Chalcoplatin, a dual-targeting and p53 activator-containing anticancer platinum(iv) prodrug with unique mode of action

Article abstract of DOI:10.1039/c4cc10409a

Complexation of cisplatin with a p53 activator as a single anticancer agent resulted in synergistically improved cytotoxicity in p53 wild-type but not p53 null human cancer cells. Mechanistic investigation was carried out on this dual-targeting Pt(iv) prodrug, chalcoplatin. The prodrug effectively entered cancer cells and arrested the cell cycle at the S and G₂/M phases, distinctive of that from cisplatin. Chalcoplatin significantly induced p53 activation as well as the subsequent apoptosis pathways. This unique mode of action renders chalcoplatin remarkably cytotoxic and makes this compound among the first examples of a Pt(iv) prodrug that directly interacts with the downstream pathway after the formation of Pt-DNA lesions. This journal is

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Chalcoplatin, a dual-targeting and p53 activator-
containing anticancer platinum(IV) prodrug with
Cite this: DOI: 10.1039/x0xx00000x
†
unique mode of action
a,d,‡
a,d,‡
b
c
a
Lili Ma,
Rong Ma,
Yiping Wang, Xiaoyue Zhu, Junliang Zhang, Hoi
a
c c b
Received 00th January 2012,
Accepted 00th January 2012
Ching Chan, Xianfeng Chen, Wenjun Zhang, Sung-Kay Chiu, and Guangyu
a,d
Zhu *
DOI: 10.1039/x0xx00000x
Complexation of cisplatin with a p53 activator as a single anticancer agent resulted in
synergistically improved cytotoxicity in p53 wild-type but not p53 null human cancer cells.
Mechanistic investigation was carried out on this dual-targeting Pt(IV) prodrug, chalcoplatin.
www.rsc.org/chemcomm
The prodrug effectively entered cancer cells and arrested the cell cycle at the S and G /M
2
phase, distinctive of that from cisplatin. Chalcoplatin significantly induced p53 activation as
well as the subsequent apoptosis pathways. This unique mode of action renders chalcoplatin
remarkable cytotoxicity and makes this compound among the first examples of a Pt(IV)
prodrug that directly interacts with the downstream pathway after the formation of Pt-DNA
lesions.
(
MDM2), has emerged as a novel approach for cancer therapy
7
Introduction
by reactivating p53 function to eradicate tumor cells.
A
number of small-molecule inhibitors of the p53-MDM2
interaction have been developed in pre-clinical and early
clinical settings.
Platinum(II)-based anticancer drugs including cisplatin [cis-
diamminedichloroplatinum(II), cDDP] have been successful in
chemotherapeutic treatments against certain types of
malignancies, although severe side effects including toxicity
8
In the present work, we design a novel dual-targeting
platinum(IV) anticancer prodrug that can not only carry the
DNA-binding platinum warhead into the tumor cells but also a
small-molecule fragment that can promote the p53 pathway.
The prodrug will likely be reduced in the cells, releasing
cisplatin and p53 activators. The former will damage DNA and
the latter will activate p53 simultaneously. Chalcone, a small-
molecule inhibitor of the p53-MDM2 interaction, was
conjugated with the platinum pharmacophore. Chalcones are
the derivatives of phenoxy acetic acid and phenoxymethyl
tetrazole, and they are known to inhibit the p53-MDM2
interaction with IC50 values in the micromolar range by binding
1
and drug resistance issues limit their efficacies. Platinum drugs
form Pt-DNA cross-links that subsequently inhibit DNA
2
metabolism and trigger apoptosis. In the search for the next
generation of platinum drugs, platinum(IV) complexes have
3
shown great promise. The platinum(IV) scaffolds allow for
tuning the oral availability, anticancer activity, and toxicity of
platinum(II) drugs, and they are reduced to platinum(II)
equivalents once inside the cells. Examples include
a
platinum(IV) carboxylate compound satraplatin that underwent
phase III clinical trials for an orally active prodrug against
prostate cancer, as well as several other platinum(IV)
complexes containing biologically active units on the axial
9
to the p53 pocket on MDM2. Thus, such a Pt(IV) complex
4
could potentially enhance the anticancer efficacy of cisplatin by
promoting the p53 pathway. The new complex is designated as
chalcoplatin (4, Scheme 1), which is among the first examples
of a Pt(IV) prodrug that directly interacts with the downstream
pathway after the formation of Pt-DNA lesions.
position. Very few examples, however, were designed based
on the mechanism of action of cisplatin using biologically
active moieties that directly interact with the pathways that
process Pt-DNA cross-links.
Failure of cisplatin-induced apoptotic pathways has been
found to be one of the major reasons for reduced anticancer
efficacy. For example, p53, the “guardian angel” of genome
Results and discussion
integrity, plays a profound role in the induction of cell-cycle
arrest or apoptosis in response to platinum-DNA damage. The Claisen-Schmidt aldol condensation protocol as previously
Chalcone (3) synthesis was preformed according to the
5
1
0
function of p53 has been correlated with cisplatin sensitivity in described. Chalcoplatin (
4
) was subsequently obtained by the
2
a
certain types of cancer cells. Thus, promoting p53 activation reaction of c,c,t-[Pt(NH ) Cl (OH) ] with acyl chloride form of
3
2
2
2
1
1
could be used as a strategy to increase the anticancer activity of
3 (Scheme 1). The compound was purified and then
1
13
195
cisplatin. On the other hand, the protein itself has become an characterized by H, C, Pt NMR, ESI-MS, and elemental
emerging and attractive drug target due to its tumor-suppressor analysis (ESI and Figures S1-S5).
6
function. Specifically, inhibiting the interaction between p53
and its negative regulator, human murine double minute 2
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chalcone exhibited IC50 values of 5.6 and 2.7 μM, respectively.
Therefore, the mixture has a 2.1-fold increased cytotoxicity. In
HL-60 p53 null cells, however, the cytotoxicity of the mixture
did not change significantly compared with cisplatin. These
data indicate that chalcone, a p53 activator, potentiates cisplatin
in p53 wild-type cells but not in p53 null cells, further
confirming our hypothesis that promoting p53 activation by
small-molecules is one way to elevate the anticancer activity of
cisplatin.
Table 1 Cytotoxicities of cDDP, chalcone, a mixture of cDDP and chalcone,
as well as chalcoplatin against different cell lines. Cells were treated with the
complexes for 72 h, and IC50 values (μM) are presented.
Scheme 1 Synthesis of chalcoplatin. Reagents and conditions: (i) 50% KOH
cell line
HeLa
p53
status
wt
cDDP
5.6±4.2
8.3±2.0
chalcone
cDDP+ a
chalcone
2.7±0.6
chalco- b
platin (FI )
1.5±0.8
solution; (ii) Diluted HCl solution; (iii) Oxalyl chloride, DMF, 50 °C, 2 h in dried
acetone; (iv) c,c,t-[Pt(NH Cl (OH) ], reflux in dried acetone for 20 mins. The
3
)
2
2
2
34.0±1.3
details for the synthesis are given in the ESI.
(3.8)
c
HCT-
wt
wt
nd
nd
0.8±0.4
(10.0)
4.8±1.0
(3.8)
1.9±0.7
(3.9)
The reduction potential of chalcoplatin was measured, since
the value is an indicator showing how easily the Pt(IV)
1
16
MCF-7
18.4±1.6 62.1±31.2
12.0±1.9
2.0±0.3
4.2±0.1
3.9±1.1
31.7±3.4
1
2
compound will be reduced to its Pt(II) equivalent. A cyclic
voltammogram was recorded at room temperature in DMF
containing 0.1 mol/L (n-Bu N)PF as the supporting electrolyte.
A549
wt
7.4±4.2
4.5±0.6
4.9±0.2
50.5±2.4
6.8
11.8±2.3
45.4±4.6
65.1±9.0
>50.0
4
6
1
3
HL-60
MRC-5
null
4.1±1.3
Trace amount of ferrocene was added as an internal reference.
(1.1)
The scan rate was set to 100 mV/s. The result showed that
chalcoplatin exhibited a single irreversible reduction peak with
9.0±0.4
(0.5)
1.6±0.5
(31.6)
0.8
+
Ep= -0.73 V vs. Fc /Fc= +0.05 V (Figure S6). The moderately
A549-
cDDP
low reduction potential of chalcoplatin indicates that its
reduction to platinum(II) species will not occur fast in the
blood, implying its potential biological activity.
d
RF
a
Cisplatin along with two equivalents of chalcone were used and IC50
We further studied the reduction ability of chalcoplatin. was expressed as the concentration of platinum
b
Chalcoplatin is in its Pt(IV) form and should be inert against
nucleophiles in DNA. The compound will be activated only
after its reduction to Pt(II) equivalent that will efficiently bind
to DNA. Thus, we measured the ability of chalcoplatin to bind
calf-thymus (ct)-DNA without and with the presence of a
reducing agent, ascorbic acid (AsA), as an indirect way to
quantify reduced chalcoplatin. Cisplatin was used as a control
in this assay. After 48 h incubation at 37 C, cisplatin bound to
DNA effectively. The levels of bound Pt on ct-DNA without
and with the presence of AsA were 37 and 29 µg-Pt/mg,
respectively (Figure S7). Without AsA, chalcoplatin displayed
a low level of DNA binding (6 µg-Pt/mg), indicating that the
compound existed mainly in the Pt(IV) form. In contrast, in the
presence of AsA, chalcoplatin had a dramatically increased
level of Pt (21 µg-Pt/mg), indicating that most of Pt bound to
DNA after reduction to Pt(II) form. These data confirmed that
chalcoplatin can be reduced, and the active Pt(II) moiety can
bind to DNA, resulting in Pt-DNA adducts.
FI (fold increase) is defined as IC50(cDDP)/IC50(chalcoplatin)
Not determined
RF (resistant factor) is defined as IC50 in A549cDDP/IC50 in A549
c
d
The cytotoxicities of chalcoplatin were examined
subsequently. Compared with a mixture of cisplatin and
chalcone, chalcoplatin has significantly increased cytotoxicities
in p53 wild-type cells. Notably, chalcoplatin showed up to
1
1
0.0-fold increased cytotoxicity compared with cisplatin (Table
). In p53 null cells, however, chalcoplatin did not show
elevated cytotoxicity with a fold increase of 1.1. These results
indicated that the complexation of cisplatin with chalcone
resulted in a synergistic effect to increase the anticancer activity
of cisplatin in p53 wild-type cells, and it is tempting to
speculate that this effect was from the promotion of p53
activity. The result in HL-60 cells further proved that our
strategy was not applicable to p53-null cells. In addition, in
normal human lung fibroblasts, chalcoplatin displayed
decreased cytotoxicity compared with cisplatin, indicating that
chalcoplatin has increased cancer-cell selectivity in the tested
cell lines. Furthermore, chalcoplatin was also very active
against cisplatin-resistant A549cDDP cells with an resistant
factor (RF) of 0.8, which was much lower than an RF of 6.8
from cisplatin, and chalcoplatin had 31.6-fold increased
cytotoxicity in the resistant cells compared with cisplatin.
Mechanistic investigations were carried out to study the
possible reasons why chalcoplatin has increased cytotoxicity
compared with cisplatin. We first tested the cellular uptake of
platinum. Decreased level of platinum uptake has been related
to reduced drug efficacy. Cells were treated with 10 μM
platinum compounds for 12 h before the measurement of
platinum levels in whole cells. In HeLa cells, the levels were
4
respectively (Figure S8A). These data demonstrated that the
complexation of chalcone moiety with the platinum
We next examined the cytotoxicities of the complexes. A
panel of human cancer cell lines including p53 wild-type HeLa
(
cervical), HCT-116 (colon), MCF-7 (breast), A549 (lung), and
p53 null HL-60 (leukaemia), as well as normal human lung
fibroblast MRC-5 and cisplatin-resistant cells A549cDDP, were
selected to evaluate the in vitro anticancer activity of the
compounds (Table 1). Cisplatin displayed cytotoxicity against
the cancer cells tested mostly in a typical low micromolar range
and the drug was also very active against normal cells.
Chalcone itself showed moderate cytotoxicities against human
cancer and normal cells, and the IC50 values were in the range
of 34.0 to 65.1 μM. We next evaluated the cytotoxicities of a
mixture of cisplatin and chalcone at a molar ratio of 1:2 to test
whether chalcone was able to potentiate cisplatin. In p53 wild-
type cells, the mixture showed increased cytotoxicities. For
example, in HeLa cells, cisplatin and a mixture of cisplatin and
14
6
.3 and 28.7 ng Pt/10 cells for cisplatin and chalcoplatin,
2
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pharmacophore resulted in an increased cellular uptake, which mildly upon treatment with cisplatin, showing that cisplatin
may also account for its higher cytotoxicity in p53 wild-type indeed triggered p53 activation, which was likely from its
1
8
cells. We also studied the Pt levels on the genomic DNA of binding to DNA and forming platinum-DNA cross-links. In
HeLa cells (Figure S8B). HeLa cells treated with 10 µM stark contrast, chalcoplatin promoted p53 expression much
cisplatin or chalcoplatin showed 1.7 and 1.4 ng Pt per mg DNA, more effectively than cisplatin at all the concentrations tested.
respectively, confirming the binding of chalcoplatin to genomic For example, the normalized p53 level upon treatment with 15
DNA in live cells.
μM chalcoplatin was 0.54, while the level was only 0.01 for the
same concentration of cisplatin. This result further corroborated
our hypothesis that the increased anticancer activity of
chalcoplatin was from promoting p53 activation, and the
chalcone moiety contributed to this effect. The highest
concentration of chalcoplatin did not induce the highest
expression level of p53 because that the treated cells undergone
severe apoptosis, leading to protein degradation, as shown by
actin expression (Figure 2B).
cDDP
chalcone
chalcoplatin
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
S
G /G1
0
G /M
2
0
0.4 2 10
0 0.4 2 10
0 0.4 2 10
Concentration (M)
Fig. 1 Cell cycle distribution upon treatment with cisplatin or chalcoplatin in HeLa
cells
Since chalcoplatin entered cells very efficiently, the ability of
chalcoplatin to affect cell cycle was examined, which will shed
further light on the mode of action of chalcoplatin. HeLa cells
were treated with 0, 0.4, 2, and 10 μM cisplatin, chalcone, or
chalcoplatin for 24 h, then cell cycle distribution was tested.
Cisplatin arrested the cell cycle at the S phase. For example, in
the untreated cells, 9.6% of the cells were in the S phase, while
this number increased to 59.0% when the cells were treated
with 10 μM cisplatin. This result is in consistent with previous
Fig. 2 Expression level of p53 upon treatment with platinum compounds. MCF-7
cells were treated with (
A
) cisplatin or (
B
) chalcoplatin and the expression levels
of p53 and actin were assessed. (
chalcoplatin or cisplatin treatment.
C) relative expression levels of p53 upon
1
5
reports. Chalcone weakly arrested the cell cycle at the G /M
2
phase. This is in agreement with other findings that breaking
the interactions between p53 and MDM2 by small-molecule
inhibitors results in G /M or G /G arrest, where activated p53
2
0
1
1
6
stops the cell cycle at the checkpoints and triggers apoptosis.
The weak cell cycle arrest from our assay was due to fact that
the concentrations we used were not high enough to strongly
affect the cell cycle, given the observation that chalcone has
low cytotoxicity. Chalcoplatin displayed S-phase arrest,
although not as strongly as cisplatin. In untreated controls, the
fraction of S-phase cells was 9.6%, and the fraction increased to
11.2%, 12.8%, and 22.4% when the cells were treated with 0.4,
2, and 10 μM chalcoplatin, respectively (Figure 1).
Remarkably, chalcoplatin-treated cells also presented
Fig. 3 Merged confocal microscopic images of HeLa cells treated with 0, 3, and 20
moderately increased levels of G /M phase (20.2% at 10 μM μM cisplatin or chalcoplatin for 24 h. The images of each channel (J-aggregates,
2
compared with 11.7% in the control). This unique cell cycle J-monomer, and DAPI) are provided in the ESI.
arrest ability indicated that the chalcone moiety in chalcoplatin
played pivotal roles in killing cancer cells, possibly by
activating p53, and that chalcoplatin has additional mode of
action compared with cisplatin.
Last but not least, we tested the ability of chalcoplatin to
induce apoptosis. HeLa cells were treated with 0, 3, and 20 μM
cisplatin or chalcoplatin for 24 h, and the mitochondrial
We next scrutinized whether chalcoplatin killed cancer cells
by activating p53. The expression levels of p53 protein upon
treatment with cisplatin and chalcoplatin were examined by
Western Blot. Since the expression level of p53 in HeLa cells is
transmembrane potential change (Δψ ) was indicated using a
m
cationic dye JC-1. Disruption of Δψm is often associated with
1
9
the early stages of apoptosis after the activation of p53. At the
lower concentration, there were apparently more JC-1
monomers, shown as in green fluorescence (Figure 3), in the
chalcoplatin-treated cells than the ones in cisplatin-treated cells,
indicating that chalcoplatin was able to strongly decrease Δψm.
Furthermore, at the higher concentration, chalcoplatin induced
severe DNA fragmentation compared with cisplatin, further
1
7
low, we used MCF-7 cells. The cells were treated with 0, 5,
5, and 25 μM platinum compounds for 48 h, and the
1
expression levels of p53 and actin were assessed. The
normalized p53 expression levels against actin control are
shown in Figure 2. The expression levels of p53 increased
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confirming its ability to induce apoptosis (Figures 3 and S9).
We also used an Annexin V/propidium iodide (PI) double-
staining assay to measure the level of apoptosis and necrosis
upon treatment. Chalcoplatin induced a large fraction of
apoptotic but not necrotic cells (Figure S10).
1
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evaluated chalcoplatin, the first example of a platinum(IV)
prodrug containing moieties of p53 activators. Compared with
cisplatin or a mixture of cisplatin and chalcone, chalcoplatin
displayed significantly increased cytotoxicity in p53 wild-type
but not in p53 null cells, showing a synergistic effect of
conjugating chalcone to cisplatin, and the compound was less
active in normal cells. Chalcoplatin was able to reduce to its
1
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Pt(II) equivalent in
a reducing environment. Profound
5
6
mechanism studies revealed that chalcoplatin effectively
entered cells, bound to genomic DNA well, arrested the cell
3
cycle at both the S and G /M phases, heavily induced p53
2
expression, and dramatically increased the apoptosis level.
These results indicated that compared with cisplatin,
chalcoplatin has a distinct mechanism of action to kill cancer
cells. Although chalcone might have other targets than p53-
MDM2, it indeed promotes the anticancer activity of cisplatin
by activating p53. We cannot exclude the possibility that the
promoted cellular uptake of platinum, due to the hydrophobic
feature of chalcone moiety in chalcoplatin, may also contribute
to the elevated apoptosis and cytotoxicity in p53 wild-type
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indicated that the chalcone moiety plays a key role in the
cytotoxicity of chalcoplatin, not only by facilitating the cell
entrance. Nevertheless, this study highlights the advantages of
conjugating cisplatin with a p53 activator as a single anticancer
agent and paves the way for further development of
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†
Electronic Supplementary Information (ESI) available:. See
DOI: 10.1039/b000000x/
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