Restraining Cancer Cells by Dual Metabolic Inhibition with a Mitochondrion-Targeted Platinum(II) Complex

Article abstract of DOI:10.1002/anie.201900387

Cancer cells usually adapt metabolic phenotypes to chemotherapeutics. A defensive strategy against this flexibility is to modulate signaling pathways relevant to cancer bioenergetics. A triphenylphosphonium-modified terpyridine platinum(II) complex (TTP)

Full text of DOI:10.1002/anie.201900387

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Accepted Article
Title: Restraining Cancer Cells by Dual-Metabolic Inhibitions with a
Mitochondrion-Targeted Platinum(II) Complex
Authors: Kun Wang, Chengcheng Zhu, Yafeng He, Zhenqin Zhang,
Wen Zhou, Nafees Muhammad, Yan Guo, Xiaoyong Wang,
and Zijian Guo
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900387
Angew. Chem. 10.1002/ange.201900387
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Restraining Cancer Cells by Dual-Metabolic Inhibitions with a
Mitochondrion-Targeted Platinum(II) Complex
Kun Wang,[a] Chengcheng Zhu,[a] Yafeng He,[a] Zhenqin Zhang,[a] Wen Zhou,[a] Nafees Muhammad,[a]
Yan Guo,[a] Xiaoyong Wang,*[b] and Zijian Guo*[a]
Abstract: Cancer cells usually adapt metabolic phenotypes to
chemotherapeutics. A defensive strategy against this flexibility is to
modulate signaling pathways relevant to cancer bioenergetics. A
triphenylphosphonium-modified terpyridine platinum(II) complex
(TTP) was designed to inhibit thioredoxin reductase (TrxR) and
multiple metabolisms of cancer cells. TTP exhibits enhanced
cytotoxicity against cisplatin-insensitive human ovarian cancer cells in
a caspase-3-independent way, and shows preferential inhibition on
mitochondrial TrxR. The morphology and function of mitochondria are
severely damaged, and the levels of mitochondrial and cellular
reactive oxygen species are decreased. As a result, TTP exerts strong
inhibition to both mitochondrial and glycolytic bioenergetics, inducing
cancer cells to enter into a hypometabolic state.
specifically regulates redox status in mitochondria, and its
inhibition may lead to a distinct pharmacodynamic profile.[14]
Among TrxR inhibitors, metal complexes, such as those of Au, Ag,
Ru or Ir, have been found to possess anticancer potency.[15,16]
Some Pt complexes also showed strong inhibition to TrxR;[17]
however, the perturbations of Pt-based TrxR inhibitors on energy
metabolism are largely unknown.
Pt-based anticancer drugs, such as cisplatin (CDDP) and
oxaliplatin, represent a class of successful metallodrugs in
chemotherapy.[18] Nuclear genome is believed to be the main
target for these drugs; however, their efficacy is often whittled
away by DNA repair mechanisms.[19] Pt complexes with
metabolism-inhibiting ability may circumvent this obstacle, but
they suffered from metabolic alterations and discrepant effective
doses between pharmacophores.[20,21] Interestingly, terpyridine
Pt complexes could inhibit TrxR effectively,[22] thus providing a
structural motif to accomplish synergistic inhibition to both
mitochondrial and glycolytic metabolisms in cancer cells.
Triphenylphosphine (TPP) has been widely used as a
mitochondrial targeting group, and TPP-tethered agents also
showed potential to influence mitochondrial metabolism.[23,24]
Herein we combined TPP and [(TPy)PtCl]+ (TPy = 2,2ʹ:6ʹ,2ʹʹ-
terpyridine) to form a mitochondrion-targeted complex TTP
(Figure 1) for the inhibition of mitochondrial TrxR. TTP causes
tremendous damages to mitochondria, disturbs mitochondrial and
cytosolic redox homeostasis, and destroys both respiratory and
glycolytic phenotypes.
Mitochondrial metabolism has long been misunderstood as a
bystander in the oncogenic process of rapidly proliferating
cells;[1,2] whereas emerging evidences reveal that it is essential
for tumorigenesis in providing metabolic intermediates through
anabolism and catabolism.[3,4] Many metal complexes have been
reported to target mitochondria and damage mitochondrial
metabolism;[5,6] however, the effect is discounted due to the
alteration of metabolic phenotypes from oxidative phosphorylation
to glycolysis for energy compensation.[7] Combination therapy
targeting both metabolic pathways has been exploited to increase
the anticancer potency of single metabolic inhibitors,[8] but optimal
proportion and toxic effects need to be settled before clinical
studies. An alternative strategy may lie in targeting the signaling
pathways that affect both mitochondrial and glycolytic
metabolisms.[9] For example, to perturb mitochondrial and
cytosolic redox homeostasis of cancer cells may induce oxidative
stress, leading to mitochondrial dysfunction and glycolytic defect,
and thereby to metabolic collapse and cell death.[10]
Thioredoxin reductase (TrxR) plays a pivotal role in defending
reactive oxygen species (ROS) in cellular compartments,[11]
which exists in three isoforms, that is, TrxR1 in cytosol, TrxR2 in
mitochondria, and TrxR3 in testicles. Overexpression of TrxR in
cancer cells has been related to drug resistances;[12] and TrxR
has been demonstrated to influence cancer metabolic state via
regulating cellular redox signaling pathways.[13] Moreover, TrxR2
Figure 1. Crystal structure of TTP. Hydrogen atoms and chloride counter anions
are omitted for clarity. Selected bond lengths (Å) and angles (°): N(1)-Pt
1.885(17), N(2)-Pt 1.971(15), N(3)-Pt 2.049(15), Cl-Pt 2.311(4); N(1)-Pt-N(2)
82.2(8), N(1)-Pt-N(3) 162.6(7), N(2)-Pt-N(3) 80.4(8), N(1)-Pt-Cl 98.0(6), N(2)-
Pt-Cl 178.0(4), N(3)-Pt-Cl 99.4(5). Detailed data are deposited at the Cambridge
Crystallographic Data Centre with the Deposition Number of CCDC 1573857.
[a]
K. Wang, Dr. C. Zhu, Dr. Y. He, Dr. Z. Zhang, Dr. W. Zhou, Dr. N.
Muhammad, Y. Guo, Prof. Dr. Z. Guo
State Key Laboratory of Coordination Chemistry
School of Chemistry and Chemical Engineering
Nanjing University
TTP was obtained by the reaction of K2PtCl4 with TPP-
modified TPy in dimethyl sulfoxide (see Supporting Information
Scheme S1). It was fully characterized by NMR, ESI-MS, and X-
ray crystallography (Figure S1 and Tables S1, 2). As shown in
Figure 1, the linker phenyl group tilts 23.96° from the TPyPt plane
formed by a Pt and three N atoms of pyridines; and the P atom
stretches out from the methylene for 1.83 Å with an angle ca
113.38°. Pt and P atoms form two cationic centres with a distance
of 11.19 Å. According to the Nernst equation, the plasma
Nanjing 210023 (P. R. China)
E-mail: zguo@nju.edu.cn
Prof. Dr. X. Wang
State Key Laboratory of Pharmaceutical Biotechnology
School of Life Sciences
Nanjing University
Nanjing 210023 (P. R. China)
E-mail: boxwxy@nju.edu.cn
[b]
Supporting information for this article is given via a link at the end of
the document.
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membrane potential (30‒60 mV) and large mitochondrial
membrane potential (MMP) (140‒180 mV) of cancer cells, being
negative inside,[25] are expected to drive the dicationic lipophilic
TTP to accumulate in mitochondria.
the control (Figure S5), suggesting that TTP caused little nDNA
damage in Caov3 cells.
Mitochondria are essential for cancer formation and
progression; and changes in mitochondrial morphology can
determine the fate of cancer cells.[2] The effect of TTP on
mitochondrial morphology in Caov3 cells was hence examined
using transmission electron microscope (TEM). As shown in
Figure 2, the mitochondria were induced into multilamellar
globules and the folds of cristae in inner membrane extended to
onion-like circles in the presence of TTP, indicating that
mitochondria were severely damaged for cellular recycling. In
contrast, the mitochondria in CDDP-treated cells remained almost
unchanged. Immuno-fluorescence confirmed that TTP induced an
increased expression of Mitofilin (IMMT) relevant to the regulation
of cristae morphology (Figure S6),[28] providing a mechanism for
the changes in mitochondrial morphology.
The cytotoxicity of TTP was tested by the MTT assay on a
panel of human cancer cell lines, including Caov3 and SKOV3
(ovarian), HeLa (cervix), MCF-7 (breast), HCT-116 (colon), A549
and CDDP-resistant A549/DDP (lung), as well as normal cell line
HK-2 (kidney), with CDDP as a reference. The half maximal
inhibitory concentration (IC50) of TTP toward most of the tested
cells is comparable to that of CDDP (Tables 1 and S3).
Nevertheless, TTP showed an enhanced cytotoxicity against
CDDP-insensitive Caov3 cells as compared to CDDP. Moreover,
the ratio of IC50 for A549 to that for A549/DDP (i.e. resistant factor)
was decreased from 4.1 for CDDP to 2.5 for TTP, indicating that
TTP can alleviate the drug resistance of A549 cells to CDDP. TTP
was less toxic to normal HK-2 cells than CDDP, suggesting that it
is much safer than CDDP as an anticancer agent.
Table 1. IC50 values (μM) of TTP and CDDP for different cancer cell lines
at 48 h. Data are shown as mean ± S.D. (n = 3).
Complex
TTP
CDDP
Caov3
9.0 ± 0.2
27.0 ± 1.3
A549
A549/DDP
24.1 ± 1.4
24.2 ± 1.3
HK-2
9.5 ± 1.2
5.9 ± 1.7
11.5 ± 0.6
7.0 ± 1.0
Cellular uptake and distribution of drug molecules are
important factors for anticancer activity.[26] The Pt uptake in
Caov3 cells was investigated using ICP-MS and shown in Table
2. It was reported that the Pt uptake is positively correlated with
the cytotoxicity;[27] however, TTP demonstrated much higher
cytotoxicity against Caov3 cells than CDDP though their total
cellular uptake is similar. Since the Pt content in mitochondria of
the TTP-treated cells is 2.65 times higher than that in the CDDP-
treated cells, while that in nuclei is almost the same, the
accumulation in mitochondria rather than the total cellular uptake
seems to play a dominant role in the anticancer activity of TTP.
Figure 2. Typical TEM images indicating the alterations of mitochondrial
morphology in Caov3 cells induced by TTP or CDDP at 37 °C for 48 h.
Mitochondrial damage may induce autophagy or
mitophagy.[29] As an indicator of autophagy, formation of vacuoles
was clearly visible in the TEM image upon treatment with TTP
(Figure S7). Immunofluorescent assessment of LC3B showed
that the fluorescence intensity and fluorescent dots increased in
the TTP-treated Caov3 cells (Figure S8), manifesting that
vacuoles of autophagosomes emerged. Further evidence of
autophagy is shown in Figure 3. The ratio of lipidated LC3B-II to
normal LC3B-I was increased after incubation with TTP, which is
a reliable marker for autophagy.[29] The down-regulation of
p62/SQSTM1 by TTP demonstrated that the protein was
degraded in autophagosomes, which also verified the autophagic
process of Caov3 cells.
Mitophagy was evaluated using TEM and confocal laser
scanning microscopy. TEM images in Figure 4 (left) show that
mitochondria are capped in the vacuoles, indicating that they were
removed from cells through autophagosomes. The encapsulation
of mitochondria in lysosomes is an important hallmark of
mitophagy,[29] which was testified by co-localization of
mitochondria and lysosomes. Remarkable overlap was observed
in the confocal images of the TTP-treated Caov3 cells labelled by
MitoTracker® Green FM and LysoTracker® Red DND-99 dyes
Table 2. Cellular distribution of Pt (ng μg−1 protein) in Caov3 cells after
incubation with TTP or CDDP (5 μM) at 37 °C for 24 h. Data are shown as
mean ± S.D. (n = 3); blank values have been subtracted.
Complex
Mitochondria
Nuclei
Cytosol
Total
TTP
CDDP
11.0 ± 0.2
4.2 ± 0.1
13.5 ± 0.1
12.6 ± 0.2
8.6 ± 0.1
12.4 ± 0.1
33.2 ± 0.1
29.2 ± 0.1
The impact of TTP on DNA was first evaluated using calf-
thymus DNA (CT-DNA). An immediate intercalation of TTP with
DNA was observed in the UV-vis titration, and the affinity constant
was calculated to be (1.30 ± 0.07) × 105 M‒1 (Figure S2). NaCl
competitive experiment manifested that the interaction between
TTP and CT-DNA is an electrostatic action (Figure S3). Hoechst
33342 staining was used to visualize the influence of TTP on
nuclear DNA (nDNA) of Caov3 cells (Figure S4). After pre-
incubation with TTP at its IC50 for 24 h, almost no genome
damage was observed. In the cell cycle assay, TTP-treated cells
only showed a slightly increased S-phase arrest compared with
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simultaneously; while no overlap was observed for the control.
The results indicate that TTP could induce mitophagy.
quantitatively the MMP in Caov3 cells. The data presented in
Figure 5 by the contour plot illustrate that the fluorescence in Q1
for high MMP is shifted to Q4 for low MMP. For TTP-treated cells,
the centres of contour plots assembled to one centre located in
Q4 (86.9%), suggesting that TTP has a strong ability to dissipate
the MMP, comparable to that of decoupler carbonyl cyanide 3-
chlorophenyl hydrazone (CCCP, 88.6% in Q4). The results also
suggest that the mitochondrial membrane was impaired by TTP.
Mitochondrial morphology is closely related to mitochondrial
respiration,[31] which was evaluated using Seahorse Extracellular
Flux analyser. Caov3 cells were pre-treated with TTP for 24 h
before recording the oxygen consumption rate (OCR), with CDDP
as a reference. As shown in Figure 6, TTP induced ca 50%
decrease in both basal respiration and ATP production; moreover,
it significantly destroyed the reverse capacity of the maximal
oxygen consumption of the cells, which is indicated by the
insensitive response to carbonylcyanide p-trifluoromethoxy
phenylhydrazone (FCCP), an uncoupler of the proton gradient
across the inner mitochondrial membrane. Mitochondrial
respiration was shut down upon the injection of rotenone (an
inhibitor of mitochondrial complex I) and antimycin A (an inhibitor
of mitochondrial complex III) as the OCR decreased to
minimum.[32] No significant change in OCR was detected for the
CDDP-treated cells, indicating that CDDP hardly affects the
mitochondrial respiration. Similar results were also found in A549
cell line (Figure S9A). Here, oligomycin was used to inhibit the
ATP synthase and get the ATP synthetic ability of the cells.
Figure 3. Immunoblotting of LC3B and p62 in Caov3 cells treated with TTP at
37 °C for 24 h and corresponding quantification of the LC3B-II to LC3B-I ratio
and p62 expression. Error bars: S.D., n = 3. Statistical significance of
differences in mean values: *P < 0.05, ***P < 0.001.
Figure 4. Representative TEM images of Caov3 cells treated with TTP at 37 °C
for 48 h, emphasizing the region of mitophagy (left), and confocal microscopy
images of Caov3 cells treated with TTP for 24 h (right).
Figure 6. Kinetic profiles of OCR in Caov3 cells after treatment with TTP or
CDDP at 37 °C for 24 h (A), and quantification of basal respiration, ATP
production, and respiratory capacity from the kinetic profiles of OCR (B). Error
bars: S.D., n = 5. Statistical significance of differences in mean values: n.s not
significant, ***P < 0.001.
Figure 5. Fluorescent microscopy images (left) and flow cytometry
quantification (right) of JC-1-labeled Caov3 cells treated with TTP or CDDP at
37 °C for 24 h (λem = 529, 590 nm) .
Dissipation of MMP is one of the important hallmarks of
mitochondrial damage. The change of MMP was detected by JC-
1, which aggregates to emit red fluorescence when MMP is high,
and remains as monomers to emit green fluorescence when MMP
is dissipated.[30] As shown in Figure 5, TTP-treated Caov3 cells
emit green fluorescence, manifesting the dissipation of MMP.
Flow cytometric analysis was also performed to identify
The ATP production in Caov3 cells was further determined by
the ATP assay kit (Figure S10). In the presence of TTP, ATP
decreased in a dose-dependent way; while CDDP only induced a
slight decrease in ATP. These results confirm that the
mitochondrial basal respiration, ATP production capacity and
respiratory capacity were severely damaged by TTP. However,
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