Multiaction Platinum(IV) Prodrug Containing Thymidylate Synthase Inhibitor and Metabolic Modifier against Triple-Negative Breast Cancer

Article abstract of DOI:10.1021/acs.inorgchem.0c01736

Multifunctional platinumIV anticancer prodrugs have the potential to enrich the anticancer properties and overcome the clinical problems of drug resistance and side effects of platinumII anticancer agents. Herein, we develop dual and triple action platinumIV complexes with targeted and biological active functionalities. One complex (PFL) that consists of cisplatin, tegafur, and lonidamine exhibits strong cytotoxicity against triple negative breast cancer (TNBC) cells. Cellular uptake and distribution studies reveal that PFL mainly accumulates in mitochondria. As a result, PFL disrupts the mitochondrial ultrastructure and induces significant alterations in the mitochondrial membrane potential, which further leads to an increase in production of reactive oxygen species (ROS) and a decrease in ATP synthesis in MDA-MB-231 TNBCs. Western blot analysis reveals the formation of ternary complex of thymidylate synthase, which shows the intracellular conversion of tegafur into 5-FU after its release from PFL. Furthermore, treatment with PFL impairs the mitochondrial function, leading to the inhibition of glycolysis and mitochondrial respiration and induction of apoptosis through the mitochondrial pathway. The RNA-sequencing experiment shows that PFL can perturb the pathways involved in DNA synthesis, DNA damage, metabolism, and transcriptional activity. These findings demonstrate that PFL intervenes in several cellular processes including DNA damage, thymidylate synthase inhibition, and perturbation of the mitochondrial bioenergetics to kill the cancer cells. The results highlight the significance of a triple-action prodrug for efficient anticancer therapy for TNBCs.

Full text of DOI:10.1021/acs.inorgchem.0c01736

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Article
Multiaction Platinum(IV) Prodrug Containing Thymidylate Synthase
Inhibitor and Metabolic Modifier against Triple-Negative Breast
Cancer
Nafees Muhammad, Cai-Ping Tan,* Uroosa Nawaz, Jie Wang, Fang-Xin Wang, Sadia Nasreen,
Liang-Nian Ji, and Zong-Wan Mao*
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ABSTRACT: Multifunctional platinumIV anticancer prodrugs have the
potential to enrich the anticancer properties and overcome the clinical
problems of drug resistance and side effects of platinumII anticancer agents.
Herein, we develop dual and triple action platinumIV complexes with
targeted and biological active functionalities. One complex (PFL) that
consists of cisplatin, tegafur, and lonidamine exhibits strong cytotoxicity
against triple negative breast cancer (TNBC) cells. Cellular uptake and
distribution studies reveal that PFL mainly accumulates in mitochondria. As
a result, PFL disrupts the mitochondrial ultrastructure and induces
significant alterations in the mitochondrial membrane potential, which
further leads to an increase in production of reactive oxygen species (ROS)
and a decrease in ATP synthesis in MDA-MB-231 TNBCs. Western blot analysis reveals the formation of ternary complex of
thymidylate synthase, which shows the intracellular conversion of tegafur into 5-FU after its release from PFL. Furthermore,
treatment with PFL impairs the mitochondrial function, leading to the inhibition of glycolysis and mitochondrial respiration and
induction of apoptosis through the mitochondrial pathway. The RNA-sequencing experiment shows that PFL can perturb the
pathways involved in DNA synthesis, DNA damage, metabolism, and transcriptional activity. These findings demonstrate that PFL
intervenes in several cellular processes including DNA damage, thymidylate synthase inhibition, and perturbation of the
mitochondrial bioenergetics to kill the cancer cells. The results highlight the significance of a triple-action prodrug for efficient
anticancer therapy for TNBCs.
as they are kinetically inert in the blood plasma compared with
their parental compounds and can be activated upon reduction
to PtII species. It is assumed that the higher concentration of
biological reductants (e.g., glutathione and ascorbic acid (AA))
inside the cells helps to reduce the octahedral PtIV complexes to
square planar cytotoxic PtII species. To date, Satraplatin is the
only bioavailable PtIV based chemotherapeutic agent that was
evaluated in a phase III clinical trial, but it failed to improve
overall survival in a patient with hormone-refectory prostate
tumor.11 Recently, an increasing number of dual or triple action
PtIV complexes have been developed, which opens up new
possibilities to target different cellular targets with a single
molecule.12−14
INTRODUCTION
■
Triple negative breast cancer (TNBC), characterized by lack of
expression of estrogen, progesterone, and human epidermal
growth factor receptor 2 (HER2), is the main cause of breast
cancer mortality.1 Conventional chemotherapy continues to
play a pivotal role in the treatment of TNBC. However, clinical
studies have proved that the efficacy of conventional drugs is
limited by poor outcomes, drug resistance, and short duration
response, which is caused by extensive intratumoral hetero-
geneity.2 Cisplatin (cDDP) and its derivatives (carboplatin and
oxaliplatin) are well-known for their high therapeutic efficacies
against breast cancer.3,4 cDDP forms varies forms of Pt-DNA
cross-links that subsequently inhibits DNA metabolism and
triggers cell death pathways.5 In spite of their success, clinically
used platinum drugs may cause severe side effects and develop
drug resistance.6−8
PtIV complexes emerge as promising candidates to ameliorate
the undesirable side effects of cDDP. The two additional axial
positions on PtIV metal center offer extra opportunities to fine-
tune the lipophilicity, bioavailability, and redox properties by
selectively tethering biovectors or other biologically active
molecules.9,10 PtIV complexes are generally regarded as prodrugs
Received: June 12, 2020
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a
Scheme 1. Synthetic Routes of PF-I, PF-II, PFD, PFB, and PFL
a
Conditions and reagents: (a) TBDMS, DCM, 12 h; (b) K2CO3, ACN, reflux; (c) Et3N·3HF, THF, 24 h; (d) succinic anhydride, ACN, 50 °C; (e)
oxoplatin, TBTU, TEA, 48 h; (f) DMSO, RT, 72 h; (g) biotin, TBTU, TEA, 48 h; (h) DCA, 24 h; (i) lonidamine, TBTU, TEA, 48 h.
Tegafur (FT) is a prodrug of the antineoplastic agent 5-
fluorouracil (5-FU) that shows mild toxicity and enhanced
survival rates in patients with malignant tumors.15 The addition
of a tetrahydrofuran (THF) moiety improves the antiangiogenic
and cytocidal performance of 5-FU.16 Upon cellular entrance,
FT is converted to 5-FU (an inhibitor of thymidylate synthase
(TS)) by the cytochrome P-450 enzyme.17 TS is crucial for the
de novo synthesis of thymidylate (dTMP) that is required for
the synthesis of DNA. TS is overexpressed in cancer cells (e.g.,
breast, prostate, kidney, and bladder), and elevated TS levels are
thought to be associated with tumor invasiveness and meta-
stasis.18−20 Biotin (vitamin B7) is an effective cancer-targeting
moiety, as cancer cells proliferate actively and they require biotin
as a growth promoter.21 Dichloroacetate (DCA) is an orphan
drug that decreases the lactate level by inhibiting the pyruvate
dehydrogenase kinase (PDK, an enzyme crucial for mitochon-
drial respiration).22 As demonstrated by Lippard et al.,
“mitaplatin” with two dichloroacetate moieties coordinated to
the PtIV metal center at the axial positions can act as a dual-
targeting molecule to overcome cDDP resistance.23 Lonidamine
(LN) is an indazole carboxylate that can selectively sensitize
tumors when used in combination with other antineoplastic
agents possibly by triggering the mitochondrial apoptotic
pathway.24−26
which could induce multiple cellular events to execute cell death
by overcoming the drug resistance associated with the use of
single agent. In the present study, we report the synthesis and
biological properties of PtIV prodrugs with FT coordinated at the
axial positions (Scheme 1). To enhance the selectivity for cancer
cells or achieve the combined effects on nuclei and
mitochondria, biotin, DCA and lonidamine are used to
construct the PtIV complexes with mixed axial ligands. We
studied the selectivity of these complexes to TNBC cells. The
anticancer mechanism of the most active agent, PFL, is
investigated, which includes its intracellular reduction, cellular
uptake and localization, effects on nuclear DNA damage and
mitochondrial functions. Finally, the effect PFL on tran-
scriptome is investigated by RNA sequencing. We demonstrate
that PFL can effectively induce TNBC cell death by activating
multicellular events.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The synthetic routes of
PF-I, PF-II, PFD, PFB, and PFL are depicted in Scheme 1.
Oxoplatin was synthesized in a manner reported previously.28
The hydroxyl group of bromoethanol was first protected with
tert-butyldimethylsilyl chloride (TBDMS) to yield intermediate
1 which then reacted with tegafur under refluxing acetonitrile
(ACN) to give 2. Deprotaction of silyl ether using Et3N·3HF
give intermediate 3 which then reacted with succinic anhydride
to give FTA. PF-II was synthesized by esterification reaction
between FTA and oxoplatin in the presence of (1H-
It remains unknown whether single-agent chemotherapy
might be good for TNBC as chemo-resistance quickly develop
after third to fourth cycle of treatment.27 Therefore, the rationale
behind the design of all complexes is to find a suitable candidate
B
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Table 1. Cytotoxicity Profile of PtIV Compounds in Comparison to cDDP
MDA-MB-231
MCF-7
A549
HepG2
MCF-10A
PF-I
PF-II
PFD
PFB
PFL
cDDP
6.1 0.1
29.4 0.4
10.3 0.4
21.5 1.2
2.9 0.2
5.1 0.2
22.4 0.5
8.2 0.4
16.8 0.5
4.8 0.2
4.1 0.2
9.2 0.4
27.5 0.8
16.3 0.5
32.2 0.5
9.8 0.4
9.8 0.2
17.5 1.2
52.3 1.1
13.2 0.3
44.6 1.2
10.7 0.9
13.5 0.4
8.1 0.4
22.2 0.2
9.1 0.3
28.8 0.7
8.3 0.1
7.8 0.3
15.2 0.1
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluorobo-
rate (TBTU) and triethylamine (TEA). To obtain a high yield
of PF-I, FTA-NHS ester (prepared by reacting FTA with 1-
Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-
hydroxysuccinimide (NHS)) was used to react with oxoplatin in
DMSO. PFB and PFL were then subsequently prepared by
reaction of PF-I with biotin or lonidamine in the presence of
TBTU and TEA. PFD was prepared by reacting PF-I with neat
dichloroacetic anhydride (DCA). The complexes were charac-
terized by (1H, 13C and 195Pt) NMR spectroscopy, ESI-MS
spectrometry (Figure S1−S25, SI), and CHN elemental
analysis. The 195Pt NMR spectra show a single resonance peak
at 1050, 1227, 1228, 1221, and 1225 ppm for PF-I, PF-II, PFD,
PFB, and PFL, respectively. Prior to the biological evaluation,
lipophilicity of PtIV complexes were measured because it might
influence the antitumor properties of the PtIV prodrugs. All
complexes were stable in phosphate buffer saline (PBS) over a
period of 24 h as confirmed by high-performance liquid
chromatography (HPLC; Figure S26). The lipophilicity (log
Figure 1. Content of Pt (ng/106 cells) in MDA-MB-231 cells after
treatment with 10 μM of PF-I, PF-II, PFD, PFB, or PFL for 24 h. (A)
Whole cell uptake of platinum compounds. (B) Subcellular distribution
of cDDP and PFL.
the uptake of PF-II is significantly (4-fold) lower than its
monosubstituted complex (PF-I), which is in agreement with
the related monosubstituted PtIV complexes.32 Unexpectedly,
the addition of biotin moiety does not significantly increase the
uptake of PFB compared with PFD, though both complexes
share slightly similar lipophilicity. It is interesting to note that
PFD shows a better cytotoxicity profile because of its
multifunctional nature compared with PFB, though the uptake
of the latter is higher than the former. This also indicates that
uptake of PtIV complexes is only one of the parameters for better
cytotoxicity. As PFL shows a broad spectrum of activity and is
more potent against MDA-MB-231 cells than cDDP, it is chosen
for further mechanistic investigations. The total platinum
content associated with MDA-MB-231 cells upon treatment
with PFL and cDDP is depicted in Figure 1B. cDDP treatment
results in a much lower uptake in MDA-MB-231 cells than PFL
(25 vs 91 ng Pt in 106 cells). The subcellular distribution studies
reveal that PFL mainly resides in mitochondria, while cDDP
mainly accumulates in nuclei. The elevated Pt uptake and
mitochondrial accumulation may contribute to the higher
cytotoxicity of PFL.
Reduction Analysis with HPLC. As PF-I and PFL display
the highest cellular uptake levels, we next examined their rate of
reduction in the absence or presence of AA, a cellular reducing
agent, over a period of 24 h using HPLC (Figures 2 and S27).
Both complexes are stable in the absence of AA, whereas in the
presence of AA, the absorption peak of PF-I decreases rapidly
and the peak of FTA increases simultaneously. In contrast, PFL
is reduced less quickly than PF-I under the same conditions.
After 6 h, the percentage of PFL remained drops to about 9%,
and the value increases to 39% after 12 h. It was suggested that
the rate of reduction depends upon the electron transfer from
the reductant to the PtIV metal center that results in the
formation of a bridge between the PtIV complex and the reducing
agent. Hydroxyl ligands in the coordination sphere would
greatly facilitate the electron transfer process compared with
P
o/w) of PF-I (−0.86), PF-II (−0.47), PFD (−0.52), PFB
(−0.56) , and PFL (−0.35) are measured using the shake flask
method in a 1-octanol/phosphate buffer system (pH 7.4). The
result indicates the monosubstituted complex (PF-I) is less
lipophilic than the disubstituted complexes (PF-II, PFD, PFB,
and PFL).
In Vitro Anticancer Activity. The cytotoxicity of the
synthesized complexes was measured on a panel of human
cancer cell lines including MDA-MB-231 (TNBC), MCF-7
(breast), A549 (lung), HepG2 (liver), and normal human
mammary epithelial (MCF-10A) cells (Table 1). The half-
maximal inhibitory concentrations (IC50) of all the tested
complexes are in the low micromolar range, whereas ligands
FTA, DCA, and LN are found to be inactive (IC50 > 60 μM).
Among the synthesized complexes, PF-I and PFL are found to
be more potent with IC50 values comparable to those of cDDP
against MCF-7 cells. Notably, PFL that consists of a TS inhibitor
(FT) and the metabolic modifier (LN) shows about 5-fold
higher cytotoxicity against MDA-MB-231 cells than cDDP. The
cytotoxicities of the other dual-action complexes (PF-II, PFD,
and PFB) are lower than that of cDDP in the cancer cell lines
tested. PFB with a biotin moiety shows slightly higher
cytotoxicity than PF-II. The low cellular uptake (vide infra)
and slow reduction rate might responsible for lower cytotoxicity
of these disubstituted complexes.29,30 In addition, in MCF-10A
cells, PF-I, PFD, and PFL display comparable cytotoxicity to
cDDP, indicating that both complexes have increased cancer cell
selectivity in the breast cancer cells.
Cellular Uptake. It has been reported that the intracellular
accumulation of platinum drugs is associated with their
cytotoxicity.31 So, we first determined the whole cell uptake of
a synthesized complex in MDA-MB-231 carcinoma cells to
interlink their cytotoxicity profile using inductively coupled
plasma mass spectrometry (ICP-MS). As shown in Figure 1A,
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with the respective accumulation of PFL in nuclei, indicating
that damaging nDNA may also play a critical role in anticancer
mechanism of PFL.
We next examined apoptosis induced by PFL using annexin-
V/propidium iodide (PI) double staining that can differentiate
intact cells (annexin V-/PI-), early apoptotic (annexin V+/PI-),
late apoptotic (annexin V+/PI+), and necrotic (Annexin V-/PI
+) cells (Figure 3A). After treatment with PFL (10 μM), the
percentage of early apoptotic cells (52.7%) significantly
increases compared with control (5.58%), which is higher
than cDDP (12.8%). The higher potency of PFL to induce
apoptosis is in line with the cytotoxicity data. Next, the impact of
PFL on cell cycle distribution is examined by flow cytometry
(Figure 3B). PFL arrests the cell cycle in S phase, which is
similar to that of cDDP, but its potency was much stronger than
cDDP.34 In untreated samples, the fraction of S-phase cells is
23%, and the fraction increases up to 60% with a concomitant
decrease in G2 phase and an increase in G0/G1 phase when the
cells were treated with 10 μM of PFL.
Figure 2. RP-HPLC analysis of remaining platinumIV complexes. 500
μM of PtIV complexes were incubated with and without the presence of
5 mM AA in a solution of PBS/MeOH (9/1, V/V, pH 7.4) at 37 °C in
the dark.
Mitochondria play vital and lethal functions in both
physiological and pathological scenarios, as they are key
regulators of energy production and crucial regulators for
apoptotic machinery.35 Since the alteration in the mitochondrial
morphology is mechanistically interlinked with its functions, we
hence examined the mitochondrial ultrastructure of MDA-MB-
231 cells using transmission electron microscopy (TEM).
Mitochondria in untreated cells show integral double membrane
structure and physical coordination of polymorphic cristae exist
within the mitochondrial matrix (Figure 4). In contrast, the
portion of intact mitochondria decreases significantly in PFL-
treated cells. Furthermore, PFL treatment induces mitochon-
drial swelling (a common feature of membrane permeabiliza-
tion) and disrupts cristae (Figure 5 inset, indicated with white
arrows). In addition, some autophagosomes which are generated
as a result of autophagy and multivascular bodies are also
observed in MDA-MB-231 cells treated with PFL (Figure 4
carboxylate;29 therefore, we observed the faster reduction rate of
PF-I compared with PFL. Both complexes are completely
reduced after incubation with AA for 24 h.
Mechanism of Cytotoxicity. It is well-known that
platinum-based anticancer drugs target nuclear DNA
(ncDNA) in initiating their anticancer effect in tumor cells.33
Therefore, we first use confocal microscopy to study the possible
DNA damage upon treatment with PFL. As shown in Figure
S28A, treatment of MDA-MB-231 cells with higher concen-
trations of PFL induces severe DNA fragmentation compared
with cDDP, which confirms its ability to cause cellular DNA
damage. We also studied the Pt levels on the genomic DNA of
MDA-MB-231 cells (Figure S28B, SI). MDA-MB-231 cells
treated with cDDP (10 μM) and PFL (10 μM) show 9.7 and 2.4
ng Pt per μg of DNA, respectively. These results are inconsistent
Figure 3. Impact of PFL on apoptosis and cell cycle analysis. (A) Flow cytometry analysis of MDA-MB-231 cells treated with cDDP and PFL for 48 h,
followed by staining with annexin-V FITC and PI. (B) Cell cycle arrest of MDA-MB-231 cells treated with cDDP and PFL for 48 h.
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Figure 4. TEM images of mitochondria structure in MDA-MB-231 cells treated with or without PFL (10 μM) for 30 h.
inset, indicated with black arrows). As demonstrated above, PFL
specifically accumulates in mitochondria and induces mitochon-
drial swelling in cells; therefore, its impact on mitochondrial
membrane potential (MMP) is examined using 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-
1) staining and flow cytometry. As shown in Figure 5A, the
MMP in control cells is high where JC-1 assembles to form J
aggregates (red fluorescence). After treatment with PFL, a
marked decrease in MMP is observed as the JC-1 fluorescence
shifts from red to green. The percentages of cells with
depolarized mitochondria increase from 3.5 1.5% (Control)
to 67.3 5.7% (PFL, 15 μM). These findings are consistent with
our WB results (vide infra), where the release of cytochrome C
linked to loss of MMP. Mitochondria are indispensable for
energy production and hence the survival of cells; therefore, as a
consequence of loss of MMP, cellular ATP production should be
perturbed. To confirm this, the impact of PFL on intracellular
ATP production in MDA-MB-231 cells is measured with an
ATP assay kit. Treatment with PFL results in a significant dose-
dependent decrease in ATP production as compared with the
control cells (Figure S29). These findings are similar to previous
reports where lonidamine alone triggers ATP depletion in breast
xenograft models.36
Destruction of mitochondrial dynamics function could also
influence the intracellular redox homeostasis; thus, intracellular
ROS level was tracked by flow cytometry using 2′,7′-
dichlorofluorescein diacetate (H2DCF-DA) ROS probe. As
shown in Figure 5B, a dose-dependent increase of ROS
production was observed in MDA-MB-231 cells after treatment
with PFL for 30 h. Compared with the control cells, a 4-fold
increase in ROS production was observed in cells treated with
PFL at a dose of 10 μM.
Next, we measured the oxygen consumption rate (OCR) in
MDA-MB-231 cells upon treatment with PFL using a Seahorse
XF24 extracellular flux analyzer. As shown in Figure 5C,
exposure to PFL results in a significant impact on mitochondrial
respiration. The data show that basal respiration dramatically
decreases upon treatment with PFL compared with the control
in a dose-dependent manner. Addition of oligomycin followed
by mitochondrial uncoupler carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP) into the media
markedly reduces ATP synthase, maximal mitochondrial
respiration and spare capacity (Figure 5D). These results
suggest that PFL has a strong impact on mitochondrial
bioenergetics of MDA-MB-231 cells.
As lonidamine can inhibit glycolysis in cancer cells, we then
determined the effect of PFL on aerobic glycolysis of MDA-MB-
231 cells by measuring the extracellular acidification rate
(ECAR). As shown in Figure 5E, ECAR is decreased in a
dose-dependent manner upon PFL treatment. Changes in
ECAR in response to sequential addition of glycolysis
modulators (glucose, oligomycin, and 2-deoxy-D-glucose (2-
DG)) to MDA-MB-231 cells were defined to determine the
indices of glycolysis, glycolytic capacity, and glycolytic reserves.
As expected, all these indices measured for PFL-treated cells
(Figure 5F) were substantially lower than those for the control
samples under the same conditions.
To further shed light on the mechanisms involved in the PFL-
induced apoptosis, we investigated the impact of PFL on the
expression of Bcl-2 family proteins including antiapoptotic (Bcl-
2) and proapoptotic (Bax) proteins that play important roles in
the mitochondrial pathway of apoptosis. The integrity of outer
mitochondrial membrane (OMM) is control by Bcl-2 family of
proteins that oligomerize into proteolipid pores and permeabi-
lize the OMM, allowing the efflux of cytochrome c and other
intermembrane space proteins to the cytosol during apoptosis.37
Western blot data was used to study the expressions of Bcl-2,
Bax, and Cyto c in MDA-MB-231 cells after treatment with PFL.
As expected, the expression of Cytochrome C and Bax is
significantly upregulated, while BCl-2 expression is down-
regulated upon treatment with PFL (Figure 6A). These results
indicate that PFL can activate apoptosis mediated by
mitochondrial pathway. In addition, we investigated the TS
expression using TS 106 monoclonal antibody (Figure 6B). PFL
treatment significantly upregulates TS expression by forming a
ternary complex (38 kDa) which confirms that FT is converted
to 5-FU in the MDA-MB-231 cells and subsequently inhibits the
TS enzyme.
RNA-seq Analysis. The impact of PFL on transcriptome
was further investigated by comparing the gene expression
profiles in TNBC cells. A correlation coefficient above 0.9 was
observed between cells from the same group containing 3
individual samples (Figure S30). More than 97% of readings are
mapped to reference genes in all samples, and 83% of readings
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Figure 5. Detection of MMP by JC-1 staining after treatment with varying concentration of PFL. (A) Generation of ROS in MDA-MB-231 cells after
incubation with PFL for 30 h. (B) Respiratory profile of PFL treated MDA-MB-231 cells after the addition of oligomycin (O), FCCP (F), and the
mixture of antimycin and rotenone (A&R). The data are presented as the mean SD (n = 3). (C) Key parameters of mitochondrial respiration. (D)
Glycolysis profile of MDA-MB-231 cells after treatment with PFL for 30 h by a Seahorse XF24 Extracellular Flux Analyzer (E and F).
were located in exons (Figure S31). Among the 2992 genes that
are significantly changed in the expression levels, 996 genes are
upregulated and 1996 genes are downregulated (Figure 7A and
Table S1). The heatmap of RNA-Seq shows that expression
patterns are very similar classes are assigned to cluster different
genes with putative functions (Figure S32). PFL treatment
causes significant overall changes in gene categories including
cellular process, biological regulation, cell part, organelle,
binding, and catalytic activity (Figure S33). The genes are
further analyzed by KEGG (Kyoto Encyclopedia of Genes and
Genomes) pathway annotation. Various pathways for cancer
progression such as PI3K-Akt signaling pathway and MPAK
signaling pathway are regulated by PFL treatment (Figure S34).
Gene Set Enrichment Analysis (GSEA) reveals that gene
expression profile of TNBC cells treated with PFL is negatively
associated with DNA synthesis, DNA damage, metabolism, and
positively related to transcriptional regulator activity (Figure 7).
Interestingly, of all the 2992 genes with significant changes after
PFL treatment, 1414 genes were related to transcription factor
(TF). Among them, 499 TF genes were up-regulated and 965
TF genes were down-regulated (Figure S34 and Table S2). Most
of these genes belong to the high mobility group (HMG, 576
genes) and zinc finger C2H2 (ZF-C2H2, 403 genes) family
(Figure 7C). The results are consistent with previous findings,
indicating that cDDP can influence the activity of transcription
factors, especially for HMG transcription factors.38−41
CONCLUSIONS
■
PlatinumIV complexes emerge as promising candidates in recent
years to ameliorate the undesirable side effects of cDDP and its
analogues. These complexes act as a prodrug in blood plasma
and can only be activated once inside the cell with the biological
reductant that are abundantly expressed in cancer cells. In this
study, we developed a series of both dual and triple action PtIV
complexes and studied their in vitro anticancer activity against a
panel of human cancer cell lines. The antiproliferative potency of
these complexes are correlated with their cellular uptake efficacy.
One of the most active complex, PFL, which consists of a
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Subsequent investigations reveal that the PFL induces cell
cycle arrest in S phase. Cellular distribution studies reveal that
PFL mainly accumulates in mitochondria; hence, we carry out in
depth mechanistic investigation to study the molecular
mechanism involved in PFL-induced apoptosis. To do so, first
we employed TEM to study the effect of PFL treatment onto
mitochondrial inner membrane topology of MDA-MB-231
carcinoma cells. PFL exposure induces sewer mitochondrial
swelling and disrupts cristae which are interlinked with a marked
decrease in MMP as observed with JC-1 fluorescence shifts from
red to green. Furthermore, treatment with PFL reduces cellular
ATP production, inhibits the mitochondrial respiration, and
elevates the level of ROS production. Western blot analysis
reveals that PFL treatment significantly changes expression level
of Bcl-2 family of proteins (Bcl-2, Bax), which eventually leads to
the release of cytochrome C from mitochondria. In addition,
Western blot analysis shows the formation of ternary complex of
thymidylate synthase, which confirms the release of tegafur from
the coordination sphere of PFL and its subsequent conversion to
5-FU inside the cells. RNA seq transcriptome analysis reveals
that PFL can perturb the pathways involved in DNA synthesis,
DNA damage, metabolism and those positively related to
transcriptional regulator activity. These results provide new
Figure 6. Expression of BCl-2, Bax, and Cyto C in MDA-MB-231 cells
after treatment with cDDP or PFL (A). Expression of TS in MDA-MB-
231 cells after treatment with cDDP, FT, FTA, and PFL for 48 h (B).
thymidylate synthase inhibitor and a metabolic modifier,
exhibits enhanced anticancer activity against TNBC cells.
Figure 7. (A) Volcano plots showing the differentially expressed genes in MDA-MB-231 cells treated with PFL (10 μM). (B) GSEA of PFL-altered
genes in various cellular processes. (C) The top 10 transcription factor families with the largest number of significantly different genes.
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149.29, 156.99, 157.25, 172.64, 179.56. 195Pt NMR (DMSO-d6): 1227
ppm. ESI-MS (negative mode, m/z): 985.46 (M-H), elemental analysis
calculated (%) for Pt(NH3)2Cl2(C28H32F2N4O14) (Mw = 986.63): C
34.08, H 3.88, N 8.52; found: C 34.15, H 3.76, N 8.45.
insight into the development of multiaction PtIV complexes that
activate multiple cellular events for the treatment of TNBC.
EXPERIMENTAL SECTION
Synthesis of PFD. To a stirred suspension of PF-I (130 mg, 0.19
mmol) in dry DCM was added DCA anhydride (140 μL, 1 mmol). The
reaction mixture was stirred at room temperature for 24 h. After
completion of reaction, addition of diethyl ether quickly precipitated
the desired product which was washed several times with diethyl ether
and dried in vacuo. Yield 70%. 1H NMR (DMSO-d6) δ (ppm) 1.91−
2.05 (m, 3H), 2.23−2.32 (m, 1H), 2.38−2.41 (m, 2H), 3.80−3.86 (q,
1H), 4.06−4.08 (d, 2H), 4.22−4.29 (m, 3H), 5.95 (s, 1H), 6.22−6.83
(m, 7H), 7.94−7.96 (d, 1H). 13C NMR (DMSO-d6) δ (ppm) 23.96,
30.15, 30.42, 32.18, 61.05 66.20, 70.10, 87.94, 124.17, 124.50, 138.65,
140.92, 149.30, 157.00, 157.25, 170.95, 172.50, 179.51. 195Pt NMR
(DMSO-d6): 1221 ppm. ESI-MS (negative mode, m/z): 769.77,
e l e m e n t a l a n a l y s i s c a l c u l a t e d ( % ) f o r [ P t -
(NH3)2Cl2(C16H17Cl2FN2O9)] (Mw = 770.29): C 24.92, H 3.01, N
7.26; found: C 24.82, H 3.11, N 7.23.
Synthesis of PFB. To a stirred suspension of PF-I (100 mg, 0.15
mmol) in dry DMSO was added biotin (40 mg, 0.16 mmol), TBTU (51
mg, 0.16 mmol), and TEA (21 μL). The reaction mixture was stirred at
room temperature for 48 h. TLC shows the completion of reaction.
Solvent was evaporated on vacuo, and the product was purified by
column chromatography using CH2Cl2/MeOH (40:60). 1H NMR
(DMSO-d6) δ (ppm) 1.27−1.67 (m, 7H), 1.92−2.05 (m-3H), 1.92−
2.05 (m, 3H), 2.21−2.30 (m, 3H), 2.37−2.41 (t, 2H), 2.57−2.60 (d,
1H), 2.81−2.85 (dd, 1H), 3.08−3.13 (dd, 1H), 3.80−3.86 (q, 1H),
4.07−4.08 (d, 2H), 4.14−4.33 (m, 5H), 5.95 (s, 1H), 6.36−6.68 (m,
8H), 7.94−7.96 (d, 1H). 13C NMR (DMSO-d6) δ (ppm) 23.96, 25.92,
28.55, 30.21, 30.74, 32.18, 35.97, 55.89, 59.68, 61.02, 61.50, 70.10,
87.94, 124.16, 124.50, 138.65, 140.91, 149.30, 156.99, 157.25, 162.80,
163.21, 172.66, 179.57, 181.27. 195Pt NMR (DMSO-d6): 1229 ppm.
ESI-MS (negative mode, m/z): 885.29, elemental analysis calculated
(%) for [Pt(NH3)2Cl2(C24H31FN4O10S)] (Mw = 886.64): C 32.51, H
4.21, N 9.48; found: C 32.31, H 4.26, N 9.45
Synthesis of PFL. To a stirred suspension of PF-I (130 mg, 0.19
mmol) in dry DMF was added lonidamine (81 mg, 0.25 mmol), TBTU
(82 mg, 0.25 mmol), and TEA (38 μL, 0.25 mmol). The reaction
mixture was stirred at room temperature for 48 h. Upon completion of
reaction, the solvent was evaporated, and the residue was purified using
CH2Cl2/MeOH (95:5). 1H NMR (DMSO-d6) δ (ppm) 1.87−2.08 (m,
3H), 2.23−2.33 (m-1H), 2.41−2.44 (t, 2H), 3.81−3.86 (q, 1H), 4.03−
4.09 (m, 2H), 4.24−4.30 (m, 2H), 5.78−5.83 (t, 2H), 5.96 (s, 1H),
6.38−6.99 (m, 7H), 7.27−7.32 (t, 1H), 7.37−7.46 (m, 4H), 7.69−7.79
(m, 2H), 7.95−7.96 (d, 1H), 8.32−8.34 (d, 1H). 13C NMR (DMSO-
d6) δ (ppm) 23.96, 30.22, 30.69, 32.19, 49.97, 61.05, 70.14, 87.96,
110.41, 122.69, 123.57, 124.05, 124.18, 127.23, 128.13, 129.51, 131.14,
133.61, 134.0, 141.12, 149.25, 157.16, 172.64, 179.63. 195Pt NMR
(DMSO-d6): 1225 ppm. ESI-MS (negative mode, m/z): 961.23,
e l e m e n t a l a n a l y s i s c a l c u l a t e d ( % ) f o r [ P t -
(NH3)2Cl2(C29H25Cl2FN4O9)] (Mw = 962.51): C 36.15, H 3.24, N
8.72; found: C 36.02, H 3.29, N 8.62.
Measurement of Lipophilicity (log Po/w). The shake flask
method was used to measure the lipophilicity of the synthesized metal
complexes.42 Briefly, saturated solutions of PF-I, PF-II, PFB, PFD, and
PFL were prepared in water presaturated with n-octanol. About half of
this stock solution was mixed with an equal volume of octanol
presaturated with water, and the mixture was shaken at room
temperature for 2 h. Centrifugation was carried out at 12 000g to
separate the phases, and the concentration of Pt was measured in initial
and final aqueous phase by ICP-MS. The log Po/w was measured using
the following equation.
■
Synthesis of 1. 2-Bromoethanol (6 g, 48 mmol) was added to a
mixture of imidazole (3.6 g, 52 mmol) and tert-butyldimethylsilyl
chloride (7.6 g, 50 mmol) in anhydrous DCM (40 mL). The reaction
mixture was stirred at 0 °C for 30 min followed by stirring at RT for 12
h. After completion, the solvent was removed by a rotary evaporator,
and the oily liquid was redissolved in ethyl acetate and then washed with
water (20 mL × 2) and saturated NaCl (20 mL × 2). The solution was
dried over Na2SO4. Evaporation of solvent yielded a colorless liquid
(yield 90%). 1H NMR (DMSO-d6) δ (ppm) 0.11 (s, 6H), 0.93 (s, 9H),
3.40−3.43 (m, 2H), 3.90−3.93 (m, 2H).
Synthesis of 2. (2-Bromoethoxy)-tert-butyldimethylsilane 1 (6 g,
25 mmol) was added to a stirred suspension of tegafur (1 g, 5 mmol),
K2CO3 (3.4 g, 25 mmol), and KI (0.5 mmol) in dry acetonitrile (150
mL). The reaction mixture was heated to reflux for 36 h, cooled to room
temperature, and filtered. The filtrate was concentrated in vacuo, and
the residue was purified by column chromatography using dichloro-
methane (100%) to yield 2 as a viscous oil (72%). 1H NMR (DMSO-
d6) δ (ppm) 0.03 (s, 6H), 0.81 (s, 9H), 1.84−2.04 (m, 3H), 2.21−2.30
(m, 1H), 3.71−3.75 (t, 2H), 3.79−3.85 (q, 1H), 3.93−3.96 (t, 2H),
4.24−4.29 (m, 1H), 5.93−5.96 (m, 1H), 7.94−7.95 (d, 1H).
Synthesis of 3. In a 50 mL single-neck flask equipped with magnetic
stirrer, 2 (1.70 g, 4.74 mmol) was dissolved in dry THF (20 mL) under
a nitrogen atmosphere. Et3N·3HF (1.69 g, 6.6 mmol) was then added
dropwise, and the reaction mixture was stirred at room temperature.
TLC shows the completion of the reaction after 24 h. Solvent was then
evaporated, and the residue was purified by column chromatography
1
using CH2Cl2/MeOH (95:5). H NMR (DMSO-d6) δ (ppm) 1.89−
2.06 (m, 3H), 2.21−2.30 (m, 1H), 3.50−3.54 (q, 2H), 3.79−3.85 (q,
1H), 3.88−3.93 (m, 2H), 4.24−4.29 (m, 1H), 4.76−4.79 (t, 1H),
5.95−5.98 (m, 1H), 7.91−7.93 (d, 1H).
Synthesis of FTA. To a solution of 3 (1.05 g, 4.30 mmol) in dry
acetonitrile was added TEA (630 μL) and succinic anhydride (520 mg,
5.17 mmol), and the reaction mixture was stirred at 50 °C overnight.
Upon completion of the reaction, the solvent was rotary evaporated,
and the residue was purified by column chromatography using CH2Cl2/
MeOH (85:15). 1H NMR (DMSO-d6) δ (ppm) 1.91−2.07 (m, 3H),
2.21−2.30 (m, 1H), 2.42−2.44 (d, 4H), 3.80−3.86 (q, 1H), 4.04−4.07
(t, 2H), 4.20−4.30 (m, 3H), 5.93−5.96 (m, 1H), 7.93−7.95 (d, 1H),
12.22 (s, 1H).
Synthesis of PF-I. To a stirred suspension of oxoplatin (200 mg,
0.59 mmol) in dry DMSO was added FTA-NHS ester (265 mg, 0.60
mmol) previously prepared by reacting FTA with EDC and NHS. The
reaction mixture was stirred at room temperature for 3 days. A clear
yellow solution was obtained upon completion of reaction. Solvent was
reduced to half, and the product was extracted by repetitive washing
with diethyl ether. 1H NMR (DMSO-d6) δ (ppm) 1.89−2.08 (m, 3H),
2.23−2.30 (m, 1H), 2.38−2.41 (m, 3H), 3.80−3.86 (q, 1H), 4.05−4.07
(t, 2H), 4.20−4.30 (m, 3H), 5.79−6.05 (m, 7H), 7.94−7.96 (d, 1H).
13C NMR (DMSO-d6) δ (ppm) 23.95, 30.52, 31.49, 32.19, 60.91,
70.10, 87.94, 124.15, 138.65, 140.91, 149.29, 156.98, 157.23, 172.92,
179.69. 195Pt NMR (DMSO-d6): 1050 ppm. ESI-MS (negative mode,
m/z): 660.12, elemental analysis calculated (%) for Pt-
(NH3)2Cl2(C14H16FN2O7)OH (Mw = 660.35): C 25.46, H 3.51, N
8.48; found: C 25.20, H 3.48, N 8.53.
Synthesis of PF-II. To a stirred suspension of oxoplatin (100 mg,
0.29 mmol) in dry DMF was added 4 (220 mg, 0.64 mmol), TBTU
(205 mg, 0.64 mmol) and TEA (82 μL). The reaction mixture was
stirred at room temperature for 48 h. A clear yellow solution was
obtained upon completion of reaction. Solvent was evaporated on
vacuo, and the product was purified by column chromatography using
log P = log(([C]initial − [C]final)/[C]final
o/w
1
CH2Cl2/MeOH (70:30). H NMR (DMSO-d6) δ (ppm) 1.88−2.08
Reduction of PtIV Complexes Analyzed by HPLC. Reduction of
PtIV complexes PF-I and PFL (0.5 mM) was carried out using AA (5
mM) in 0.1 M PBS (Prepared by using 1.37 M NaCl, 27 mM KCl, 100
mM Na2HPO4, 18 mM KH2PO4 in 1 L of doubly distilled water, and
pH is adjusted with NaOH) /MeOH (9/1, V/V, pH 7.4), and the
(m, 8H), 2.21−2.30 (m, 2H), 2.31−2.46 (m, 6H), 3.79−3.84 (q, 2H),
4.03−4.06 (t, 4H), 4.20−4.28 (m, 6H), 5.92−5.95 (m, 2H), 6.34−6.57
(m, 6H), 7.93−7.95 (d, 2H). 13C NMR (DMSO-d6) δ (ppm) 23.95,
30.18, 30.68, 32.19, 61.02, 70.10, 87.94, 124.15, 124.49, 138.65, 140.92,
H
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Inorg. Chem. XXXX, XXX, XXX−XXX

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reduction product was examined at 37 °C by RP-HPLC at different
time intervals. Water and methanol was used as a mobile phase for a
gradient elution at a flow rate of 1 mL/min. The HPLC elution program
was as follows: 5% B (0 min) → 100% B (linear increase in 15 min).
The injection volume was 5 μL.
Limited) according to the manufacturer’s instructions. The Pt content
was quantified by ICP-MS.
Western Blot. The assays were performed according to similar
procedures previously reported.43 For TS expression, MDA-MB-231
cells (106) were treated with FT (140 μM), FTA (140 μM), PFL (10
μM), and cDDP (10 μM) for 48 h. For Bcl-2, Bax, and Cytochrome C
expression, MDA-MB-231 cells (106) were treated with cDDP (10 μM)
or PFL (5 and 10 μM) for 48 h. The cells were collected and lysed by
ice-cold RIPA lysis buffer supplemented with protease and phosphatase
inhibitor. Protein samples were separated on SDS-PAGE on 12% gel
and transferred to a PVDF membrane. The membrane was incubated
with primary monoclonal antibodies (Cyto c, Bax, Bcl-2, and α-tubulin)
at 4 °C overnight. Subsequently, the membrane was incubated with
peroxidase-labeled goat anti-rabbit HRP secondary antibody for 2 h.
Western blots were visualized by an enhanced chemiluminescence
detection system.
TEM. MDA-MB-231 cells were treated with 10 μM of PFL at 37 °C
for 30 h. The cells were collected and fixed overnight at 4 °C in PBS (pH
7.4) containing 2.5% glutaraldehyde. The cells were then treated with
osmium tetroxide, stained with uranyl acetate and lead citrate, and
visualized under a transmission electron microscope (JEM 100 CX,
JEOL, Tokyo, Japan).
Measurement of MMP. MDA-MB-231 cells were seeded into a 6-
well plates at a density of 1 × 106 cells per well. After an incubation for
20 h, the cells were treated with PFL (10 or 15 μM) for 30 h. The cells
were collected, and the pellets were resuspended in JC-1 staining
solutions for 20 min. Fluorescence intensity changes in MDA-MB-231
cells were measured using flow cytometry (FACS CaliburTM, Becton
Dickinson, NJ). Data were analyzed by FlowJo software (Tree Star,
OR).
Mitochondrial Bioenergetics. MDA-MB-231 cells were treated
with PFL (5, 10, or 15 μM) for 30 h at 37 °C. The assay was performed
according to the method previously reported.44 The key parameters of
mitochondrial function were assessed using the XF Cell Mito Stress
Test Kit with the Seahorse XFe24 analyzer (Seahorse Bioscience,
Billerica) by directly measuring the oxygen consumption rate (OCR)
and ECAR according to the manufacturer’s instructions.
Intracellular ATP Level. Cellular ATP levels were measured using
the Cell Titer-Glo Luminescent Cell Viability assay kit (G7570,
Promega, U.S.A.) according to the manufacturer’s instructions. MDA-
MB-231 cells were seeded in 96-well plates and treated with PFL (10 or
15 μM) for 30 h. The Cell Titer-Glo reagent was added, and the plate
was incubated for 10 min. The luminescence was recorded using a
microplate reader (Infinite M200 Pro, Tacan, Switzerland).
Cellular ROS Detection. MDA-MB-231 cells were seeded in 6-well
cultural plates at a density of 2 × 105 cells per well and cultivated for 20
h. The cells were treated with PFL (10 or 15 μM) for 30 h. The cells
were then washed twice with PBS and resuspended in serum-free
medium. Cells were stained with H2DCF-DA (10 μM) at 37 °C for 20
min in the dark. The fluorescence intensity in MDA-MB-231 cells was
measured by flow cytometry (FACS CaliburTM, Becton Dickinson,
U.S.A.). λex = 485 nm, λem = 520 nm.
RNA-seq and Bioinformatics. MDA-MB-231 cells were treated
with PFL (10 μM) for 30 h. Total RNA was purified using RNeasy mini
kit (Qiagen, Hilden, Germany). RNA purity was checked using the
kaiaoK5500Spectrophotometer (Kaiao, Beijing, China). The integrity
and concentration of RNA were assessed using the RNA Nano 6000
Assay Kit (Agilent Technologies, CA). A total amount of 2 μg RNA per
sample was used as input. Sequencing libraries were generated using
NEBNext Ultra RNA Library Prep Kit for Illumina (#E7530L, NEB,
U.S.A.). RNA concentration of the library was measured using Qubit
RNA Assay Kit in Qubit 3.0 to preliminarily quantify and then diluted
to 1 ng/μL. Insert size was assessed using the Agilent Bioanalyzer 2100
system (Agilent Technologies, CA), and qualified insert size was
accurate quantification using StepOnePlus Real-Time PCR System.
The libraries were sequenced on an Illumina platform and 150 bp
paired-end reads were generated. Sequencing reads were mapped to
reference human genome sequence (NCBI 36.1 [hg19] assembly by
TopHat (Version 2.0.6). Genes with false discovery rate (FDR) of
<0.05 and >200 bp were considered as differentially expressed. Gene
Stability of PtIV Complexes Analyzed by HPLC. The stability of
PF-I, PF-II, PFD, PFB, and PFL was analyzed in PBS and investigated
by HPLC. All complexes were incubated in PBS buffer (pH = 7.4) at 37
°C, and the analysis is performed over a period of 24 h. Reversed-phase
HPLC was implemented on a 250 × 4.5 mm ODS column, and the
HPLC profiles were recorded on UV detection at 220 and 266 nm. The
percentage of remaining PtIV compounds were calculated using the
ratio of peak area at 266 nm. Water and methanol were used as the
mobile phase for a gradient elution at a flow rate of 1 mL/min. The
samples were taken for HPLC analysis after filtration by a 0.45 μm filter.
Cytotoxicity. The cytotoxicity of PF-I, PF-II, PFD, PFB, PFL were
assessed using MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazo-
lium bromide] assay. Briefly, cells were seeded in a 96-well plate at a
density of 2000 cells/well, in 100 μL of growth medium and were
preincubated for 24 h before exposure to the drugs. A stock solution of
cDDP was prepared in PBS while PF-I, PF-II, PFD, PFB, and PFL
stock solutions were prepared in DMSO. The stock solutions were
diluted in a complete medium and then added in aliquots of 100 μL per
well (DMSO concentration <0.5%). After continuing exposure for 72 h,
the cells were treated with MTT (20 μL, 5 mg mL−1 in PBS) for 4 h.
The medium was removed, and DMSO (200 μL) was added to dissolve
the purple formazan crystals. The plates were shaken for 10 min and the
absorbance of the solution was measured on a Varioskan flash
multimode reader (Tokyo, Japan) at 570 nm. Each test was performed
in triplicate.
Intracellular Distribution. MDA-MB-231 cells (106) were seeded
in 10 cm cell culture dishes using RP-1640 cultural media supplemented
with 10% fetal bovine serum. The cells were incubated with PF-I, PF-II,
PFD, PFB, PFL or cDDP at a concentration of 10 μM for 24 h. The cells
were washed twice with PBS, collected by trypsin, and resuspended in
PBS (1 mL). Mitochondria isolation kit (Beyotime Institute of
Biotechnology, China) was used to separate the nuclei, mitochond-
rion-free cytoplasm, and mitochondria. The isolated fractions were
digested with concentrated nitric acid (100 μL) at 95 °C for 1 h. The
solutions were diluted to 1 mL by doubly distilled water. The content of
Pt was tested by ICP-MS, and the control value was subtracted.
Annexin V-FITC and PI Staining. MDA-MB-231 cells were
seeded in a 6-well cultural plate at a density of 2 × 105 cells per well.
After 20 h of incubation, the medium was replaced with fresh one
containing PFL (10 μM) or cDDP (10 μM). After an incubation for 48
h, the cells were stained with the Annexin V-FITC assay kit and
detected by flow cytometry. Data were analyzed by the Flowjo 7.6.1
software.
Cell Cycle Analysis. MDA-MB-231 cells were seeded in a 6-well
plate at a density of 2 × 105 cells per well. After 20 h of cultivation, the
medium was replaced with the fresh one containing PFL (10 μM) or
cDDP (10 μM). After 48 h, the cells were collected by trypsinization
and washed with PBS. The cells were then fixed in ice-cold ethanol
(70%) for 6 h, treated with RNase A, and stained with PI in PBS for 15
min. The samples were analyzed by flow cytometry.
Hoechst Staining. MDA-MB-231 cells were treated with cDDP or
PFL (15 μM) for 30 h. After they were fixed with 4% paraformaldehyde
for 10 min at room temperature, the cells were washed with PBS twice
and stained with Hoechst 33342 (10 μg/mL, 15 min) in the dark. The
samples were washed with PBS again and observed by confocal
microscopy. λex = 405 nm; λem = 460 20 nm.
DNA Platination. MDA-MB-231 cells were seeded in a 10 cm cell
cultural dishes at a density of 2 × 106 cells. After they were incubated at
37 °C for 20 h, the cells were treated with cDDP (15 μM) or PFL (15
μM) for 30 h. The attached cells were washed twice with PBS,
trypsinized, and washed again with 1 mL of PBS. NucDNA were
extracted and purified by commercial spin column quantification kits
(AxyPrep Blood gDNA MiniPrep kit). The quantification and purity of
DNA were measured by NanoDrop (MD2000, Gene Company
I
https://dx.doi.org/10.1021/acs.inorgchem.0c01736
Inorg. Chem. XXXX, XXX, XXX−XXX

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pubs.acs.org/IC
Article
Set Enrichment Analysis (GSEA) was performed following the standard
procedure (http://www.broadinstitute.org/gsea/doc/
GSEAUserGuideFrame.html) as described by the GSEA user guide.
(2015A030306023), and the Fundamental Research Funds for
the Central Universities.
REFERENCES
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01736.
■
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AUTHOR INFORMATION
Corresponding Authors
■
Cai-Ping Tan − MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China; Email: tancaip@
mail.sysu.edu.cn
Zong-Wan Mao − MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China; orcid.org/0000-0001-
7131-1154; Email: cesmzw@mail.sysu.edu.cn
Authors
Nafees Muhammad − MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China
Uroosa Nawaz − Department of Surgery, P.O.F. Hospital, Wah
Cantt 47040, Pakistan
Jie Wang − MOE Key Laboratory of Bioinorganic and Synthetic
Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China
Fang-Xin Wang − MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China
Sadia Nasreen − Department of Environmental Engineering,
University of Engineering & Technology (UET), Taxila 47080,
Pakistan
Liang-Nian Ji − MOE Key Laboratory of Bioinorganic and
Synthetic Chemistry, School of Chemistry, Sun Yat-sen University,
Guangzhou 510275, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01736
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This study was supported by the National Natural Science
Foundation of China (nos. 21778078 and 21837006), the
innovative team of Ministry of Education (no. IRT_17R111),
the G uangdong Natural Science Foundation
J
https://dx.doi.org/10.1021/acs.inorgchem.0c01736
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Inorganic Chemistry
pubs.acs.org/IC
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