CAIXplatins: Highly Potent Platinum(IV) Prodrugs Selective Against Carbonic Anhydrase IX for the Treatment of Hypoxic Tumors

Article abstract of DOI:10.1002/anie.202005362

Hypoxia and the acidic microenvironment play a vital role in tumor metastasis and angiogenesis, generally compromising the chemotherapeutic efficacy. This provides a tantalizing angle for the design of platinum(IV) prodrugs for the effective and selective killing of solid tumors. Herein, two carbonic anhydrase IX (CAIX)-targeting platinum(IV) prodrugs have been developed, named as CAIXplatins. Based on their strong affinity for and inhibition of CAIX, CAIXplatins can not only overcome hypoxia and the acidic microenvironment, but also inhibit metabolic pathways of hypoxic cancer cells, resulting in a significantly enhanced therapeutic effect on hypoxic MDA-MB-231 tumors both in vitro and in vivo compared with cisplatin/oxaliplatin, accompanied with excellent anti-metastasis and anti-angiogenesis activities. Furthermore, the cancer selectivity indexes of CAIXplatins are 70–90 times higher than those of cisplatin/oxaliplatin with effectively alleviated side-effects.

Full text of DOI:10.1002/anie.202005362

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Accepted Article
Title: CAIXplatins: highly potent Pt(IV) prodrugs selectively against
hypoxic tumors via microenvironment and metabolism regulation
Authors: Qian Cao, Dan-Jie Zhou, Zheng-Yin Pan, Gang-Gang Yang,
Hang Zhang, Liang-Nian Ji, and Zong-Wan Mao
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
CAIXplatins: highly potent Pt(IV) prodrugs selectively against
hypoxic tumors via microenvironment and metabolism regulation
a†
a†
a†
a
a
a
Qian Cao, Dan-Jie Zhou, Zheng-Yin Pan, Gang-Gang Yang , Hang Zhang, Liang-Nian Ji and
a*
Zong-Wan Mao
Abstract: Hypoxia and acidic microenvironment play a vital role in
tumor metastasis and angiogenesis, generally compromising the
chemotherapeutic efficacy. This provides a tantalizing angle for the
Pt(IV) prodrugs design to achieve effective and selective killing of
solid tumors. Herein, two carbonic anhydrase IX (CAIX)-targeted
platinum(IV) prodrugs have been developed, named as CAIXplatins. agents, including cisplatin.
gene and protein expression patterns, such as the expression of
components in mismatch repair system and in double strand
breaks checkpoint, resulting in a deficit in mismatch repair and
accumulation of genomic instabilities and mutations, which
negatively affects the anticancer activity of DNA interacting
[18]
Moreover, hypoxia and low pH
Based on the strong affinity and inhibitory activity for CAIX,
CAIXplatins can not only overcome hypoxia and acidic
microenvironment, but also inhibit metabolic pathways of hypoxic
cancer cells responsible for energy supply and biomacromolecules
synthesis, resulting in significantly enhanced therapeutic effect on
hypoxic MDA-MB-231 tumors both in vitro and in vivo compared
with cisplatin/oxaliplatin, accompanied with excellent anti-metastasis
and anti-angiogenesis activities. The cancer selectivity indexes of
CAIXplatins are 70~90 times higher than that of cisplatin/oxaliplatin,
and the systemic side-effects of platinum drugs are effectively
alleviated, thus showing great potential in further clinical use of
cancer therapy.
creates a bio-reductive milieu that can influence the stability,
penetration and bioprocess of most chemotherapeutic drugs,
[
19]
thus reducing their anticancer efficacy. In this case, even if the
hypoxic (reducing) condition may be beneficial for the Pt(IV)→
Pt(II) reduction, the activity of the released Pt(II) drugs will be
subsequently hindered in the same milieu. Keppler and
coworkers have found that reducing the oxygen partial pressure
to establish a more reducing environment does not increase the
cytotoxicity of Pt(IV) complexes. They claimed that that hypoxia,
independent of the induction method, was not sufficient to
[
20]
activate platinum(IV) complexes to a considerable extent.
Thus, regulation of tumor hypoxia and acidic microenvironment
may be a promising strategy to enhance the therapeutic efficacy
of platinum drugs and has profound clinical significance in
cancer therapy. However, only a few Pt(IV) prodrugs modified by
HIF-1α inhibitor or PPARα agonist have been reported capable
of regulating the adaptation of cells to hypoxia, thus exhibiting
improved anticancer activity in hypoxia with respect to
Introduction
Nowadays, despite the promising prospects of targeted
therapy and immunotherapy in cancer treatment, conventional
cytotoxic chemotherapy, especially cisplatin, is still the first-line
[
21,22]
normoxia.
Unfortunately, these Pt(IV) prodrugs show similar
[
1,2]
in vitro toxicity to cancer cells and normal cells, and their in vivo
therapeutic effect on solid tumors is not superior to cisplatin.
weapon against solid tumors.
Continuous efforts have been
made to improve anticancer efficacy of platinum drugs and
[
3,4]
Carbonic anhydrase IX (CAIX) is
a well-known trans-
reduce their side-effects.
The promising alternatives are
membrane protein over-expressed in developing and aggressive
tumors (e.g. colon, cervical, esophagus, lung, bone, and breast
cancer, etc) rather than normal tissues, and its expression is
platinum (IV) prodrugs which are kinetically inactive prior to
[
5,6]
reduction.
Many inspiring platinum (IV) prodrugs have been
reported, the modified axial ligands of which can provide
significant bioactivity functioning in a manner synergistic to that
of Pt(II) drugs, thus improving curative effect through various
ways, including tuning lipophilicity and kinetics, changing cellular
uptake mechanisms, providing cancer-specific targeting,
releasing multiple bioactive substances, inhibiting DNA repair
pathway, activating chemo-immunotherapy, controlling on-site
[
23]
promoted by hypoxia through HIF-1α.
CAIX is to catalyze the hydration of CO
bicarbonate, thus responsible for the regulation of both extra-
The main function of
2
to proton and
[
24]
and intracellular pH equilibrium.
For example, rhenium
arylsulfamide conjugates that can selectively inhibit CAIX have
been found able to effectively reduce extracellular acidification of
[25]
[
7-16]
tumors. Recently, Chen et al. has reported the construction of
short peptide-based self-assembled CAIX inhibitors, which can
remarkably enhance the anticancer efficacy in doxorubicin
photo-activation, etc.
Despite these encouraging efforts,
hypoxia is still an important issue seriously limiting the
therapeutic efficacy of Pt(IV) prodrugs on solid tumors, which
has not been extensively investigated.
[
26]
administration of aggressive breast tumors.
CAIX-targeted
photosensitizers have also been reported, capable of
overcoming hypoxia caused by photodynamic therapy thus
Hypoxia and acidic niche are the characteristics of solid
tumors, displaying a vital role in promoting tumor growth,
angiogenesis and metastasis. Hypoxia can also interfere with
[
27]
[17]
leading to superior inhibition of breast tumors.
On the other
hand, hypoxia and acidic microenvironment are known closely
related to the remodeling of tumor metabolism, and it has been
proposed that destroying both pH-regulating proteins and
bioenergy metabolic pathways is an effective means to induce
metabolic catastrophe in aggressive and rapidly developing
[a]
Dr. Q. Cao, D.-J. Zhou, Z.-Y. Pan, Dr. G.-G. Yang, H. Zhang, Prof.
L.-N. Ji, Prof. Z.-W. Mao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry, Sun Yat-Sen University
Guangzhou 510275, China
[28]
tumors.
Therefore, inhibition of CAIX is not only an effective
E-mail: cesmzw@mail.sysu.edu.cn
way to regulate the unique hypoxia and acidic microenvironment
of tumor tissues, but also can interfere with tumor metabolism.
This proposes a tantalizing angle for designing Pt(IV) prodrugs
†
These authors contributed equally to this work
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
10.1002/anie.202005362
RESEARCH ARTICLE
corresponding to the axial release of L (7.813 min), and nearly
all Pt1 has already been reduced in 1 h. Under the same
condition, the ratio of peak areas indicates that only ca. 50% of
Pt2 (9.676 min) has been reduced in 1 h, and all Pt2 can be
reduced in 24 h. This suggests that both Pt(IV) compounds can
be effectively reduced for Pt(II) drugs release, and Pt2 may be
more stable in the blood circulation than Pt1. Furthermore, after
20 h reaction of Pt1 (0.2 mM) and ascorbic acid (20 mM) in
aqueous solution in the presence of guanosine (0.8 mM), ESI-
MS of the reaction mixture shows a new peak at m/z 548.3,
which has been assigned to the formation of cis-
[30b]
[
Pt(NH3)2Cl(guanosine)]+ adduct,
further demonstrating the
Scheme 1. Chemical structures of ligand L and Pt(IV)-CAIXi hybrid complexes
release of cisplatin from Pt1 (Fig. S10, ESI†).
Firstly, we evaluated in vitro cytotoxicity of
designed in this work; SLC-0111 (a CAIX inhibitor on trail) is also present.
L, Pt1 and Pt2
against various cancerous cell lines (triple negative breast
cancer MDA-MB-231, HeLa, HepG2) and normal cell lines
to achieve effective and selective killing of solid tumors, as well
as angiogenesis and metastasis inhibition.
(
MCF-10A, LO2, HLF) by MTT assay (72 h), using
cisplatin/oxaliplatin as positive controls (Table 1 . Under
normoxia (20% O ), both Pt1 and Pt2 exhibit moderate
In the present work, CAIX inhibitors (CAIXi) are introduced as
axial ligands of Pt(IV) prodrugs (named as CAIXplatins), aiming
at regulating tumor microenvironment and metabolism to
enhance the therapeutic effect of platinum drugs on solid tumors.
Such design possesses several striking characteristics: (i) due to
the over-expression of CAIX on membranes of hypoxic cancer
)
2
cytototxicity against cancer cell lines, which is similar to that
of cisplatin/oxaliplatin. However, the cytotoxicity of Pt1 and
Pt2 against normal cell lines is extremely low (IC50 > 100μM)
giving a cancer selectivity index (SI) 8-folds higher than that
of cisplatin/oxaliplatin.
,
[
23,29]
cells,
CAIXplatins can markedly facilitate cancer-specific
targeting, resulting in extremely low toxicity (IC50 >100 μM, 72 h)
to normal cell lines, and the cancer selectivity indexes (SI) are
Under hypoxia (1% O
against cancer cell line
2
), the cytotoxicity of Pt1 and Pt2
is significantly enhanced with
s
70~90 times higher than that of cisplatin/oxaliplatin in hypoxia; (ii)
respect to normoxia while that of cisplatin/oxaliplatin is
reduced, particular in hypoxic MDA-MB-231 cells, Pt1 and
Pt2 are 10-times more toxic than cisplatin/oxaliplatin.
Accordingly, the cancer selectivity indexes of Pt1 and Pt2
are substantially elevated in hypoxia, which is 70~90 folds
higher than the SI of cisplatin/oxaliplatin. These results
demonstrate that Pt1 and Pt2 can overcome hypoxia, thus
selectively and effectively killing hypoxic cancer cells
based on the strong affinity and inhibitory activity for CAIX,
CAIXplatins can overcome hypoxia and acidic microenvironment,
resulting in excellent anti-metastasis and anti-angiogenesis
activities, as indicated by the inhibition of wound healing,
zebrafish blood vessel formation, and CD31 (an angiogenesis
marker) expression in tumor tissues; (iii) CAIXplatins can also
interfere with metabolic pathways of hypoxic cancer cells
responsible for the energy supply and biomacromolecules
synthesis, thus significantly enhancing the therapeutic effect on
hypoxic tumors both in vitro and in vivo; (iv) CAIXplatins
effectively alleviate side-effects e.g. bone marrow hematopoietic
system toxicity, hepatotoxicity, nephrotoxicity, ototoxicity of
platinum drugs, thus showing great potential in further clinical
use of cancer therapy.
Table 1. Cytotoxicity (IC50, μM) of tested compounds against cancerous cell
lines (MDA-MB-231, HeLa, HepG2) and normal cell lines (*MCF-10A, LO2,
[a]
HLF) for 72 h.
Pt1
Pt2
L
Cisplatin
Oxaliplatin
Normoxia (20% O
2
)
MDA-MB-231
HeLa
18.0 ± 1.8
12.6 ± 1.2
28.6 ± 0.6
158.5 ± 3.4
102.6 ± 8.6
153.7 ± 3.6
8.5
27.6 ± 2.3
41.7 ± 2.3
30.4 ± 0.8
>200
>200
>200
>200
>200
>200
>200
/
14.6 ± 0.8
15.9 ± 1.3
17.9 ± 1.9
12.0 ± 1.5
10.7 ± 2.9
17.9 ± 1.9
1.3
35.4 ± 2.0
28.1 ± 2.6
8.1 ± 0.8
31.0 ± 5.3
23.7 ± 1.9
8.9 ± 0.4
1.0
Results and Discussion
HepG2
LO2
As shown in Scheme 1, the axial ligand L was designed
containing benzene sulfonamide, a common active moiety of
CAIX inhibitors, and two CAIXplatins defined as Pt1 and Pt2
were developed with cisplatin and oxaliplatin core, respectively.
Their structures were fully characterized by elemental analysis,
HLF
>200
*MCF-10A
>200
[
b]
SI
>7.3
2
Hypoxia (1% O )
1
13
195
ESI-MS, H, C,
Pt NMR and HPLC (Fig. S1-S8, ESI†).
[
30a]
MDA-MB-231
HeLa
1.9 ± 0.9
3.6 ± 0.3
8.3 ± 1.8
80.9
5.8 ± 2.1
15.8 ± 2.6
5.6 ± 1.5
>34.5
>200
>200
>200
/
21.9 ± 5.9
28.2 ± 1.7
20.2 ± 3.8
0.9
65.7 ± 5.3
>50
Considering that there are many reducing agents in cells,
we
measured their stability in cell culture medium, showing that
both Pt1 and Pt2 are stable for at least 48 h (Fig. S9A, ESI†).
Then the reduction of Pt1 and Pt2 (50 μM) was investigated in
PBS containing 10.0 mM of ascorbic acid, which was a reducing
substance that exists naturally in living organisms.
in Fig. S9B (ESI†), the HPLC signal for Pt1 (8.576 min)
decreases in tandem with the appearance of peak
HepG2
18.1 ± 2.1
0.5
[
c]
SI
[a] Data are presented as the means ± standard deviations (SD) of three
repeated measurements. [b-c] SI (cancer selectivity index) is defined as ratio
of IC50(*MCF-10A) in normoxia / IC50(MDA-MB-231) in hypoxia.
[
30b]
As shown
a
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
without affecting normal cells. We also measured the IC50
values of SLC-0111 in 2:1 mixture with cisplatin or oxaliplatin,
showing that their cytotoxicity under hypoxia is much lower than
that under normoxia (Table S1, ESI†). This further demonstrates
that attaching CAIX inhibitor to Pt(IV) has a great advantage
compared with the combination therapy using the mixture of
CAIX inhibitor and Pt(II) drugs.
Pt
Pt
B
C
We also investigated the anti-metastasis and anti-
angiogenesis of Pt1 and Pt2 in hypoxic MDA-MB-231 cells
2
(1% O ). As shown in wound healing assay (Fig.1A-B), the
wound borders in control medium are well defined
immediately after scratching the monolayer, but migrate to
the centre to narrow the wound area after 24 h incubation,
indicating the good migration ability of MDA-MB-231 cells
under hypoxia. However, upon treatment with Pt1 and Pt2 (5
μM, 24 h), cell migration is significantly suppressed by
D
81.2% and 77.0%, respectively, as indicated by the less
closure of the wound borders, while treatment with cisplatin
and oxaliplatin under the same conditions only suppresses the
migration by 27.1% and 4.5%, respectively. This indicates the
E
excellent anti-metastasis activity of Pt1 and Pt2
.
Simultaneously, Western blotting (Fig. 1C) shows that Pt1
and Pt2 treatment (20 and 40 μM, 36 h) can substantially
decrease the expression level of vascular endothelial growth
Figure 1. (A-B) Representative pictures in wound healing assay and the
2
factor (VEGF) in hypoxic MDA-MB-231 cells s (1% O ) in a
migration area of hypoxic MDA-MB-231 cells treated with or without Pt drugs
dose-dependent manner. Furthermore, the formation of blood
vessels in zebrafish (Is5Tg:mCherry) was monitored (Fig. S11,
ESI†). Blood vessels are well-distinguished at the head and
trunk intersection of untreated zebrafish as indicated by the
intensive red fluorescence from the reporter protein. Pt1 and Pt2
treatment (10 μM, 48 h) can significantly quench the red
fluorescence, similar to the case of AAZ (a FDA approved CA
inhibitor), indicating that zebrafish angiogenesis is effectively
inhibited. All these results suggest the excellent anti-metastasis
(
10 μM, 24 h). ** means p< 0.01. (C) Western blot analysis of VEGF
expression in MDA-MB-231 cells upon 36 h treatment of Pt(IV) compounds
under hypoxia(1% O2). GADPH is an internal reference. (D) Cellular
accumulation of Pt contents after 24 h treatment of MDA-MB-231 or MCF-10A
cells with various compounds (10 μM). (E) Western blot analysis of CAIX
expression in: 1. MCF-10A cells, 20% O
MDA-MB-231 cells, 1%O
2 2
; 2. MDA-MB-231 cells, 20%O ; 3.
2
.
cisplatin/oxaliplatin exhibit negligible uptake differences
between cancer and normal cell lines. Moreover, the
accumulation of Pt1 and Pt2 in hypoxic MDA-MB-231 cells is
and anti-angiogenesis activity of Pt1 and Pt2
.
Based on the structure design, the action mechanism of
Pt1 and Pt2 is expected to be closely related to CAIX
binding and intervention. Molecular docking (Fig. S12, ESI†)
was firstly carried out, showing that both Pt1 and Pt2 can enter
higher
than
cisplatin/oxaliplatin,
but
lower
than
cisplatin/oxaliplatin in MCF-10A cells (Fig. 1D). 24 h and 48 h
organelle distributions of these Pt(IV) and Pt(II) drugs further
demonstrated these findings (Fig. S13B, ESI†). In addition,
pre-treatment of MDA-MB-231 cells (1% O
(
uptake of Pt1 and Pt2 by 45.6% and 72.4% with respect to
untreated cells, whereas the uptake of cisplatin and
oxaliplatin is not substantially affected by SLC-0111 (Fig.
S14, ESI†); Simultaneously, pre-treatment with carbonyl
cyanidem-chlorophenyl hydrazone (CCCP, a metabolic inhibitor,
2
+
the pocket of CAIX (PDB code: 3IAI), coordinating with Zn
2
) with SLC-0111
a CAIX inhibitor, 50 μM, 1 h) can greatly reduce the cellular
active center and hydrogen bonding with THR199 and THR200
residues. This binding mode is similar to AAZ (a FDA approved
CA inhibitor), but Pt1 and Pt2 have more hydrogen bonds with
amino acids (HIS64, GLN92, ASN62, GLN67, and TRP5). The
binding energies are also determined as L (-6.6 kcal/mol), Pt1 (-
7.5 kcal/mol) and Pt2 (-8.1 kcal/mol), indicating the good binding
capacity of Pt1 and Pt2 to CAIX.
CAIX binding capacity of Pt1 and Pt2 is benefiting their
cancer-specific cellular uptake. As shown in ICP-MS (Fig. 1D),
3
0 μM, 1 h) or chloroquine (a endocytosis modulator, 50 μM, 1
h) also leads to significantly decreased (ca. 40-70%) cellular
uptake of Pt1 and Pt2 (Fig. S14, ESI†). These results
demonstrate that Pt1 and Pt2 can selectively accumulate in
hypoxic cancer cells instead of normal cells mainly due to their
CAIX binding capacity and CAIX-mediated active transport
and endocytosis
selectivity and cytotoxicity to hypoxic cancer cells
Then the inhibition effects of Pt1 and Pt2 on CAIX were
studied both in vitro kit test and at cellular level. As shown in kit
test using human CAIX as the enzyme source and 4-NPA as
after treatment with various platinum drugs (10
accumulation of Pt1 and Pt2 in MDA-MB-231 cancer cells (20%
is substantially higher than that in MCF-10A normal cells,
which is further elevated in MDA-MB-231 cells under hypoxia
1% O ). This is consistent with the CAIX expression level
in MDA-MB-231 cells (1% O ) > MDA-MB-231 cells (20%
) > MCF-10A cells measured by western blot (Fig. 1E).
M, 24 h) the
2
O )
,
accordingly explaining their excellent
(
2
.
2
O
2
Semi-quantitative analysis of the CAIX expression level is also
obtained from western blot (Fig. S13A, ESI†). By comparison,
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Angewandte Chemie International Edition
10.1002/anie.202005362
RESEARCH ARTICLE
normoxia (20% O
2
)
, indicating a positive correlation between
hypoxia and extracellular acidification of cancer cells (Fig. 2C).
After 24 h of Pt1 and Pt2, the pH values of hypoxic cells are
increased by 0.15~0.2, similar to the case of SLC-0111
treatment (a CAIX inhibitor, 50 μM, 1 h but unaffected by
e
)
cisplatin/oxaliplatin under the same conditions, indicating
that Pt1 and Pt2 can weaken extracellular acidification via the
action of CAIXi moiety (Fig. 2C). In addition, we also noted that
MDA-MB-231 cells (1% O
2
)
were arrested in G2 phase after
M, 48 h) whereas cisplatin
Pt1 and Pt2 treatment 10 and 20
(

and oxaliplatin treated cells were mainly arrested in S phase (Fig.
S16, ESI†). This may be due to the abnormal pH regulation
mediated shortage of bicarbonate content, which is the
[
4]
necessary material for protein and nucleotide synthesis.
All these results demonstrate that Pt1 and Pt2 can
effectively bind CAIX and inhibit its activity, thus
overcoming the hypoxia and weakening the extracellular
acidification of hypoxic cancer cells, which contributes to
their excellent anti-metastasis and anti-angiogenesis activities.
To further understand the action mechanism of Pt1 and
Pt2, differentially expressed proteins among Pt1
/Pt2-treated
and control cells (MDA-MB-231, 1% O ) were characterized
2
Figure 2. (A) Inhibitory IC50(nM) of CAIX activity by complexes Pt1, Pt2, L,
AAZ (a FDA approved CAi with CAIX inhibitory IC50 of 25 nM). (B) Western
blot analysis of CAIX expression in MDA-MB-231 cells upon 24 h treatment of
using iTRAQ-based proteomics. According to stringent
criteria of having ≥1 unique peptide per protein present, p-
value <0.05, and fold-change >1.2 or <0.83, 386 and 333
2
compounds Pt1, Pt2 (20 μM, 40 μM) under hypoxia (1% O ). (C) Impact of
compounds on the pH of MDA-MB-231 extracellular environment under
different conditions. (D) Representative confocal images of cells after
incubation with compounds (10 μM) for 48 h. Scale bar = 50 μm. The RDDP
Pt1
control
Pt2
control
A
probe (red) was used to detect the O
2
. (E) HIF-1α expression(green) of cells
1.5
after incubation with compounds (10 μM) for 36 h under hypoxia (1% O
2
).
1
Scale bar = 100 μm.
0.5
0
substrate (Fig. 2A and Fig. S15, ESI†), both Pt1 and Pt2 can
effectively inhibit CAIX activity with inhibitory IC50 values at
nanomolar levels (251.2 and 77.6 nM), and the inhibitory activity
of L < Pt1 < Pt2 is in good agreement with their calculated
binding capacity. In hypoxic MDA-MB-231 cells (1% O ), the
2
expression of CAIX is not affected by cisplatin/oxaliplatin
-0.5
-1
-1.5
B
Pt2
Pyrimidine metabolism
Pt1
Pt2
but substantially inhibited by Pt1 and Pt2 treatment in a
Oxidative phosphorylation
Metabolic pathways
dose-dependent manner (20 and 40

M, 24 h)
，
and the
Pt1
Pt2
inhibition effect of Pt2 is better than Pt1 (Fig. 2B). This is
consistent with the in vitro kit test results.
Considering the role of CAIX on maintaining hypoxic and
Pt1
Pt2
HIF-1 signaling pathways
Pt1
Pt2
acidic microenvironment, the extracellular pH values (pH
and content in MDA-MB-231 cells was monitored. As
shown in Fig. 2D-E, hypoxic cells (1% O have low O content
as indicated by the strong red fluorescence from RDDP (
commercial O fluorescent probe), which is accompanied with
high expression level of HIF- as indicated by green
Immunofluorescence. After Pt1 and Pt2 (10 M) treatment, the
RDDP red fluorescence is substantially quenched,
indicating an increase in the cellular O concentrations,
while the expression level of HIF- is found reduced By
comparison, cellular content is not affected by cisplatin or
e
)
O
2
Glycolysis/ Gluconegenesis
Carbon metabolism
Pt1
Pt2
2
)
2
a
Pt1
Pt2
2
Biosynthesis of amino acids
Pt1

0
2
4
6
-
log10 (p-value)
2
.
Figure 3. (A) Hierarchical cluster analysis of 383 and 333 proteins expressed
with statistically significant differences (P<0.05, and fold change >1.2 or <0.83)
in Pt1 and Pt2 treated and untreated (control) hypoxic MDA-MB-231 cells.
Three independent experiments were performed in Pt1 and Pt2 while two
independent in control. (B) EGCC Pathway analysis: seven pathways showing
obvious enrichment (−log (p-value) > 2) were selected. Count: number of
differentially expressed proteins mapped to this pathway.
O
2
oxaliplatin, indicating that Pt1 and Pt2 can effectively
overcome hypoxia mainly through the action of CAIXi
moiety rather than the Pt(II) drugs.
Simultaneously, pH
incubated under hypoxia (1% O
e
values of MDA-MB-231 cells
is found lower than that under
2
)
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
differentially expressed proteins are screened out from Pt1
and Pt2 treated cells, respectively. All expression values of
these differentially expressed proteins are converted to a
log form and input as hierarchical clustering algorithms (Fig.
A
B
3
A), characterizing 174 up-regulated and 112 down-
regulated proteins in the case of Pt1, while 102 up-
regulated and 221 down-regulated in the case of Pt2
.
Pt1
Pt2
According to gene ontology (GO), the most prevalent
biological processes in which these differentially expressed
proteins participate are oxidation-reduction and biomolecule
metabolic processes, including nucleobase-containing small
molecule, nucleotide, nucleoside phosphate, ribose
phosphate, ribonucleotide, rRna metabolism etc. (Fig. S17,
ESI†).
Cisplatin
Oxaliplatin
Control
Then EGCC pathway analysis was conducted to directly
reflect which pathways had significant enrichment (−log P
value > 2) of differentially expressed proteins. As present in
Fig. 3B, several important metabolic pathways have been found
systematically affected in both Pt1 and Pt2 treated cells. For
example, Glycolysis/Gluconeogenesis, Carbon metabolism,
and HIF-1 signaling pathway all present down-regulation of
C
glycolysis-related
key
proteins,
including
PGK1
(
(
(
phosphoglycerate kinase-1), ENO2 (enolase-2), HK2
Hexokinase-2), LDHA (lactate dehydrogenase-A), PDK1
Pyruvate dehydrogenase kinase), resulting in reduced
D
production of lactic acids and ATP. It is known that most cells
has plasticity in their ATP generating ability from either
glycolysis or respiration, whereby blockage of one metabolic
[
28]
pathway rapidly shifts cells to the other. However, in Pt1 and
Pt2 treated hypoxic cancer cells, Oxidative phosphorylation
pathway is also inhibited, presenting down-regulation of key
enzymes for electron transfer (from NADH to ubiquinone) of
mitochondrial respiratory chain, such as NADH
dehydrogenase, cytochrome c reductase and oxidase, etc.
Moreover, Pyrimidine metabolism and Biosynthesis of
amino acids are also seriously affected, resulting in serious
shortage of raw materials for biosynthesis (Table S2, ESI†).
These proteomics analysis suggest that Pt1 and Pt2 can not
only regulate hypoxia and acidic microenvironment, but also
drastically affect metabolic functions of hypoxic cancer cells,
particularly those responsible for energy supply and
biomacromolecular synthesis.
Figure 4. (A) Changes of the tumor volumn and body weight. The indicated
compounds (5 mg/kg) were i.v. injected every three days for 4 times totally,
including vehicle (1% DMSO in 0.9% NaCl) as control. Data are presented as
means ± SD (n = 4) (B) Tumor images after 24 days treatment. (C) CAIX and
CD31 expression on the tumor after 24h drug treatment (5 mg/kg i.v.). Scale
bar = 50 μm. (D) Impact of the on hair cells of zebrafish larvae. Scale bar =
200 μm.
Finally, the in vivo anti-tumor efficacy was evaluated using
BALB/c nude mice bearing MDA-MB-231 tumors with initial
The in vivo anticancer efficacy is consistent with their in vivo
bio-distributions. As shown in Fig. S18 (ESI† , the accumulation
3
)
volumes of ca. 80 mm . They were randomly divided into five
-1
of Pt1 and Pt2 in tumor sites reaches the highest at 8 h and 24 h
post-injection (i.v), respectively, which is 4~7 folds higher than
that of cisplatin and oxaliplatin. This is mainly due to the strong
targeting and affinity of Pt1 and Pt2 to CAIX. Simultaneously,
the expression level of CAIX and CD31 (angiogenesis markers)
in tumor tissues was measured using immunofluorescence
groups (n = 4) for tail vein injection (i.v. 120
L, 5 mg kg ) with
vehicle and various platinum drugs. The tumor volumes and
mice weights were measured every 3 days, and after 24 days all
the mice were sacrificed and the tumors in each group were
collected (Fig. 4A-B). It is found that after four consecutive
injections both Pt1 and Pt2 can inhibit tumor growth by 57.9%
and 65.3%, respectively, compared with control, while cisplatin
and oxaliplatin only inhibit tumor growth by 43.4% and 32.0%,
thus giving in vivo anticancer efficacy of Pt2 > Pt1 > cisplatin >
oxaliplatin. It is worth mentioning that all platinum drugs were
-1
assay 24 h after various platinum drugs (i.v. 120
L, 5 mg kg )
administration. Compared with the control group, the
fluorescence from CAIX (green) and CD31 (red) is not affected
by cisplatin or oxaliplatin but substantially suppressed after
Pt1 and Pt2 treatment (Fig. 4C). Combining with the
forementioned MTT assay, wound healing assay, VEGF
expression and zebrafish blood vessels formation experiments,
Pt1 and Pt2 have been demonstrated with excellent
-1
administrated at the same dose (5 mg kg ), meaning that the
actual Pt content of Pt1/Pt2 was only ca. 1/4 of
cisplatin/oxaliplatin according to their molecular weight.
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Angewandte Chemie International Edition
10.1002/anie.202005362
RESEARCH ARTICLE
therapeuticefficacy and anti-angiogenesis activity on CAIX
highly-expressed tumors.
We are grateful for financial support from the National Natural
Science Foundation of China [21837006, 91953117 and
21401217], the Ministry of Education of China (No. IRT-17R111),
Natural Science Foundation of Guangdong Province
Additionally, the side-effects of Pt1 and Pt2 were thoroughly
assessed by bodyweight tracing, H&E staining of main organs,
complete blood panel and biochemical analysis, and ototoxicity
evaluation. Firstly, cisplatin group exhibits severe bodyweight
loss since Day 12 during the in vivo anti-tumor investigations
whereas no mouse death or bodyweight loss is found in Pt1 and
Pt2 groups (Fig. 4A and S19, ESI†); Secondly, after 24 days
treatment cisplatin and oxaliplatin groups exhibit obvious
damage to major organs (Fig. S20, ESI†), significantly
decreased hematocrit (HCT), red blood cells (RBC), hemoglobin
[2017A030313041],
2013b051000047, 207999], and Fundamental Research Funds
for the Central Universities.
Guangdong
Provincial
Government
[
Keywords: Pt(IV) prodrugs • carbonic anhydrase IX • hypoxia •
side-effects elimination • anti-tumor efficacy
[
[
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+
+
We propose carbonic anhydrase IX-
targeting mediated microenvironment
Qian Cao, Dan-Jie Zhou, Zheng-Yin
+
Pan, Gang-Gang Yang, Hang Zhang,
Pt(IV)-CAIXi prodrug
cisplatin
and metabolism regulation as a novel
approach for treating hypoxic and
aggressive tumors, e.g. triple negative
breast cancer, capable of greatly
increasing cancer selectivity index,
enhancing therapeutic efficiency,
eliminating side-effects of platinum
drugs, as well as displaying excellent
anti-angiogenesis activity.
Liang-Nian Ji, Zong-Wan Mao*
HCO + H+
2
2
3-
H
O + CO
pH
e
Page No. – Page No.
CAIXplatins: highly potent Pt(IV)
prodrugs selectively against hypoxic
tumors via microenvironment and
metabolism regulation
nucleotides
Synthesis
O
2
HIF-1
Glycolysis
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Title
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