cells, along with the
molecules of the platinum component, also the molecules of
other biologically active compounds.
To prepare compounds having biological effects capable of
reducing both the tumorigenic capability of CSCs and the
proliferation of differentiated tumor cells, we have synthesized
two Pt(IV) derivatives bearing axial ligand(s) of cinnamic acid
Introduction
Cinnamic acid is a naturally occurring compound, composed
of a phenyl ring substituted with an acrylic acid group, which
commonly appears as the trans isomer (trans-CA, Figure 1).
Cinnamic acid has a number of pharmacological activities
[
1]
including anti-microbial,
anti-inflammatory and anti-oxidant
[1b, 2]
(
complexes 1 and 2, Figure 1). Compounds were designed to
activity.
Moreover, it was found to have an anti-tumor activity
release, upon their intracellular activation, molecules of cisplatin-
like moiety along with one (complex 1) or two (complex 2)
equivalents of trans-CA (Figure 1) that would be able together to
carry out their biological functions in a synergistic manner.
Moreover, the presence of trans-CA ligands in complexes 1 and
in vitro against a broad spectrum of solid human tumors at
doses that have no significant effect on normal cells. Cinnamic
acid induces apoptosis and cell differentiation accompanied by
modulation of expression of genes implicated in growth control,
[3]
tumor metastasis and immunogenicity. Some studies have
also suggested the effects of cinnamic acid on the inhibition of
invasive activity along with the induction of cell cycle arrest and
2
could give rise to more hydrophobic species, facilitating their
transport across cell membranes. Hence, they might efficiently
accumulate in tumor cells treated at the concentrations
significantly lower than those required for their constituents.
Here, we report the remarkable antiproliferative activity of
new Pt(IV) derivatives of cisplatin bearing cinnamate as axial
ligands in both CSCs and bulk, differentiated cancer cells
simultaneously. The quite exceptional anticancer effects of these
compounds were also found against rhabdomyosarcoma cells
[4]
cytoskeleton disruption in malignant tumor cell lines. More
recently, it was shown that the treatment of sphere-derived
cancer stem cells (CSCs) with cinnamic acid could effectively
diminish their CSC-like properties and increase their sensitivity
to chemotherapeutic drugs through apoptosis.
It is important to emphasize that
a large number of
anticancer chemotherapeutics, including clinically used cisplatin
and its platinum(II) derivatives, effectively kill differentiated tumor
(
RD cells), one of the most problematic and poorly treatable
pediatric tumors. Additionally, our data indicate that cinnamic
acid present in the molecule of the Pt(IV) complexes in axial
positions is responsible for a unique mechanism of action of
these prodrugs. It not only enhances the accumulation of these
prodrugs in tumor cells but strikingly also via processes
promoting differentiation of CSCs, making these cells more
sensitive to killing by the platinum complex. Our findings also
suggest that strategies based on Pt(IV) prodrugs in combination
with trans-CA may synergistically enhance antitumor activity.
[
a]
Dr. J. Zajac, Dr. V. Novohradsky, Dr. L. Markova, Prof. V. Brabec,
Prof. J. Kasparkova
Czech Academy of Sciences, Institute of Biophysics
Kralovoposlka 135, CZ-61265 Brno, Czech Republic
E-mail: jana@ibp.cz
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
#
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
constituents possibly released after activation of complexes 1
and 2, respectively, in a cellular environment.
As shown in Table 1, the IC50 values of complex 1, bearing
one axial cinnamato ligand, were found to be lower than those of
cisplatin, on average by 40% in all tumor cell lines tested.
Complex 2, having two cinnamato ligands, was shown to be
extremely effective against the tumor cells, exhibiting
approximately two orders of magnitude higher antiproliferative
activity than the parental cisplatin, with the IC50 values in a
submicromolar range. Noteworthy, the introduction of
a
cinnamato ligand(s) to the axial position of the investigated
Pt(IV) derivatives of cisplatin led to significant activity in selected
tumor cell lines notoriously poor sensitive to platinum drugs. On
the contrary, free trans-CA was proved to be hardly effective,
with the IC50 values in a millimolar range, in accordance with the
[
4a]
previously reported data.
Table 1: IC50 mean values (μM) of the investigated compounds, evaluated by
[
a]
using MTT assay
Cells
HeLa
.
cisplatin
1
2
trans-CA
Figure 1. Schematic representation of cisplatin, trans-cinnamic acid (trans-
24 ± 3
21 ± 1
34 ± 3
43 ± 3
13 ± 2
17 ± 1
6.0 ± 0.7
16 ± 1
8.5 ± 0.4
12 ± 1
27 ± 2
8.7 ± 0.5
9 ± 2
0.48 ± 0.04
0.31 ± 0.03
0.45 ± 0.02
0.39 ± 0.05
0.23 ± 0.06
0.03 ± 0.01
0.9 ± 0.1
10100 ± 1000
11800 ± 1200
15400 ± 900
11900 ± 700
11000 ± 1700
8400 ± 300
3 2
CA), and Pt(IV) complexes cis,trans,cis-[Pt(NH ) (OH)(cinn)Cl
2
] (1) and
+
/+
cis,trans,cis-[Pt(NH ) (cinn) Cl ] (2) (cinn = cinnamate) used in the present
3 2 2 2
HCT116
HCT116
study.
-
/-
MDA-MB-231
MCF-7
Results and Discussion
RD
Synthesis and Characterization of the
MRC-5 pd30
12 ± 3
14400 ± 900
Compounds
.
[
a]
The drug-treatment period was 48 h. The results are expressed as mean
Two platinum(IV) derivatives of cisplatin with axial cinnamato
ligand(s) (cinn), cis,trans,cis-[Pt(NH (OH)(cinn)Cl (1) and
cis,trans,cis-[Pt(NH (cinn) Cl ] (2), were synthesized according
to the standard procedures. In brief, cisplatin was oxidized with
the hydrogen peroxide to yield cis,trans,cis-[Pt(NH (OH) Cl
oxoplatin). Oxoplatin was after reacted with approximately 1.1
values ± SD from three independent experiments performed in a quadruplicate.
3
)
2
2
]
3
)
2
2
[
2
6]
The present study was also carried out to investigate the
antiproliferative activities of complexes 1 and 2 in normal lung
fibroblasts MRC-5 pd30. Notably, the selectivity factors of 2,
calculated as a ratio of IC50(non-tumorigenic cell line)/IC50(tumor
3
)
2
2
2
]
(
equivalent of the trans-CA anhydride in DMSO, affording a non-
symmetric monocarboxylated complex 1 (Supporting Information,
Scheme S1A). Since the second hydroxo group of oxoplatin is
less prone to carboxylation, symmetrically biscarboxylated
complex 2 was prepared by the reaction of oxoplatin with an
excess of the trans-CA anhydride (4 eq.) in DMF (Supporting
Information, Scheme S1B). Both compounds were purified by
preparative HPLC, and their purity was ascertained by analytical
HPLC and by elemental analysis. The compounds were further
characterized by NMR spectroscopy and mass spectrometry as
outlined in the Supporting Information.
cell line), showed
a pharmacologically prospective values
ranging from 2 to 30. Thus, the complex 2 exhibits significant
degree of selectivity towards the tumor cells relative to non-
tumorigenic MRC-5 pd30 fibroblasts. Moreover, the data on
antiproliferative activity of the mixtures of free cisplatin with
trans-CA in a molar ratio of 1:1 and 1:2 (Supporting Information,
Table S1) and the results of the stability studies (Supporting
Information, Figure S11) support the view that the unique
antiproliferative activity and selectivity of the investigated
compounds, particularly 2, could be attributed to the presence of
cinnamato ligands, accumulated inside the cells as an integral
part of Pt(IV) conjugates.
Antiproliferative Activity
Importantly, complex 2 exhibits a superior activity in RD cells,
being ca. one order of magnitude more active as compared to
other cancer cell lines (Table 1). Rhabdomyosarcoma
represents the most frequent malignancy in children, accounting
The antiproliferative activity of complexes 1 and 2 was
assessed by the MTT assay after 48 h of treatment in a panel of
human cell lines, namely cervical adenocarcinoma (HeLa), colon
carcinoma (HCT116, both p53 positive (+/+) and p53 non-
expressing (-/-)), breast cancers (invasive ductal carcinoma
MDA-MB-231 and adenocarcinoma MCF-7), and muscle
rhabdomyosarcoma, along with lung fibroblast (MRC-5 pd30)
non-tumorigenic cells. The results were compared with the
activity of parental cisplatin and trans-CA. For comparative
purposes, the mixtures of both cisplatin and trans-CA in 1:1 and
[7]
for 5% of all pediatric tumors. Rhabdomyosarcoma is derived
from mesenchymal stem cells, and rhabdomyosarcoma tumors
could be located in a variety of anatomic sites in the childhood
[
8]
body. Despite the current treatment approaches, notably high
proportion of patients suffer recurrence with subsequent survival
[
9]
rates no more than 20%. Thus, the development of potent
treatment for this aggressively recurrent disease represents an
important task. Moreover, the high potency of Pt(IV) derivatives
with cinnamate ligands in RD cells accords with the important
1:2 molar ratios were also included, both representing free
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
role of cancer stem cells (CSCs) in this childhood
rhabdomyosarcoma, derived from muscle cells that failed to fully
differentiate. Therefore, our further studies were focused on
understanding the mechanism of action of 1, and particularly 2 in
this type of cancer cells.
Table 2: Antiproliferative activity in a pair of Chinese hamster ovary cells [IC50
mean values (μM)] of the investigated compounds. Cell death was evaluated
[
a]
by using a system based on the tetrazolium compound MTT
.
Compound
MMC-2
CHO-K1
36.7 ± 3.1
19.7 ± 1.9
1.26 ± 0.15
Cisplatin
4.62 ± 0.35
4.17 ± 0.27
0.29 ± 0.02
Cellular Uptake
1
The accumulation of platinum-based chemotherapeutics in
2
tumor cells represents one of the key factors determining their
[10]
trans-CA
17500 ± 1500 18400 ± 1900
biological activity.
assessed following 24-h treatment with equal doses (0.5 μM) of
compounds and 2. The results show (Supplementary
The platinum content in RD cells was
[
a]
The drug-treatment period was 48 h. The results are expressed as mean
1
values ± SD from three independent experiments performed in a quadruplicate.
Information, Table S2) that the amount of platinum accumulated
in the cells treated with complexes 1 and 2 was approximately 4-
fold and 100-fold higher, respectively than that found in the cells
treated with the same dose of cisplatin. These findings suggest
However, enhancement of the antiproliferative activity (IC50
values, Table 1) of 2 in RD cells in comparison with cisplatin was
markedly higher than the enhancement of the amount of
platinum bound to nuclear DNA in RD cells (Table 2). Thus, this
comparison supports the hypothesis that Pt(IV) complexes after
their accumulation in tumor cells undergo reduction resulting in
the release of molecules of cisplatin and trans-CA. The released
cisplatin then mediates the antiproliferative activity via DNA
binding, and simultaneously trans-CA likely potentiates its
antiproliferative effects.
that the enhanced cellular accumulation of
1 and 2 in
comparison with that of cisplatin significantly contributes to the
antiproliferative activity of 1 and 2 in RD cells.
Our finding also demonstrates that the accumulation of Pt
from 2 in RD cells was higher than that from cisplatin ca. 100-
fold (Supporting Information, Table S2). Contrastingly,
antiproliferative activity of 2 in RD cells (IC50 value) was higher
than that determined for cisplatin even ca. 600-fold (Table 1).
Hence, important implication of these estimations is that a Cancer Stem Cells (CSCs) Targeting
considerably higher antiproliferative activity of 2 was not only a
consequence of its higher accumulation in cells. In other words,
yet another factor in addition to enhanced cellular accumulation
had to be responsible for enhanced antiproliferative activity of 2
in comparison with that of cisplatin.
As indicated in the introductory part, the Pt(IV) complexes
bearing cinnamate as axial ligands were designed with an
intention to prepare compounds capable of inhibiting not only
proliferation of the differentiated tumor cells, but also reducing
the tumorigenic capability of CSCs. Therefore, the
antiproliferative activity of 1 and 2 was also tested in the tumor
MCF-7 and RD cells (the cancer cells most sensitive to 1 and 2,
Table 1) enriched by the CSC subpopulation (preparation of
these cells enriched by CSCs is described in the Supporting
Information).
Molecular Mechanism of Action
For cisplatin and other platinum-based drugs, the nuclear
DNA is considered a primary cellular target responsible for the
[
11]
biological response. To assess the role of DNA interactions in
antiproliferative effects of complexes 1 and 2, we determined
their antiproliferative activity in a pair of Chinese hamster ovary
cells, differently responsive to DNA-damaging agents (Table 2).
CHO-K1 is a parental cell line proficient in nucleotide excision
repair, while its mutant cell line MMC-2 carries the ERCC3/XPB
mutation making these cells deficient in DNA repair. As shown in
Table 2, the MMC-2 cells were significantly more sensitive to the
treatment with both complexes, with the IC50 values being
approximately quarter of those in parental cells. The results
were similar to the data found for cisplatin and indicate that the
unrepaired DNA damage caused by the investigated complexes
contributes markedly to their antiproliferative activity. Thus, DNA
is likely one of the critical cellular targets of this newly
synthesized class of Pt(IV) compounds. Interestingly, free trans-
CA induced no sensitivity in repair-deficient cells. Hence, the
DNA-damaging capacity of the complexes might be assigned to
their cisplatin-like moiety.
The sorted cells were cultured for 96 h to form spheroids and
then treated with the investigated compounds for another 72 h.
The viability of cells was determined by using Cell TiterGlo 3D
assay. The 3D-culture was selected because the tumor spheres
closely reflect the tumor microenvironment and possess several
features as nutrient and oxygen gradients, intracellular and cell-
cell interactions, and drug penetration that more closely mimic
tumors in vivo, being more predictive for in vivo results than
[
12]
conventional 2D cell cultures. Cells cultured as 3D spheroids
could bring more significant data about anticancer effects
[
13]
without the need of animal models for the initial tests.
Conventional cyclophosphamide, a well-established, clinically
used agent (also in combination with other chemotherapeutics)
to treat the majority of childhood solid tumors including
[
14]
rhabdomyosarcoma, was used as a control compound.
The results (Table 3) revealed that particularly 2 showed the
lowest IC50 values in all of the investigated cell lines, being
active at sub-micromolar concentrations. Overall, both fractions
of RD tumorspheres were more sensitive to the treatment with 2
than the tumorspheres formed from MCF-7 cells, in agreement
with the data obtained for the unsorted cells grown in 2-D
cultures (Table 1). In contrast, conventional cyclophosphamide
showed relatively high IC50 values ranging from 41 μM on
RDCD133- up to 71 μM on MCF-7CD44+ thus being 88-187-fold
less potent than complex 2. Antiproliferative activity of cisplatin
was also in μM range; moreover, cisplatin was more effective in
To support the findings showing the ability of complexes 1
and
2 to trigger cell death via DNA-damage mediated
mechanism, we also assessed the amount of platinum bound to
DNA in RD cells after exposure to 1, 2, or cisplatin (Supporting
Information, Table S2).
The enhanced amount of platinum bound to nuclear DNA in
RD cells exposed to the investigated Pt(IV) complexes is very
likely also a consequence of their higher cellular accumulation.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
differentiated cancer cells (MCF-7CD44- and RDCD133-) than in
CSC cells (MCF-7CD44+ and RDCD133+), in accord with the
with trans-CA, in particular 2, may represent promising
candidates for anticancer drugs able to target both stem and
already differentiated cancer cells simultaneously, a quality not
[
15]
known no CSC-potency of cisplatin.
Importantly, the data
shows that 1 and 2 can kill both CSC-enriched and CSC-
achievable
by
conventional
antitumor
therapy.
depleted cells with similar efficiency. Thus, Pt(IV) complexes
CD44-
CD44+
CD133-
CD133+
Table 3: IC50 values of the investigated compounds determined in 3D spheroids generated from MCF-7
, MCF-7
, RD
and RD
[a]
cells
.
CD44-
CD44+
CD133-
CD133+
Compound
MCF-7
MCF-7
RD
RD
Cisplatin
19 ± 2
33 ± 5
12 ± 3
8.4 ± 0.1
0.22 ± 0.04
3000 ± 100
41 ± 4
23 ± 2
1
12 ± 2
0.6 ± 0.1
17 ± 1
0.8 ± 0.1
10300 ± 400
71 ± 9
12.0 ± 0.7
0.31 ± 0.06
2700 ± 300
58 ± 6
2
trans-CA
11000 ± 1000
53 ± 6
Cyclophosphamide
[
a]
®
IC50 values were obtained by using CellTiter-Glo 3D cell viability assay on 96 h-old spheroids and subsequent 72 h of treatment. Data are the means ±SDs
calculated from three independent experiments.
using qRT-PCR (Figure 4; for primer sequences see Supporting
Information, Table S3).
After the treatment with 2, the relative expression of MyoD
and myogenin was significantly elevated, in contrast to cisplatin
and cyclophosphamide. MyoD and myogenin are transcription
factors responsible for coordinating myoblasts to terminal
To examine the anti-CSC activity of 2 more thoroughly, we
investigated the formation of rhabdospheres from the
suspension of RDCD133+ single cells (Figure 2). The number
and size of the formed rhabdospheres were markedly lower after
the treatment of single-cell suspension of CSC-enriched
RDCD133+ cells with the submicromolar concentrations of 2,
compared to the untreated cells or cells treated with cisplatin
differentiation. The deregulation of MyoD activity accounts for
[16a]
the block of differentiation of RD cells.
Thus, an increase of
(
Figure 2). Moreover, free trans-CA had an insignificant impact
expression of MyoD and myogenin induced by 2 should trigger
on the number and size of rhabdospheres up to 500 µM
concentrations (Supporting Information, Figure S14). Notably, 2
reduced the number and size of rhabdospheres much more
effectively (ca. 1000-times) than cyclophosphamide (an
established RD-potent agent) (Figure 2).
The effect on the morphology of rhabdospheres formed from
the suspension of RDCD133+ single cells is shown in Figure 3.
Tumorspheres formed by the control, untreated cells were of
round-shape morphology with a well-defined surrounding edge
the differentiation of RDCD133+ stem cells. The observation that
cisplatin has
a negligible effect on MyoD and myogenin
expression suggests the important role of axial cinnamates that
can accelerate myoblasts differentiation through induction of
MyoD and myogenin, as already reported for 4-hydroxy-3-
[16b]
methoxy cinnamic acid (although in higher concentrations).
To further explore the possibility that 2 stimulates the loss of
stem-like character of RD cells and activate their differentiation,
RD cells treated with the investigated compounds were also
(
Figure 3A). However, spheroids generated after the treatment
[9,
analyzed for the stem cell markers, such as NanoG and Sox2.
with 2 displayed heterogeneous morphology with high number of
dissociated cell clumps (Figure 3D). These dissociated cell
clumps were found to a much greater extent than in the samples
treated with cisplatin, combination of cisplatin+trans-CA (1:2),
free trans-CA, and cyclophosphamide (Figures 3B, C, E, and F,
respectively).
Hence, our findings have important implications indicating
superior antiproliferative and cytotoxic activity of 2 against RD
stem cells.
The design of the cisplatin-based Pt(IV) compounds with
axial cinnamate ligand(s) described in this article was driven by
the hypothesis that trans-CA may trigger differentiation of CSCs,
thereby making the cells more sensitive to cisplatin.
17]
As shown in Figure 4, noticeable differences in mRNA
expression were observed for the transcription factor homeobox
protein NanoG, which is highly expressed in CSCs and may thus
[18]
function as an oncogene to promote carcinogenesis.
The
expression of NanoG was significantly elevated after the
treatment with cisplatin and cyclophosphamide, suggesting their
anti-differentiation efficiency on CSCs, which could result in the
enrichment of the CSC population, in agreement with the
[19]
previously published results.
Importantly, NanoG was down-
regulated after the treatment with 2, indicating a diversion of the
cell population from stemness. NanoG functions in concert with
SOX-2, both together are the critical factors for maintaining
Thus, to confirm this hypothesis, the effect of 2 on the
expression of the markers characteristic for RD stem cells and
differentiation was tested. The expression of several common
stem cell markers (NanoG and SOX-2, differentiation markers
[20]
[21]
pluripotency
and the self-renewal ability of CSCs. Despite
this fact we were unable to detect a statistically significant
decrease in the expression of SOX-2 after treatment with 2. On
the other hand,
cisplatin
a significant difference between parental
[
16]
(
MyoD, myogenin)
was quantified on transcription level by
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
CD133+
Figure 3. Morphology of the rhabdospheres generated from RD
single
cells treated with the investigated compounds for 96 h. A) Control (untreated)
sample, B) cisplatin (1 μM), C) cisplatin (1 μM)+trans-CA (2 μM), D) 2 (0.1 μM),
E) free trans-CA (1 mM), F) cyclophosphamide (50 μM). Scale bars represent
200 μm.
Figure 2. The effect of the investigated compounds on the formation of
rhabdospheres. A suspension of single RDCD133+ cells was treated for 96 h
with the increasing concentrations of the investigated compounds and
evaluated for the number (panel A) and diameter (panel B) of the generated
spheroids. Statistical analysis was calculated by using student t-test; star (*) at
the top of the bars denotes a significant difference from the untreated (control)
samples with p≤0.05. Non-detectable (n.d.) samples were not possible to
include in the analysis due to the high destructive effects of the applied
treatment. Experiments were done in triplicate, and the error bars are the SDs.
CD133+
Figure 4. qRT-PCR of the markers of differentiation or stemness in RD
cells treated with the tested compounds. Cells were treated for 24 h at the
concentration corresponding to IC50,72h Relative quantification of gene
method; GAPDH gene was used as
the endogenous reference control and samples isolated from the untreated
cells were used as the arbitrary calibrators. Data were subjected to statistical
analysis by using students´ t-test, and a significant difference (*p≤0.001) from
untreated control samples was marked with star at the top of the bar. The error
bars are the standard deviations calculated from three independent
experiments with quadruplicate in each run.
and trans-CA on one site and 2 on the other site was observed.
This correlates with the ability of 2 to affect both stem and
differentiated cells, whereas cisplatin attacks preferentially non-
stem cells, increasing the level of CSCs (and CSC-markers as
well) in the cell population.
To further explore the mode of cell death evoked by 2, the
progression of cell death was monitored in RDCD133+ cells.
The cells were treated for 24 h with the equitoxic concentrations
of the investigated compounds corresponding to IC50,72h
.
∆∆Ct
-
expression was calculated using the 2
(
Supporting Information, Figure S16). These results show that 2
compounds. This finding is in a good congruence with the
hypothesis that the trans-CA, released from 2 after intracellular
reduction, sensitizes CSCs to cisplatin so that the cells lose their
stemness, and cisplatin moiety is responsible for resulting
antiproliferative effect.
exhibits antiproliferative activity and induces cell death via both
apoptotic and necrotic pathways, with apoptosis prevailing.
Interestingly, there was no qualitative difference observed
between 2 and parental
mechanisms responsible for cell death are very similar for both
cisplatin,
indicating
that
the
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
Conclusion
Y.-L. Chiang, S.-H. Lu, C.-J. Weng, Eur. J. Pharm. Sci. 2011, 44, 281-
287.
[
5] a) M. Ravera, E. Gabano, M. J. McGlinchey, D. Osella, Inorg. Chim.
Acta 2019, 492, 32-47; b) D. Gibson, J. Inorg. Biochem. 2019, 191, 77-
In summary, we present new Pt(IV) cisplatin derivatives
bearing one (complex 1) or two (complex 2) cinnamate(s) as
axial ligand(s). These complexes were designed to prepare
prospective Pt-based drugs killing both CSCs and bulk,
differentiated cancer cells simultaneously. We demonstrate
superior antiproliferative activity of the complexes against panel
of cancer cell lines of various origin, particularly in the case of
complex 2, which displays antiproliferative activity at sub-
micromolar concentrations. Interestingly, an outstanding activity
was found against rhabdomyosarcoma cells, one of the most
problematic and poorly treatable pediatric tumor. The results
also confirm the hypothesis that cinnamic acid present in the
molecule of the investigated Pt(IV) complexes in axial positions
enters the cells altogether with Pt-moiety and after intracellular
reduction, it can trigger processes leading to differentiation,
making the CSCs more sensitive to killing by the platinum part of
the complex. The conjugation of both into one molecule of the
Pt(IV) complex also provides an advantage of synergy in cellular
accumulation so that both Pt and trans-CA parts can enter the
cell simultaneously in doses higher than those attainable by
individual constituents.
84.
[
6] a) J. J. Wilson, S. J. Lippard, Chem. Rev. 2014, 114, 4470-4495; b) R.
Faggiani, H. E. Howard-Lock, C. J. L. Lock, B. Lippert, B. Rosenberg,
Canad. J. Chem. 1982, 60, 529-534.
[7] D. Egas-Bejar, W. W. Huh, Adolesc. Health Med. Ther. 2014, 5, 115-
25.
1
[
8] V. Soleimani, M. Rudnicki, Cancer Cell 2011, 19, 157-159.
9] F. Dela Cruz, Front. Oncol. 2013, 3, 168.
[
[
10] M. D. Hall, M. Okabe, D.-W. Shen, X.-J. Liang, M. M. Gottesman, Annu.
Rev. Pharmacol. Toxicol. 2008, 48, 495-535.
[
11] a) D. Wang, S. J. Lippard, Nature Rev. Drug Discov. 2005, 4, 307-320;
b) V. Brabec, O. Hrabina, J. Kasparkova, Coord. Chem. Rev. 2017,
351, 2-31.
[12] a) E. Fennema, N. Rivron, J. Rouwkema, C. van Blitterswijk, J. de
Boer, Trends Biotechnol. 2013, 31, 108-115; b) C. J. Lovitt, T. B.
Shelper, V. M. Avery, Biology 2014, 3, 345-367.
[
13] D. Antoni, H. Burckel, E. Josset, G. Noel, Int J Mol Sci 2015, 16, 5517-
527.
5
[
14] a) P. A. Carpenter, L. White, G. B. McCowage, V. Nayanar, I. Toogood,
P. J. Shaw, L. Lockwood, K. Tiedemann, f. t. A. Group, N. Z. C. s. C.
Study, Cancer 1997, 80, 489-496; b) C. A. S. Arndt, J. A. Stoner, D. S.
Hawkins, D. A. Rodeberg, A. A. Hayes-Jordan, C. N. Paidas, D. M.
Parham, L. A. Teot, M. D. Wharam, J. C. Breneman, S. S. Donaldson,
J. R. Anderson, W. H. Meyer, J. Clin. Oncol. 2009, 27, 5182-5188.
Conventional platinum-based drugs, although being widely
used to treat various solid tumors, are ineffective against CSCs
[22]
[15] K. Laws, K. Suntharalingam, ChemBioChem 2018, 19, 2246-2253.
in their therapeutic doses.
Thus, there is an urgent need for
[
16] a) C. Keller, D. C. Guttridge, FEBS J. 2013, 280, 4323-4334; b) P.
Kuppusamy, I. Soundharrajan, D. H. Kim, I. Hwang, K. C. Choi,
Phytomedicine 2019, 60, 152873.
new therapeutics acting in novel mechanisms of action and
capable of preventing tumor reoccurrence via targeting CSCs. In
this respect, the conjugation of cisplatin with cinnamic acid in the
molecule of Pt(IV) complex with axial cinnamates could pave the
way for the development of new, therapeutically relevant
compounds that would be able to overcome CSCs resistance
and induce death in both CSCs and bulk cancer.
[
17] a) D. Walter, S. Satheesha, P. Albrecht, B. Bornhauser, V.
D'Alessandro, S. M Oesch, H. Rehrauer, I. Leuschner, E. Koscielniak,
C. Gengler, H. Moch, M. Bernasconi, F. Niggli, B. Schäfer, PLoS ONE
2011, 6, e19506; b) J. Neradil, R. Veselska, Cancer Sci. 2015, 106,
803-811.
[
18] J. Kim, S. Yu, Y. Xu, Mol. Cell. Oncol. 2018, 5, e1074334.
19] S. Martins-Neves, D. Oliveira, P. M. Wijers-Koster, A. Abrunhosa, C.
Ribeiro, J. Bovee, A.-M. Cleton-Jansen, C. Gomes, Cancer Lett. 2015,
370, 286-295.
[
Acknowledgements
[
20] A. Gagliardi, N. P. Mullin, Z. Ying Tan, D. Colby, A. I. Kousa, F.
Halbritter, J. T. Weiss, A. Felker, K. Bezstarosti, R. Favaro, J.
Demmers, S. K. Nicolis, S. R. Tomlinson, R. A. Poot, I. Chambers,
EMBO J. 2013, 32, 2231-2247.
This work was supported by the Czech Science Foundation,
Grant No. 17-05302S. The authors are indebted to Dr. Karel
Kubicek from the Masaryk University in Brno for his help to
record and interpret the NMR spectra and to Assoc. Prof. Jan
Vanco from the Regional Centre of Advanced Technologies and
Materials, Faculty of Science, Palacky University in Olomouc for
his help to record and interpret ESI-MS spectra and to perform
elemental analysis.
[21] V. Kashyap, N. C. Rezende, K. B. Scotland, S. M. Shaffer, J. L.
Persson, L. J. Gudas, N. P. Mongan, Stem Cells Dev. 2009, 18, 1093-
1108.
[
22] a) K. Suntharalingam, W. Lin, T. C. Johnstone, P. M. Bruno, Y.-R.
Zheng, M. T. Hemann, S. J. Lippard, J. Am. Chem. Soc. 2014, 136,
14413-14416; b) M. Flamme, P. B. Cressey, C. Lu, P. M. Bruno, A.
Eskandari, M. T. Hemann, G. Hogarth, K. Suntharalingam, Chem. Eur.
J. 2017, 23, 9674-9682.
Keywords: antitumor agents • cancer • cancer stem cells • drug
design • platinum
[
[
[
1] a) P. N. Ramanan, M. N. A. Rao, Ind. J. Exp. Biol. 1987, 25, 42-43; b)
M. Sova, Mini-Rev. Med. Chem. 2012, 12, 749-767.
2] D. Hadjipavlou-Litina, E. Pontiki, Methods Mol. Biol. 2015, 1208, 361-
377.
3] a) L. Liu, W. R Hudgins, S. Shack, M. Q Yin, S. Darawsheh, Int. J.
Cancer 1995, 62, 345-350; b) G. Qi, J. Chen, C. Shi, Y. Wang, S. Mi,
W. Shao, X. Yu, Y. Ma, J. Ling, J. Huang, Cell. Physiol. Biochem. 2016,
40, 589-596; c) L.-T. Ng, S.-J. Wu, Evidence-based complementary
and alternative medicine : eCAM 2009, 2011, 492148.
[4] a) E. L. Niero, G. M. Machado-Santelli, J. Exp. Clin. Cancer Res. 2013,
32, 31; b) J. Zhang, A. Xiao, T. Wang, X. Liang, J. Gao, P. Li, T. Shi,
Biomed. Res. 2014, 25, 405-408; c) G.-C. Yen, Y.-L. Chen, F.-M. Sun,
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201913996
RESEARCH ARTICLE
Entry for the Table of Contents
RESEARCH ARTICLE
Juraj Zajac Vojtech Novohradsky,
Lenka Markova, Viktor Brabec and
Jana Kasparkova*
The design of the new Pt(IV)
derivatives with cinnamate axial
ligans could pave the way for the
development of new, therapeutically
relevant compounds that would be
able to overcome cancer stem cells
Page No. – Page No.
Platinum (IV) Derivatives with
Cinnamate Axial Ligands Are Potent
Agents Against Both Differentiated
and Tumorigenic Cancer Stem
Rhabdomyosarcoma Cells
(
CSCs) resistance and induce death
in both CSCs and bulk cancer. An
outstanding activity of this new
Pt(IV) complex was found against
rhabdomyosarcoma cells, one of the
most
problematic
and
poorly
treatable pediatric tumors.
This article is protected by copyright. All rights reserved.