Novel hypoxia-targeting Pt(iv) prodrugs

Article abstract of DOI:10.1039/c7cc01320e

Novel Pt(iv) prodrugs targeting hypoxia inducible factor HIF-1α were prepared for evaluating their antitumor activity on hypoxic cancer cells for the first time. The resulting Pt(iv) prodrug exhibited effective inhibition on tumor growth in the HCT-116 xe

Full text of DOI:10.1039/c7cc01320e

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DOI: 10.1039/C7CC01320E
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Novel hypoxia-targeting Pt(IV) prodrugs
Zichen Xu‡, Jian Zhao‡, Shaohua Gou*, and Gang Xu
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Novel Pt(IV) prodrugs targeting hypoxia inducible factor HIF-1α PI3K/Akt/mTOR/4E-BP pathway at the post-transcriptional
were prepared for evaluating their antitumor activity on hypoxic level without any influence on HIF-1α protein half-life or its
cancer cells for the first time. The resulting Pt(IV) prodrug mRNA level, and thereby inhibited the HIF-1 function in
exhibited effective inhibition on tumor growth in the HCT-116 hepatoma cells cultured under hypoxia.13 A mechanistic
xenograft mouse model with low toxicity in vivo.
research indicated that YC-1 speeded up HIF-1α degradation
by targeting its region containing amino acids 720 to 780 or
inhibiting murine double minute 2 (Mdm2),14 and stimulated
factor inhibiting HIF (FIH)-dependent p300 dissociation from
HIF-1α.15 In vivo, low HIF-1α protein expression together with
tumor growth inhibition was perceived in YC-1-treated mice.16
From then on, the anticancer activity and anti-HIF property of
YC-1 were widely explored both in vitro and in vivo.17
In view of clinical application, the formation of hypoxic
microenvironment in tumors can decrease curative effect of
cytotoxic chemotherapeutic drugs compared with that in
normoxic tumors.18 Thus, we would like to obtain novel
promising anticancer prodrugs, which can deliver a HIF-1α
inhibitor and an active drug unit to hypoxic tumor cells.
Cisplatin (CDDP) was widely used in cancer treatment as a
classic cytotoxic drug for its excellent antitumor effects on
numerous cancers. However, hypoxia is one of the most
important issues to limit its chemotherapeutic efficacy in solid
tumors in addition to the well known side effects of cisplatin.
Upon the unique structural features and reactive inertness of
Pt(IV) complexes, two hypoxia-targeting Pt(IV) prodrugs (YCC-1
and YCC-2) were designed and prepared as shown in Scheme 1,
in which YC-1 was attached to a Pt(IV) moiety derived from
Hypoxia, caused by an inadequate oxygen supply, is a hallmark
of solid tumors.1 In some solid tumors, the median oxygen (O2)
concentration is about 4% and may even decrease to 0%
locally.2 The cellular response to hypoxia is primarily mediated
by hypoxia inducible factor
1
(HIF-1).3 Under hypoxic
conditions, the HIF-1α subunit stabilizes, accumulates, and
then translocates into the nucleus, in which it dimerizes with
HIF-1β4 and activates the transcription of genes involved in
pivotal steps of tumorigenesis, including metabolism,
angiogenesis, metastasis, proliferation, and differentiation.5 To
date, overexpression of HIF-1α has been reported in more
than 70% of human cancers and is associated with poor
patient prognosis,6 making HIF-1α an attractive target for the
development of novel cancer drugs.7
In the past a few years, there have been only a limited
number of small molecules targeting HIF-1α expression or HIF-
1 transcriptional activity.8 Among those reported HIF-1α
inhibitors,
YC-1,
1-benzyl-3-(5-hydroxymethyl-2-
furyl)indazole,9 has been regarded as one of the most
potential drug candidates due to its potent inhibitory activity
against HIF-1α with a novel advantaged indazole structure,
which was found in a variety of chemotherapeutics with
diverse pharmacological effects such as antiplatelet,10
neuroprotection11 and antitumor activity.12 YC-1 was found to
completely block HIF-1α expression by suppressing the
Pharmaceutical Research Center and School of Chemistry and Chemical
Engineering, Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research,
Southeast University, Nanjing 211189, (P.R.) China.
E-mail: sgou@seu.edu.cn
†Electronic Supplementary Information (ESI) available:Synthesis of the compounds
and their characterization by 1H, 13C and 195Pt-NMR together with ESI mass spectra
and HPLC chromatograms. Cytotoxicity profiles of measured samples on the tested
cancer cell lines and biological assays as well as the inhibitory effects of YCC-2 on
HCT-116 tumor xenograft mice model. See DOI: 10.1039/x0xx00000x
‡ These authors contributed equally.
Scheme 1 Preparation of Pt(IV) prodrugs.
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cisplatin via a succinate linker. Such obtained two Pt(IV)
prodrugs, characterized by mass and NMR spectra (Fig. S1-S12),
are expected to own the ability of releasing cisplatin and YC-1
for their respective biological actions that can not only carry
the DNA binding platinum warhead into the tumor cells but
also have a functional molecule to target the tumor cells and
inhibit HIF-1α under hypoxia. Definitely, the prodrugs are
anticipated to be more sensitive under a hypoxic condition
than under a normoxic case.
View Article Online
DOI: 10.1039/C7CC01320E
Fig.2 Contrast of the cytotoxicity of two Pt(IV) prodrugs under normoxia and hypoxia
against (a) A549 with cisplatin and YC-1 as controls, and (b) HCT-116 with cisplatin, YC-1,
oxaliplatin as controls.
YCC-2 was typically examined by HPLC at different time
(Fig.S13), which was stable in PBS over 48 h. As shown in Fig.
S14, YCC-2 was found to be reduced in the presence of
ascorbic acid, confirming it could be reduced to its Pt(II)
equivalent and release YC-1 in a slow process.19 It is reported
that tumor tissues are less susceptible to oxidative stress as a
result of the higher level of ascorbic acid in hypoxic tumor
tissue compared with normal tissue.20 This can explain why
reduced oxygen concentration causes an increase in the rate
of reduction of Pt(IV) compounds. To investigate the effect of
the Pt(IV) complexes against human cancer cells, HCT-116,
A549, MCF-7, SGC7901 and cisplatin-resistant SGC7901/CDDP
together with normal cell LO2 were first incubated with YCC-1
and YCC-2 under normoxic condition and evaluated via MTT
assay, using cisplatin and YC-1 as references. As shown in Fig.1
and Table S1, YC-1 displayed negligible cytotoxicity against five
cancer cell lines under normoxia, while YCC-1 and YCC-2
showed stronger activity than cisplatin against all tested
cancer cells. In particular, YCC-2 was about 24.7 and 7.7 times
more effective than cisplatin against human lung cancer (A549)
and colon cancer (HCT-116) cell lines, respectively, with IC50
values of 0.80 and 3.51 μM in comparison to 19.79 and 27.10
μM of cisplatin. Both Pt(IV) complexes derived from cisplatin
with YC-1 as an axial ligand exhibited similar potency toward
the sensitive and the resistant cancer cells (SGC7901 and
SGC7901/CDDP). They were found to circumvent the cisplatin
resistance of SGC7901/CDDP cells with an IC50 value of 1.45
μM (YCC-1) and 2.78 μM (YCC-2), respectively, in comparison
to 13.09 μM of cisplatin. YCC-1 was 9.0 fold more potent than
cisplatin against SGC7901/CDDP cells, while YCC-2 was 4.7 fold
as potent as cisplatin, indicating their forceful ability to
circumvent resistance of cisplatin. Significantly, two Pt(IV)
complexes exhibited weak activity toward normal liver cells
(LO2) compared with cisplatin.
the clinic application of oxaliplatin in treating human
colorectaltumor, it was also used as a positive control. As
shown in Table S2 and Fig.2, under hypoxic conditions, YC-1
exhibited improved cytotoxic activity in A549 (IC50 = 20.13 μM)
and HCT-116 (IC50 = 43.26 μM) cells, owing to the intrinsic
function of inhibition of HIF-1α. The cytotoxicity of cisplatin
was similar to that under normoxia against A549 cancer cells,
but had a medium promotion in HCT-116 cancer cells,
indicating cisplatin was more sensitive to HCT-116 than A549
under hypoxia. Remarkably, YCC-2 exhibited significantly
promoted cytotoxicity in both tested cancer cell lines under
the same condition. It was about 92.6 and 37.6 times more
potent than cisplatin against HCT-116 and A549 cancer cells,
respectively, compared with 7.7 and 24.7 times in normoxic
state. Yet, YCC-2 was even 16.7-fold more potent than
oxaliplatin against HCT-116 cancer cells. Taken together, these
results validated that YCC-2 showed selective and markedly
increased antiproliferative activity against hypoxic cancer cells.
It is noted that two Pt(IV) prodrugs had big differences in
cytotoxicity between normoxia and hypoxia, particularly, in
HCT-116 cells, indicating that YC-1 has played a significant role
in the platination of DNA with our Pt(IV) complexes. However,
the substitution of the axial ligand from chlorido to hydroxyl
group can obviously result in different biological properties
between YCC-1 and YCC-2.
The platinum contents of cisplatin, oxaliplatin and YCC-2 in
HCT-116 cells under both normxic and hypoxic conditions were
analyzed using ICP-MS after 12 h of incubation with different
platinum complexes. As shown in Table S3, the cellular uptake
of YCC-2 in normxia was nearly comparable to that of
oxaliplatin, but 1.66 fold higher than that of cisplatin. While in
hypoxia, the platinum content of YCC-2 dramatically increased,
which was 4.12 fold greater than that of cisplatin and even
1.82 fold higher than that of oxaliplatin, despite the platinum
In contrast to the cytotoxicity under normoxia, antitumor
activity of two Pt(IV) complexes were further evaluated on
A549 and HCT-116 cells under hypoxic condition. Considering
Fig.3 Intracellular accumulation of cisplatin, oxaliplatin, and YCC-2 (15 μM) in HCT-116
cells after 12 h. Each value is in nanograms of platinum per 106 cells. Results are
expressed as the mean ± SD for three independent experiments.
Fig.1 IC50 values of cisplatin, YCC-1, YCC-2 against different cell lines under normoxia..
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contents of cisplatin and oxaliplatin in cells were promoted in
contrast to those in normxia (Fig.3). These data were almost
compatible to their IC50 values in HCT-116 cells. Obviously,
introduction of the HIF-1α inhibitor, YC-1, to the Pt(IV) moiety
derived from cisplatin can remarkably promote the cellular
uptake of the resulting Pt(IV) prodrug in the cancer cells under
hypoxic condition, leading to the potent antitumor activity of
YCC-2 in vitro.
View Article Online
DOI: 10.1039/C7CC01320E
To address whether the Pt(IV) complex could inhibit HIF-1α
protein accumulation or not, we tested YCC-2 in HCT-116
cancer cells with YC-1 as positive control under hypoxia by
western blot analysis. As Fig.4 showed, YCC-2 could
downregulate the level of HIF-1α protein expression under
hypoxic condition in a dose dependent manner as YC-1, with
about 50% decrease at 1 μM although HIF-1α seemed slightly
susceptible to YCC-2 inhibition at 0.1 μM. The result implied
that YCC-2 had a pronounced effect on HIF-1α expression.
HCT-116 cells, stained with Annexin-V FITC and PI, were
used to confirm whether YCC-2 was responsible for the
induction of apoptosis under both normoxic and hypoxic
conditions. As demonstrated in Fig. 5, the tested compounds
were incubated with HCT-116 cells for 72 h at 1 μM of YCC-2
with cisplatin and oxaliplatin as positive controls. Under
normoxic condition (Fig. 5a), few (4.73%) apoptotic cells were
present in the control panel; in contrast, the apoptotic
population rose to 13.19% and 20.12% after treatment with
cisplatin and oxaliplatin at 1 μM, respectively. However,
moderate increase to 25.41% occurred after treatment with
YCC-2 at the same concentration, higher than those of positive
controls. In view of the oxygen-dependent nature of YCC-2, 1%
O2 was set in cell apoptosis study to be close to the
physiologically hypoxic condition. As illustrated in Fig. 5b, once
hypoxia was conducted, basic cell viability was generally
reduced. Meanwhile, the cell lethality was largely enhanced by
the effect of YCC-2 with the population of apoptotic cells rising
to 67.94%. Even after subtracting the blank control, the
enhanced apoptosis rate of YCC-2 was much greater than
those of cisplatin and oxaliplatin, indicating that YCC-2 had
great advantages over the marketed Pt(II) drugs. The above
results confirm the effect of YCC-2 as an antitumor agent
under hypoxia.
Fig.5 Representative flow cytometric diagrams of apoptotic HCT-116 cells after 72 h
treatment with cisplatin, oxaliplatin and YCC-2 under (a) normoxia and (b) hypoxia. The
cells were harvested and labeled with annexin-V-FITC and PI, and analyzed by flow
cytometry. Data are expressed as the mean ± SEM for three independent experiments.
(12 mg/kg); (5) vehicle. All of the drugs were given
intravenously with the above-mentioned formulations once
every 7 days for 21 days. The administration of YCC-2
efficiently inhibited the growth of HCT-116 tumor in a dose-
dependent manner (Fig. 6). The growth of HCT-116 tumor
xenograft was significantly suppressed by 50.2% and 61.5%
after administration of YCC-2 at 5 and 12 mg/kg compared
with the vehicle group, respectively (Fig. 6A, 6B). At an
equimolar dosage, the drug efficacy of YCC-2 (12 mg/kg) was
comparable to cisplatin. The measurement of tumor weight
and volume at the end of assay confirmed this result. Similar
drug effectiveness was observed for YCC-2, cisplatin and
oxaliplatin, but YCC-2 exhibited significantly lower toxicity
based on the weight measurement of mice (Fig. 6C), because
YCC-2 had a little effect on the weight of mice even at a high
dosage. The histological findings observed with hematoxylin
and eosin (H&E) staining also confirmed the result (Fig. S15,
S16).
In summary, we successfully developed two hypoxia-
targeting Pt(IV) prodrugs containing a HIF-1α inhibitor for the
first time. Each Pt(IV) complex showed higher cytotoxicity than
its mother complex, cisplatin, against all the tested cancer cell
lines including cisplatin resistant SGC7901/CDDP. Notably,
The in vivo tumor therapeutic effect of YCC-2 on the HCT-
116 tumor xenograft mice model together with cisplatin and
oxaliplatin was studied. The mice were randomly divided into
five groups with 5 mice in each treated group: (1) cisplatin (5
Fig.6 In vivo antitumor activity of drugs in mice bearing HCT-116 xenograft after
administration with YCC-2 at the dose of 5 and 12 mg/kg, cisplatin and oxaliplatin at
the dose of 5 mg/kg, for 21 days. (A) Tumor volume of the mice in each group. (B)
Tumor weight of the mice from each group at the end of the observation period. (C)
Body weight of the excised mice of each group. The data were presented as the mean ±
SEM. *P< 0.05.
Fig.4 Effects of YC-1 and YCC-2 on HIF-1α expression. HCT-116 cells were pretreated
with or without different concentrations of YC-1 and YCC-2, respectively, under hypoxia.
HIF-1α proteins were detected via western blotting with specific antibodies. GAPDH
was used as a loading control.
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