Developing an Anticancer Copper(II) Multitarget Pro-Drug Based on the His146 Residue in the IB Subdomain of Modified Human Serum Albumin

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00045

Designing a multitarget anticancer drug with improved delivery and therapeutic efficiency in vivo presents a great challenge. Thus, we proposed to design an anticancer multitarget metal pro-drug derived from thiosemicarbazone based on the His146 residue i

Full text of DOI:10.1021/acs.molpharmaceut.8b00045

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Article
Developing an anticancer copper(II) multi-target pro-drug based on the
His146 residue in the IB sub-domain of modified human serum albumin
Jun Wang, Yi Gou, Zhenlei Zhang, Ping Yu, Jinxu Qi, Qipin
Qin, Hongbin Sun, Xiaoyang Wu, Hong Liang, and Feng Yang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00045 • Publication Date (Web): 03 May 2018
Downloaded from http://pubs.acs.org on May 4, 2018
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Graphical Abstract
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4x31mm (300 x 300 DPI)
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Developing an anticancer copper(II) multi-target pro-drug based on
the His146 residue in the IB sub-domain of modified human serum
albumin
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§
1,2§
1§
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Jun Wang , Yi Gou , Zhenlei Zhang , Ping Yu , Jinxu Qi , Qipin Qin , Hongbin Sun ,
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Xiaoyang Wu , Hong Liang *, Feng Yang *
1
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Guangxi Normal University, Guilin, Guangxi, China.
2
School of Pharmacy, Nantong University, Nantong, Jiangsu, China.
3
Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA.
*
Corresponding author:
Hong Liang, hliang@mailbox.gxnu.edu.cn
Feng Yang, fyang@mailbox.gxnu.edu.cn
Phone/Fax: 86ꢀ773ꢀ584ꢀ8836
Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541003
§
J Wang, Y Gou and ZL Zhang pay the same contribution for the article.
KEYWORDS: albumin; metal complex; proꢀdrug; drug carrier; target therapy.
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Abstract
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Designing a multiꢀtarget anticancer drug with improved delivery and
therapeutic efficiency in vivo presents a great challenge. Thus, we proposed
to design an anticancer multiꢀtarget metal proꢀdrug derived from
thiosemicarbazone based on the His146 residue in the IB subꢀdomain of
palmitic acid (PA) modified human serum albumin (HSAꢀPA). The
structureꢀactivity relationship of six Cu(II) compounds with
6
ꢀmethylꢀ2ꢀformylpyridineꢀ4Nꢀsubstituted
thiosemicarbazones
were
investigated, and then the multiꢀtarget capability of 4b was confirmed in
cancer cell DNA and proteins. The structure of the HSAꢀPAꢀ4b complex
(HSAꢀPAꢀ4b) revealed that 4b is bound to the IB subꢀdomain of modified
HSA, and that His146 replaces the nitrate ligand in 4b, coordinating with
2
+
Cu , whereas PA is complexed with the IIA subꢀdomain by its carboxyl
forming hydrogen bonds with Lys199 and His242. In vivo data showed that
4
b and the HSAꢀPAꢀ4b complex inhibit lung tumor growth, and the targeting
ability and therapeutic efficacy of the PAꢀmodified HSA complex was
stronger than 4b alone.
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1
Introduction
Designing and synthesizing metal compounds that are derived from
organic ligands with anticancer activities has been the focus of many studies.
This is a promising strategy for the development of novel anticancer agents
since metal compound may have stronger biological and chemical activities
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than organic compound alone.
Metal anticancer agents with individual anticancer mechanism not only
produce severe side effects and drug resistance, but also are not sufficient for
achieving tumour recession since cancers are very complicated and
3
multigenic disease. Currently, multiꢀtarget anticancer drugs are promising
for overcoming the shortcoming of anticancer drug acting against single
4
target. In the last few decades, a lot Cu compounds derived from
thiosemicarbazones lignads were synthesized, and many Cu
thiosemicarbazone compounds have considerable anticancer activity against
5
ꢀ20
murine and human cultured cells.
Indeed, the studies of Cu
thiosemicarbazone compounds are probably one of the most heavily areas of
2
1ꢀ24
medicinal inorganic chemistry.
Interestingly, they also kill cancer cells
2
5ꢀ27
possibly using multiple different mechanisms.
This suggests the
possibility that a multiꢀtarget anticancer Cu agent could be developed, which
destroys cancer cells by several different means, thus avoiding the resistance
2
8
of cancer cells to drugs that treat cancer via a single mechanism.
Meanwhile, increasing the in vivo delivery efficiency and target ability as
well as decreasing the side effects of metal agents should also be
29
addressed. To achieve these goals, Yang et al. proposed developing metal
proꢀdrugs that are based on special Nꢀdonor residue(s) in the human serum
30ꢀ33
albumin (HSA) IIA subdomain.
This strategy, however, does not work
well when metal agents bind weakly to the HSA IIA subdomain. The reasons
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for this are the following: 1) most metal agents can be released from HSA
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during in vivo blood circulation , and 2) fatty acids are the main
endogenous ligands that bind to HSA. Therefore, when the binding affinity
of the drug is weaker than that of fatty acids, the fatty acids will displace the
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drug and bind with the HSA IIA subdomain.
These potential risks can
lead to side effects in vivo; thus, developing a novel proꢀdrug strategy based
on the other properties of HSA is necessary. Currently known structures of
HSA complexes have revealed that metal agents bind to the IB subdomain
36ꢀ38
via the His146 residue of HSA, replacing one ligand in the metal agent.
Taking into consideration the above factors, to increase the target ability
and therapeutic efficacy as well as delivery efficiency of metal agent in vivo,
we proposed to design metal proꢀdrug based on the His146 residue of
modified HSA: 1) modify HSA through palmitic acid (PA)ꢀsaturated binding
to HSA, thus blocking the HSA IIA subꢀdomain, and 2) design a metal
compound with a leaving group. The His146 residue of HSA will then
replace the leaving group and coordinate to a metal ion, forming a stable
HSA complex upon the metal compound bound to the IB subꢀdomain of
PAꢀmodified HSA (HSAꢀPA complex). Subsequently, the coordination
ability of His146 to the metal compound dramatically decreases because the
Nꢀdonor residue is protonated in the acidic environment of the cancer cell
lysosomal compartment, which allows release of the metal compound from
HSA (Figure 1).
Interestingly, West et al. synthesized a series of Cu(II) compounds
4
derived from 6ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted thiosemicarbazones,
9,39
and characterized them mainly by elemental analyses. In addition, Ali et
al. also synthesized several Cu(II) compounds derived from
4
6
ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted
thiosemicarbazones,
and
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characterized them by Xꢀray diffraction.
Obviously, their studies
provided a possibility for developing potential anticancer metal compounds
4
with 6ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted thiosemicarbazones. Thus, to
further study the anticancer function and mechanism of Cu(II) compounds
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with 6ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted thiosemicarbazone, and
investigate their structureꢀactivity relationship, we reꢀsynthesized a series of
4
Cu
compounds
with
6ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted
thiosemicarbazones (1bꢀ6b) reported by West and Ali groups, and
39,40
characterized them by Xꢀray crystallography (Figure 2).
In addition, to
confirm our hypothesis the aboveꢀmentioned by us, we used 4b and lung
cancer (A549) cells to conduct the following studies: First, the multiꢀtarget
capability of these compounds was confirmed in A549 cells. Second, the
development of a Cu proꢀdrug based on the His146 residue in the IB
subdomain of the HSAꢀPA was shown to be feasible. Third, the side effects,
in vivo targeting ability, and therapeutic efficacy of the HSAꢀPA complex
were compared with the Cu compound. Fourth, the release behavior of the
Cu compound from HSAꢀPA and the possible mechanism of the HSAꢀPA
complex’s penetration of cancer cells were investigated.
2 Experimental sections
HSA and palmitic acid (PA) were purchased from SigmaꢀAldrich. All
other solvents and chemicals were of high purity, and were purchased from
commercial sources. Distilled H O was used in all reactions. Elemental
2
analyses (C, N, H, and S) were performed on a PerkinꢀElmer 2400 analyzer
for all the samples.
2
.1 Synthesis and characterization of 1a -6a ligands
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A 10 mL methanol solution of 6ꢀmethylpicolinaldehyde (10 mmol) was
added to a 10 mL methanol solution of thiosemicarabazide (10 mmol). The
acetic acid (1ꢀ2 drops) was then added to the mixture and the yellow solution
was stirred at 60 °C for 6 h. Subsequently, the mixture was filtered, and the
white or offꢀwhite precipitate was placed in a freezer. The characterization of
compounds has been deposited into Supporting Information.
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2.2 Synthesis and characterization of Cu(II) compounds
The Cu(II) compounds 1bꢀ6b were synthesized according to literature
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9,40
procedures.
In brief, the relevant ligands (1 mmol) and the Cu(II) salts (1
mmol) were dissolved in an aqueous methanol solution (20 mL),
respectively, and stirred for 2 h. Dark blue single crystals suitable for Xꢀray
diffraction analysis were obtained after 5 days. The characterization of Cu
compounds has been deposited into Supporting Information.
2
.3 Binding characterization of 4b to HSA-PA
We determined the binding characterization of metal proꢀdrug to
HSAꢀPA by MatrixꢀAssisted Laser Desorption Ionization TimeꢀofꢀFlight
Mass Spectrometry (MALDIꢀTOFꢀMS), fluorescence quench (Shown in
Supporting Information) and Xꢀray crystallography. In addition, the binding
efficiency of Cu(II) agent with HSAꢀPA and Cu agentꢀloading capacity of
HSAꢀPA were determined by the [1] and [2] equations: Binding efficiency
(%) = (Cu agent binding/Cu agent ) × 100% [1]; Cu agentꢀloading capacity
total
(%) = (weight of Cu agentbinding/weight of HSAꢀPAꢀCu agent complex) ×
100% [2].
X-ray crystallography: Briefly, HSA (100 µL and 100 mg/mL) and
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palmitic acid (PA) (960 µL, 2.5 mM) were incubated for 2 h, and then 60 µL
of the Cu compound (2.5 mM and 5 mM) were added to the mixture for 24 h.
The mixture solution had a concentration of 100 g/L with a Millipore spin
filter (10,000 Da cutꢀoff). Crystallization was performed according to a
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previous study.
The data of HSA complexes were collected at 100 K using a BL17U
beam line of the Shanghai Synchrotron Radiation Facility and then
42,43
processed with HKL2000.
The HSA complex structure was determined
by molecular replacement method using the PHASER program in PHENIX
suites with an initial HSAꢀMYR (PDB:1bJ5) model. However, the ligand
was stripped as an initial searching model. All ligands were built into the
model with the LigandFit program in PHENIX suites and manually PA
44,45
modified and adjusted in COOT.
The figures that depict the structures
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were prepared by PyMOL software. Data collection details and unit cell
parameters are listed in Table 1.
2.4 Determining the possible mechanism for uptake of the HSA-PA-4b by
cells
The possible mechanism of uptake of the HSAꢀPAꢀ4b complex by cells
was determined according to previous methods (shown in Supporting
30ꢀ33
Information).
2
.5 Cu pro-drug release from the HSA or HSA-PA complex
The releasing behavior of the Cu compound from the HSA or the
HSAꢀPA complex was assessed according to a previously reported
30ꢀ33
method.
Briefly, 2 mL of the HSAꢀPAꢀ4b suspension in a dialysis pocket
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were dispersed in a tube containing 50 mL of pH 7.4 and 4.7 buffers for 48 h,
respectively. The amount of the Cu compound that was released from the
HSAꢀPA complexes was examined with a graphite furnace atomic
absorption spectrometer (GFꢀAAS).
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.6 In vitro anticancer activity
The cytotoxicity assay (MTT) of ligands and Cu compounds to cells was
performed according to a previous protocol (shown in Supporting
30ꢀ33
Information).
2
.7 Determine of multi-target anticancer mechanism of 4b and the
HSA-PA-4b complex
Some potential anticancer mechanism of 4b and the HSAꢀPAꢀ4b
complex were designed and conducted according to a previous protocol
30ꢀ33
(shown in Supporting Information).
4
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2
.7.1 In vitro proteasome activity assay
Purified 20S proteasome (17.5 ng) was incubated for 1 h at 37˚C in 100
L of the assay buffer (pH 7.5, 50 mM TrisꢀHCl) with or without different
ꢁ
concentrations of 4b and 10 ꢁmol/L fluorogenic peptide substrate
SucꢀLLVYꢀAMC. After incubation, production of hydrolyzed AMC groups
was measured using a multiꢀwell plate Wallac Victor 3 multilabel counter
with an excitation filter of 365 nm and an emission filter of 460 nm.
48
2
.7.2 TRAP-silver staining assay
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A telomerase extract was prepared from the A549 cells. A total of 1.0 ×
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0 A549 tumor cells that were untreated or treated with the complexes 4b (1
µM) and HSAꢀPAꢀ4b (1 µM) were pelleted. The cells were washed with 5.0
mL of PBS. Then the cells were scraped and lysed for 30.0 min on ice.
Finally, the lysate was centrifuged at 13,000 rpm for 0.5 h at 4.0 °C. The
supernatant was collected and subsequently stored at −80 °C before use. A
modified version of the TRAP assay was utilized. Afterward, PCR was
performed in a reaction solution composed of reaction mix (45.0 mL)
containing TrisꢀHCl (pH 8.0, 20.0 mM), deoxynucleotide triphosphates
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(
50.0 mM), BSA (20.0 mg/mL), EGTA (1.0 mM), MgCl (1.5 mM), KCl
2
(
63.0 mM), Tweenꢀ20 (0.005%), primer TS (18.0 pmol;
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’ꢀAATCCGTCGAGCAGAGTTꢀ3’),
’ꢀG [T AG ] ꢀ3’), primer
primer
Cxext
H21T
(22.5
(3.5
pmol;
pmol;
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’ꢀGTGCCCTTACCCTTACCCTTACCCTAAꢀ3’), primer NT (7.5 pmol;
’ꢀATCGCTTCTCGGCCTTTTꢀ3’), TSNT internal control (0.01 amol;
’ꢀATTCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGATꢀ3’),
Taq
DNA polymerase (2.5 U), and telomerase (100 ng), with a final volume of
0.0 mL. Distilled water (5.0 mL) was added. Finally, PCR was carried out
5
in an Eppendorf Mastercycler equipped with a hot lid. The reaction was
incubated for 30.0 min at 30.0 °C, followed by 92.0 °C for 30 s; 52.0 °C for
3
0 s, and 72.0 °C 30.0 s for 30 cycles. Loading buffer (8.0 mL; 5 × TBE
buffer, 0.2% xylene cyanol, and 0.2% bromophenol blue) was added to the
reaction after amplification. An aliquot (15.0 mL) was loaded onto a
nondenaturing acrylamide gel (16.0%; 19:1) in 1 × TBE buffer. It was
resolved at 200.0 V for 1.0 h. Gels were fixed and subsequently stained with
silver nitrate. Commonly, the integrated optical density (IOD) data of
TRAPꢀsilver staining assay were captured using Gel pro4.0, and the
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inhibitory rates of complexes 4b and the HSAꢀPAꢀ4b on the telomerase
could be described by the following: Inhibitory rates (%) = (IOD control group
ꢀIOD treatment group)/IOD control group × 100%.
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2
.7.3 Cell migration inhibition assay
A scratching wound healing assay was implemented in the A549 cell line
to study the effect of the complexes on cancer cell migration. In brief, A549
cells were seeded in six well plates and grown to 90% confluence. Then the
monolayer of the cells was scratched using a sterile microtip. After being
washed three times with iceꢀcold PBS, the new medium was used and
incubated with 4b (0.6 µM) or the HSAꢀPAꢀ4b complex (0.6 µM). The phase
images of the cells were captured at different time points (0, 12 and 24 h)
using a bright field microscope.
2
.7.4 Molecular docking
Docking studies were carried out using Autodock Vina. The crystal
50
structures of the B–DNA dodecamer d(CGCGAATTCGCG) (PDB ID:
2
1BNA), DNA Topoisomerase 1 (PDB ID: 1T8I) and BclꢀxL (PDB ID: 2YXJ)
were downloaded from the protein data bank. The DNA and protein
structures were modified to include polar hydrogen atoms. The coordinates
of the metal complex were taken from its crystal structure. During docking
studies, the DNA and protein structures were kept rigid. Rotation in the Cu
complex was permitted about all single bonds.
2
.7.5 Gene expression profiles were generated by Agilent Human Gene
Expression Array
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To evaluate which genes were upꢀ or downꢀregulated in response to the
different treatments, we created a set with a log foldꢀchange cutoff of ≥2
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and realized a hierarchical clustering of these differentially expressed gene
sets, using a rowꢀnormalized dataꢀset that was prepared using the Feature
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Extraction software Preꢀprocess Dataset.
2.8 In vivo animal studies
2
.8.1 Animal subject and tumor models
Athymic nude mice and Kunming mice (4 weeks of age) were obtained
from the Shanghai Laboratory Animal Center and used at 6 weeks of age.
The Animal Management Rules (document NO. 55, 2001) of the Ministry of
Health of the People’s Republic of China as well as the Animal Care
Committee of the Institute approved all animal experiments.
2.8.2 Acute toxicity study
The acute toxicity studies of 4b and the HSAꢀPAꢀ4b complex were
5
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performed on healthy mice as described previously. In brief, 24 Kunming
mice were placed into three groups of eight mice each. The mice were
treated with 20 µmol Cu/kg 4b or the HSAꢀPAꢀ4b complex. Blood samples
from each mouse were obtained after three days with intravenous (i.v.)
injection to prepare the serum samples. The serum biochemical parameters
were aspartate aminotransferase (AST), creatinine kinase (CK), alanine
aminotransferase (ALT), and blood urea nitrogen (BUN).
2
.8.3 In vivo anti-tumor activity study
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A total of 4 × 10 A549 cells were injected subcutaneously in the right
flank region of the nude mice. The 24 mice were randomly divided into 3
groups for the antiꢀtumor activity study when the tumor volume was about
3
1
00 mm . The mice in the different treatment groups were intravenously (i.v.)
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injected every two days with normal saline (vehicle), free 4b or the
HSAꢀPAꢀ4b complex at a dose of 3.0 µmol Cu/kg body weight. The body
weight of the mice and the size of the tumors were measured before every
injection before the end of experiment. The tumor volume (V) was
2
calculated with the following equation: V = 1/2 × width × length. Mice
were sacrificed, and major organs and tumor tissues were excised for
histopathological analysis after 18 days of treatment.
2.8.4 H&E staining
Tumor samples and major organs were subjected to a routine
histopathological examination using standard hematoxylin and eosin (H&E)
staining. The tissue specimens were collected and placed in
paraformaldehyde (4%) for proper fixation. They were subsequently
dehydrated, cleared and embedded in paraffin wax. The tissue sections were
then stained with H&E and visualized under an Eclipse E800 Nikon (Nikon,
Japan).
2
.8.5 TUNEL assay
At 18 days after the final injection, the mice were sacrificed and the
tumor tissues were excised. They were then fixed with paraformaldehyde for
48 h and embedded in paraffin. Each tissue section was cut into 5ꢀꢁm slices.
Apoptosis in the neuromasts was assayed with a TUNEL assay and an in situ
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cell detection kit (Aꢀ23210; Molecular Probes, Eugene, OR), per the
manufacturer’s protocol. Samples were then visualized under a fluorescence
microscope (AxioCam MRc5; Carl Zeiss).
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2
.8.6 In vivo targeting ability study
At the end of the in vivo experiment, the tumor tissues and major organs
of the mice were homogenized. 0.5 g of the sample was then placed in a
Teflon container and mineralized in a microwave device under pressure
(system Milestone MSL 1200) in the presence of 7 mL of concentrated nitric
acid and 1 mL of 30% H O . ICPꢀAES was used to determine the contents of
2
2
Cu in the tumors and major organs.
2
.9 Statistical analysis
A Student’s t test was used to evaluate the significance of the differences
that were measured. The representative results are expressed as the mean ±
standard deviation (SD), and they were considered to be significant when p
<
0.05.
3
Results
3
.1 Synthesis and structure of Cu pro-drugs
Since thiosemicarbazone itself is a promising anticancer agent, a
thiosemicarbazone was designed to be the primary ligand (pharmacophore)
in the Cu compound, and another ligand, without anticancer activity, was
53ꢀ55
designed to be a leaving group (Figure 2).
compounds with
To optimize anticancer Cu(II)
4
6ꢀmethylꢀ2ꢀformylpyridineꢀ Nꢀsubstituted
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thiosemicarbazone, and further study their structureꢀactivity relationship, we
39, 40
synthesized the six Cu(II) compounds according to the previous method.
Single crystals that were suitable for Xꢀray diffraction were obtained from
the compounds (1bꢀ6b).
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As shown in Figure 2, singleꢀcrystal Xꢀray diffraction revealed that all
Cu(II) compounds were mononuclear, and each thiosemicarbazone ligand
was coordinated in a tridentate fashion, forming two 5ꢀmembered bisꢀchelate
rings around the Cu(II) center. All Cu−N/S bond lengths fall in the range of
1
.927−2.290 Å, which compare well with those observed for similar Cu(II)
12,13,40
compounds.
In compounds 1b, 2b, and 4bꢀ6b, the coordination
geometry surrounding all of the Cu(II) centers formed a distorted square
pyramid geometry (τ = 0.03 for 1b, 0.51 for 2b, 0.35 for 4b, and 0.16 for 5b
and 0.18 for 6b,). In compound 3b, the Cu(II) metal center, which is
coordinated by one Cl atom and two nitrogen atoms of 3a ligand, adopts a
squareꢀplanar coordination geometry.
3.2 Structure-activity relationship of Cu compounds
The Cu compounds showed higher cytotoxicity against many types of
2
+
cancer cells compared with the ligands (1aꢀ6a) and Cu alone (Table 2).
2
+
This may imply that the chelation of ligands to Cu had a synergistic effect
with respect to cytotoxicity. Six Cu compounds showed higher cytotoxicity
against A549 cells (≤ 13.69 ± 0.97 µM) than cisplatin (17.36 ± 0.75 µM)
(
Table 2). Compound 1b had the lowest cytotoxic activity against A549 cells
13.69 ± 0.97 µM) of the six Cu compounds. The cytotoxic activities of 2b
(
and 3b were enhanced (6.77 or 2.75 µM) compared with 1b, and an even
higher cytotoxicity was observed (0.88−1.47 µM) for 4bꢀ6b (Table 2).
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Therefore, we can keep the pharmacophore of the ligand attached to Cu
intact and modify the lipophilic group(s) of the ligand to regulate the Cu
agent’s anticancer activity.
Importantly, the cytotoxicity of the HSAꢀPAꢀCu (II) complexes was
approximately 1.0ꢀ3.0ꢀfold higher in A549 cells relative to the Cu II)
compounds alone. However, the cytotoxicity of the complexes was not
higher in the normal HL7702 cells (Table 2). The HSAꢀPAꢀ4b complex
showed higher cytotoxic activity in A549 cells compared with the other Cu
compounds in vitro. The above complex was selected for further
investigation.
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3.3 Feasibility of the Cu pro-drug design based on the IB sub-domain of
the HSA-PA
The intrinsic fluorescence properties of HSA are attributable to the single
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Trp214 residue in IIA subdomain of the HSA. Compounds that bind to IIA
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subdomain would diminish the fluorescence emission of HSA. The
fluorescence of HSA was gradually quenched upon increasing the
concentration of Cu compound (Figure S1A), indicating that the Cu
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compound binds to the subdomain IIA of HSA. The binding constant of
HSA for Cu compound was estimated from the Stern−Volmer equation
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(
Figure S1B). The binding constant of Cu compound to HSA was
3
−1
estimated to be K = 6.38 ± 0.07 × 10 M .
Owning to Cu compound weakly bound to HSA IIA subdomain, the HSA
was modified by creating a palmitic acid (PA)ꢀsaturatedꢀHSA complex in
vitro to protect the metal compound from being replaced by fatty acids in
vivo. The matrixꢀassisted laser desorption/ionization timeꢀofꢀflightꢀmass
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spectrometry (MALDIꢀTOFꢀMS) spectrum showed an increase of ～280 Da
for the HSAꢀPA complex, which was equivalent to a molecular weight of
one Cu compound (Figure S1C).
The structure of the HSAꢀPAꢀ4b complex was resolved to provide solid
evidence of the feasibility of developing a Cu(II) proꢀdrug based on the
His146 residue of the HSAꢀPA IB subꢀdomain. The electron density maps of
the HSAꢀPA complex indicate clearly that there was one Cu compound
molecule at the IB subꢀdomain and one PA molecule at the IIA subꢀdomain
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(Figure 3A and B). The HSAꢀPAꢀ4b complex structure indicated that 4b
could not compete with PA to bind in the IIA subꢀdomain. The structure of
the HSAꢀPAꢀ4b complex was heartꢀshaped overall (Figure 3C). The 4b
portion binds to the subꢀsite of the narrow and long hydrophobic pocket in
the IB subdomain of HSA. It was in contact with several hydrophobic
residues of HSA, away from PA1. Nitrate is replaced by His146 of HSA in
4b and forms a coordination bond with the Cu ion (Figure 4A and C). PA7
binds to a large hydrophobic pocket in the IIA subdomain of HSA. This was
delimited by Trp214, Phe223, Leu219, His242, Leu238, Ile264, Arg257,
Ser287, Leu260, Ala291, and Ile290. Its carboxyl group forms hydrogen
bonds with His242 and Lys199 (Figure 4B and D).
Furthermore, although the incubation ratio of 4b to HSAꢀPA ranges from
1:1 to 1:3, just one 4b bound to per HSAꢀPA, indicating the binding
efficiency of 4b to HSAꢀPA can reach ～100%, and the 4bꢀloading capacity
to HSAꢀPA is ～0.6%.
3
.4 Potential mechanism for HSA-PA-4b complex absorption by cancer
cells
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The uptake efficiency of 4b and HSAꢀPAꢀ4b in the cancer cells was
investigated by treating A549 cells with 4b (0.6 ꢁM) and HSAꢀPAꢀ4b (0.6
ꢁ
M) for 6 and 12 h. The results showed that the Cu content in A549 cells
treated with the HSAꢀPAꢀ4b is higher than that of in cells treated by 4b
Figure 5A), suggesting A549 cells can absorb more HSAꢀPAꢀ4b than 4b. In
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(
addition, A549 cells at 4°C internalized less Cu than A549 cells incubated
with the HSAꢀPAꢀ4b complex at 37°C, indicating that the uptake of the
HSAꢀPAꢀ4b complex was energyꢀdependent. Glucose deprivation
significantly decreases the uptake of the HSAꢀPAꢀ4b complex in cells. The
uptake of HSAꢀPAꢀ4b complex by cells was inhibited in the presence of
sodium azide or sodium cyanide relative to the control (Figure 5B). These
data suggested the HSAꢀPAꢀ4b complex absorbed by the cells was reliant on
57
metabolic energy. Phenylglyoxal distinctly inhibited the uptake of the
HSAꢀPAꢀ4b complex in cells. Also, methylꢀβꢀcyclodextrin, which is a
known endocytosis inhibitor, stopped the albuminꢀinduced increase in
58
transendothelial HSA permeability (Figure 5C). The results suggested that
the HSAꢀPAꢀ4b complex possibly penetrated the A549 cells by energyꢀ and
temperatureꢀsensitive endocytosis.
3.5 Releasing behavior of 4b from the HSA-4b and HSA-PA-4b complexes
We examined the releasing behavior of the Cu compound from the
HSAꢀ4b complex without PA. Approximately 50% of the loaded 4b was
released from the HSA within 72 h in the pH 7.4 buffer (Figure 5D). This
implied that the HSAꢀ4b complex was unstable in the circulating blood in
vivo, and the majority of the Cu compound was released from the HSA
before delivery into the cancer cells. The reason is that the IIA subdomain is
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available in unmodified HSA, and the Cu complex bound in the IIA
subdomain is easily released at a pH of 7.4.
The releasing behavior of the Cu compound from the HSAꢀPA inside
cancer cells needs to be explained. Thus, we simulated the environment
inside the cancer cells and the amount of Cu compound released from the
HSAꢀPA was measured in pH 7.4 and pH 4.7 buffers. Approximately 5% of
the loaded 4b was released from the HSAꢀPA within 72 h in the pH 7.4
buffer. Furthermore, about 7% of the loaded 4b was released from the
HSAꢀPA in the Dulbecco's Modified Eagle's medium (DMEM) with 10%
FBS during 0−72 h. About 80% of the 4b was released from the HSAꢀPA in
the pH 4.7 buffer during the same time period (Figure 5D).
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3.6 Confirmation of the multi-target anticancer Cu pro-drug
The primary intracellular targets of many drugs used to treat cancer are
5
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cancer cell DNA or proteins.
Herein, we investigated the interaction of Cu compound with
calfꢀthymus (CT)ꢀDNA and supercoiled pBR322 plasmid DNA by multiple
techniques. As shown in Figure 6A, upon the addition of CTꢀDNA, the
absorption band of the 4b observed at 395 nm exhibited a hypochromism of
about 5.1% with 1 nm red shift in the band position. Competitive binding
studies with ethidium bromide (EB)ꢀbound DNA were performed to further
elucidate the binding nature of the Cu compound. On addition of the 4b to
the EBꢀDNA system, the emission was quenched by about 56% (Figure 6B),
6
−1
and the observed binding constant (K ) value of the 4b is 2.2 × 10 M .
app
The viscosity of CTꢀDNA increased on increasing the concentration of the
4
b (Figure 6C). Docking studies further revealed that the Cu compound fit
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snugly into the curved contour of the targeted DNA (Figure 6D). These
results suggested the Cu compound involve in intercalative DNA interaction
61ꢀ63
with moderate binding affinity.
Particularly, to evaluate the DNA
cleavage properties of the Cu compound, the pBR322 DNA as a substrate
was incubated with the 4b, and the cleavage reactions on pBR322 DNA were
investigated by agarose gel electrophoresis. The 4b is capable of converting
supercoiled pBR322 DNA into Form II (open circular form) in a
doseꢀdependent fashion (Figure 6E). The cleavage efficiency of the 4b kept
unaffected in the presence of hydroxyl radical scavengers DMSO and MeOH
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Lane 3 and Lane 4) and singlet oxygen scavenger NaN (Lane 5) (Figure
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•
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F), which means that HO and/or O were not involved in the DNA
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cleavage.
When a DNA mimic phosphates bis(2,4ꢀdinitrophenyl)
phosphate (BDNPP) was added (Lane 6), the reaction of DNA cleavage was
partially inhibited, suggesting that the hydrolysis pathway for the DNA
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cleavage process is possible. Notably, when hydrogen peroxide scavenger
KI (Lane 7) was added, the DNA cleavage was significantly inhibited,
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implying that H O was the reactive species in the cleavage process.
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Furthermore, EDTA (Lane 8) efficiently inhibited DNA cleavage, implying
2
+
that the complexation of 4a ligand with Cu is crucial to the cleavage.
Taken together, these results implied the compound bound to DNA by
moderate intercalative binding mode, and efficiently cleaved pBR322 DNA
probably via an oxidative mechanism with the involvement of H O .
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Topoisomerase I (Topo I), as isomerase enzyme, plays an essential role
7
3
in the topology of DNA. The Cu compound showed significant inhibition
at 15 µM (Figure 7A). The docking model further revealed that the Cu
compound was wellꢀfitted into the site of DNA cleavage of the Topo I
enzyme (Figure 7B). In addition, the Cu compound not only inhibited 20S
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proteasome activity (Figure 7C) and telomerase activity (Figure 7D) but also
inhibited A549 cell cycle progression at the G0/G1 phase and promoted cell
apoptosis (Figures S2 and S3) by regulating the activity of proteins related to
the cell cycle and apoptotic progression of the cancer cell (Figure 8AꢀC).
Interestingly, the Cu compound not only inhibited the transfer of cancer cells
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(Figure S4) but also the activity of metastasisꢀassociated in colon cancerꢀ1
74
(
MACC1) protein related to tumor transfer (Figure 8D). Furthermore, gene
array results showed that the Cu compound regulated the gene expression of
cancerꢀrelated proteins (Figure 8E).
Together, these results demonstrated that the Cu compound kills cancer
cells by targeting DNA and proteins in cancer cells at the same time.
3.7 Evaluating the therapeutic efficiency of 4b and the HSA-PA-4b
complex in vivo
A lung cancer A549 xenograft mouse model was established to further
evaluate whether the HSAꢀPA complex enhanced the therapeutic efficiency
of the Cu compound in vivo.
3
.7.1 Acute toxicity of 4b and the HSA-PA-4b complex in vivo
The acute toxicities of 4b and the HSAꢀPAꢀ4b complex were analyzed in
the kidneys, hearts, and livers of normal mice by measuring levels of blood
urea nitrogen (BUN), creatine kinase (CK), alkaline phosphatase (ALT), and
aspartate aminotransferase (AST) 3 days after intravenous (i.v.) injection of
drugs (Table S1).
Compound 4b caused significant nephrotoxicity, which was indicated by
a higher BUN value in the treated mice (23.6 ± 1.8 mmol/L) compared with
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the control mice (8.6 ± 1.0 mmol/L). CK levels in mice treated with the
HSAꢀPAꢀ4b complex were similar to the levels in the control mice. This
indicated there was low cardiotoxicity. In contrast, nephrotoxicity was
significantly lower in the mice that were treated with the HSAꢀPAꢀ4b
complex, which indicated there was lower nephrotoxicity. The mice that
were treated with 4b had significantly elevated levels of AST and ALT.
However, the AST and ALT levels in mice treated with the HSAꢀPAꢀ4b
complex were similar to those in control mice, which indicated a lower level
of liver damage in the mice treated with the HSAꢀPAꢀ4b complex compared
with those treated with 4b.
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3.7.2 Anti-tumor activities of 4b and the HSA-PA-4b complex in vivo
The tumor volumes in mice treated with 4b and NaCl were larger than the
tumor volumes in mice that were treated with the HSAꢀPAꢀ4b complex
(Figure 9A). This suggested that the HSAꢀPAꢀ4b complex was better at
inhibiting tumor growth than 4b. The tumor weights in mice treated with 4b
were higher than in mice treated with the HSAꢀPAꢀ4b complex and
compared with the NaCl group (Figure 9B).
To further evaluate the antiꢀtumor effects of 4b and the HSAꢀPAꢀ4b
complex in mice, the tumor tissues were excised with terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.
The TUNEL stained tissue sections indicated clear differences in tumor
tissue morphology between the control groups and treated groups. The tumor
cells of mice in the vehicleꢀtreated group were normal, and showed no clear
apoptosis (Figure 9C). However, different degrees of tumor cell apoptosis
were observed in the 4b and the HSAꢀPAꢀ4b treated groups (Figures 9C and
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S5). The HSAꢀPAꢀ4b complex was particularly more effective at promoting
cell apoptosis relative to 4b.
3
.7.3 Comparing the side effects and targeting ability of 4b and the
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HSA-PA-4b complex
Mice that were treated intravenously with the HSAꢀPAꢀ4b complex
showed no significant weight loss compared with the control mice (p > 0.05).
There was, however, an approximate 9% reduction in body weight in the
mice that were treated with 4b (Figure 10A). In addition, 4b caused damage
to the liver (vacuolation and hepatic cord loss), heart (vacuolation), and
kidneys (focal abnormalities) in mice. The damage to these tissues was less
in mice treated with the HSAꢀPAꢀ4b complex (Figure 10B). Together, the
results demonstrated that the side effects induced by the HSAꢀPAꢀ4b
complex were lower compared with the side effects induced by 4b.
Inductively coupled plasma atomic emission spectrometry (ICPꢀAES)
indicated that the increase in Cu content in the tumors of mice that were
treated with the HSAꢀPAꢀ4b complex was approximately 1.3ꢀ higher than in
the mice that were treated with 4b alone. The HSAꢀPA, however, was
helpful for decreasing 4b accumulation in the kidney and liver (Figure 10C).
In conclusion, the HSAꢀPA enabled the Cu compound to selectively
accumulate in the lung tumor in vivo.
4
Discussion
Anticancer functions and mechanisms may be diversified by combining
25
metal ions and ligands with anticancer activities to form metal compounds.
Indeed, many studies have confirmed that, compared with a ligand alone, the
anticancer activity of metal compounds are optimized, and anticancer
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mechanisms are diversified, especially in regard to regulating the activity of
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proteins related to cancer. Thus, a promising strategy is to rationally
design a multiꢀtarget metal agent that can work on different intracellular
targets in cancer cells at the same time. Indeed, the results of the current
study showed that Cu compounds derived from thiosemicarbazones could
kill cancer cells by targeting DNA and proteins in cancer (Figures 6ꢀ8).
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Telomerase, in particular, is a promising target for novel anticancer drugs
76
(
Figure 7D). Developing drugs that inhibit tumor migration are especially
important and challenging since tumor migration is the main cause of death
due to cancer. The MACC1 protein plays an important role in tumor
metastasis. The Cu compound can inhibit the activity of the MACC1 protein
and prevent cell migration (Figure 8D). Therefore, the Cu compound derived
from thiosemicarbazone shows promise for development as a multiꢀtarget
64
2+
anticancer metal lead drug. Excitingly, many studies revealed that Cu can
be used as a costꢀeffective probe for positron emission tomography (PET)
7
7ꢀ80
imaging of various types of cancers.
It suggested that we can design
6
4
2+
multiꢀfunction Cu compound derived from thiosemicarbazide that
simultaneously diagnose and treat cancer.
HSA is the most abundant protein in plasma, which has been promising
drug carrier because HSA is a nonꢀtoxic, nonꢀantigenic, biocompatible, and
8
1
biodegradable endogenous protein. There are two commonly applied
strategies for design HSA to deliver metal agent, namely, forming
albuminꢀdrug conjugate by chemical linker and encapsulating metal agent
29,81ꢀ83
into albumin nanoparticles.
However, the aboveꢀmentioned two
methods can introduce exogenous chemicals or change albumin’s
conformation. To overcome drawbacks associated with HSAꢀbased delivery
strategies for metal agent, we developed a novel approach of HSA carrier for
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metal agent by the specific residue(s) in HSA IIA subdomain coordinated to
30ꢀ33
the metal centre, forming tightly complex of Cu compound and HSA.
Unfortunately, our results revealed that the majority of 4b can escape from
the HSA carrier in the blood circulation because of their weak bonds to the
IIA subdomain of HSA (Figure 5D). Therefore, developing a novel strategy
to render drugs tightly bound to HSA is necessary in order to avoid the
release of the Cu agent from HSA prior to its delivery to the target site. We
therefore proposed to develop a metal proꢀdrug based on the Nꢀdonor of the
HSAꢀPA IB subꢀdomain by taking into account the nature of metal
compounds and the cancer cells. The results of the current study support the
feasibility of this idea. The HSAꢀPAꢀ4b complex structure revealed that the
His146 in the IB subdomain replaced the nitrate of the Cu compound and
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coordinated with Cu (Figures 3 and 4). Afterwards, about 5% of the Cu
compounds were released from the HSAꢀPA at pH 7.4. Approximately 80%
of the Cu compounds were released from the HSAꢀPA in an acidic
environment (pH 4.7) (Figure 5D). These release profiles indicated that the
HSAꢀPA complex was stable in the circulating blood in vivo, and the Cu
compound was released after accumulation in the lysosomes of cancer cells.
Importantly, the HSAꢀPA complex has better therapeutic efficiency than
the metal agent alone in vivo (Figures 9 and 10). The tumor inhibition rate
(TIR) of the HSAꢀPAꢀ4b complexꢀtreated mice (almost 65.3%) was
approximately 1.6 times higher than in the 4bꢀtreated mice. The HSAꢀPAꢀ4b
complex not only caused less weight loss and was well tolerated but damage
to the liver, heart, and kidney was less relative to 4b alone (Figure 10A, B).
Furthermore, the HSAꢀPA enabled the Cu compound to selectively
accumulate in the tumor in vivo (Figure 10C). The HSAꢀPA was helpful by
improving the therapeutic efficiency of the Cu agent. This was due to
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enhanced permeability the retention effect (EPR), and endocytosis of the
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HSAꢀPA in tumors.
5
Conclusions
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Due to the rational design of the metal ion and ligand, the Cu proꢀdrug
derived from thiosemicarbazone kills cancer cells mainly by targeting the
DNA and proteins in cancer. Also, the delivery efficiency of the Cu proꢀdrug
was improved when the leaving group of Cu proꢀdrug was replaced with
2
+
His146 and coordinated with Cu to form the HSAꢀPA complex. The
HSAꢀPA complex showed better tolerance compared with the Cu compound
alone and a higher drug accumulation in the tumor, a stronger capacity for
inhibiting tumor growth, and lower toxicity in other tissues. These results
may be used as a guide to rationally design an HSA carrier to deliver
multiꢀtarget metal agents for targeted cancer therapy.
ACKNOWLEDGMENTS
This work received financial support from the Natural Science Foundation
of China (21431001, 31460232), the Natural Science Foundation of Guangxi
(2017GXNSFEA198002, AD17129007), National Basic Research Program
of China (IRTꢀ16R15), Ministry of Education of China (CMEMR2017ꢀA03),
Guangxi “Bagui” scholar program to HB Sun, and Highꢀlevel innovation
team and distinguished scholar program of Guangxi universities to F Yang.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is free of charge on the ACS Publications
website.
Characterization of 1aꢀ6a ligands and 1bꢀ6b compounds, determination of
binding affinity of HSA for Cu compound, in vitro anticancer activity,
DNAꢀbinding and cleavage experiments, topoisomerase I inhibition assay, in
vitro proteasome activity assay, western blot analysis, determining the
possible mechanism of uptake of HSAꢀPAꢀ4b by cells, serological and organ
weights analysis, crystal data for Cu compounds, selected bond lengths (Å)
and angles (deg) for Cu compounds, cell cycle distribution and apoptosis
analysis, cell migration inhibition assay, apoptosis in tumors, 4b and
HSAꢀPAꢀ4b uptake by A549 cells, the change of mitochondrial membrane
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potential, intracellular ROS measurements, H and C NMR spectra of
1
aꢀ6a.
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the
Xꢀray
crystal
structure
of
the
chloro
(
copper(II)
complex.
(
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14) Belicchi Ferrari, M.; Bisceglie, F.; Gasparri Fava, G.; Pelosi, G.;
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