Synthesis of a series of novel In(iii) 2,6-diacetylpyridine bis(thiosemicarbazide) complexes: Structure, anticancer function and mechanism

Article abstract of DOI:10.1039/d0dt02266g

The anticancer function and anticancer mechanism of indium (In) complexes still remain mysterious to date. Furthermore, it is greatly challenging to design a multi-functional metal agent that not only kills cancer cells but also inhibits their invasion an

Full text of DOI:10.1039/d0dt02266g

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Synthesis of a series of novel In(III) 2,6-
diacetylpyridine bis(thiosemicarbazide) complexes:
Cite this: DOI: 10.1039/d0dt02266g
structure, anticancer function and mechanism†
Shanhe Li,‡a Muhammad Hamid Khan,‡a Xiaojun Wang,‡a Meiling Cai,a
Juzheng Zhang,a Ming Jiang,a Zhenlei Zhang, a Xiao-an Wen,b Hong Lianga and
Feng Yang*a
The anticancer function and anticancer mechanism of indium (In) complexes still remain mysterious to
date. Furthermore, it is greatly challenging to design a multi-functional metal agent that not only kills
cancer cells but also inhibits their invasion and metastasis. Thus, to develop novel next-generation anti-
cancer metal agents, we designed and synthesized a series of novel In(III) 2,6-diacetylpyridine bis(thiose-
micarbazide) complexes (C1–C4) for the first time and then investigated their structure–activity relation-
ships with human urinary bladder cancer (T-24) cells. In particular, C4 not only showed higher cytotoxicity
to cancer cells and less toxicity toward normal cells relative to cisplatin but also inhibited cell invasion and
metastasis of T-24 cells. Interestingly, C4 acted against T-24 cells exhibiting multiple mechanisms: (1)
arresting the S-phase of cell cycle via regulation of cytokine kinases, (2) activating the mitochondrial-
mediated apoptosis, endoplasmic reticulum-stress-mediated cell death, PERK and c-Jun N-terminal
kinase 1 (JNK) cell signaling pathways, and (3) inhibiting the expression of telomerase via the regulation of
c-myc and h-TERT proteins. Our results suggested that C4 may be developed as a potential multi-func-
tional and multi-targeting anticancer candidate.
Received 26th June 2020,
Accepted 29th October 2020
DOI: 10.1039/d0dt02266g
rsc.li/dalton
Recently, novel N-heterocyclic metal complexes showed potent
cytotoxicity against numerous cancer cells. These complexes
1 Introduction
Cancer has become one of the most serious diseases which can make stable coordination bonds with numerous metal
pose a great threat to human health around the world. A ions that could be more stable under physiological conditions
number of new cancer diagnoses have been introduced, but and increase their target ability to cancer cells.8–10 On the
the mortality rate in developed and developing countries is other hand, drugs that act against multiple targets of cancer
increasing.1–3 About half of all the patients who receive anti- cells can enhance therapeutic efficacy and lower their resis-
cancer chemotherapy are treated with cisplatin and its tance relative to the single target-based therapy.11–13
derivatives.4,5 However, increased drug effluence, drug inacti-
Thiosemicarbazones that have extensive pharmacological
vation, variations in the drug target, and processing of drug- flexibility and their application as antitumor, antiviral
induced damage limited anticancer applications of platinum- and antimicrobial agents have been widely studied.14
based anticancer drugs.6 This has encouraged researchers to Thiosemicarbazones, particularly N-heterocyclic thiosemicar-
employ different approaches in the development of new metal- bazides, have low π-electron density at the side chain and the
based anticancer agents with different mechanisms of action.7 ring N-atom has an electron-pair donor to transition metals to
form coordination compounds.15–18 Numerous studies have
revealed that the coordination of thiosemicarbazones to metal
ions increased the activity of the overall complex, enhanced
aState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China, Guangxi Normal University,
the efficacy and overcame the deficiency of organic parent
compounds.19,20
Guilin, Guangxi, China. E-mail: fyang@mailbox.gxnu.edu.cn;
Fax: +86-773-584-8836; Tel: +86-773-584-8836
bJiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, Nanjing, Jiangsu, China
In general, “In” donates three outermost electrons to
become the In(III) oxidation state. It could form stable In(III)
complexes with ligands under physiological conditions and
showed remarkable biological applications.21,22 Owing to the
advantages of the In complex, it may be a promising alterna-
†Electronic supplementary information (ESI) available. CCDC 1961543, 1961545,
1961546 and 1961553. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/d0dt02266g
‡These authors contributed equally to this article.
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tive of Pt agents. However, only a few studies of In compounds were identified by X-ray crystallography and solved by the Olex-
have been reported to date.23,24 Their anticancer functions and 2 application (Table 1) (Fig. 1).
mechanisms still remain mysterious.
C1 is crystallized in the monoclinic space group p21/n with
To develop novel next-generation anticancer metal agents, one In(III) per complex. The In atoms of the dimer are hepta-
we focused on designing and synthesizing a series of In(III) 2,6- coordinated, binding to three N-atoms and two S-atoms of the
diacetylpyridine bis(thiosemicarbazide) complexes for the first ligands and two terminal chlorine atoms of the complex. The
time (Scheme S1†). In(III) complexes were modified at the N-4 central In atom of each of the ligand is surrounded by coordi-
position of the ligands and thoroughly analyzed by IR spec- nation polyhedra, which could be described as having penta-
troscopy, mass spectroscopy, and 1H NMR spectroscopy and gonal bipyramid geometry. The metal displacements of In1–
their multifunctional mechanisms were investigated by bio- Cl2, In1–Cl1, In1–S2, In1–S1, In1–N5, In1–N3, and In1–N4 are
logical experiments. Subsequently, we not only investigated ranging from 2.328 Å to 2.6296 Å. The distinct In(III) dimer of
the structure–activity relationships of In complexes to human the complex having bond angles of Cl2–In1–S2, Cl2–In1–S1,
urinary bladder cancer (T-24) cells and their metastasis inhi- Cl1–In1–Cl2, Cl1–In1–S2, Cl1–In1–S1, S1–In1–S2, N5–In1–Cl2,
bition activity but also confirmed their multi-targeting mecha- N5–In1–Cl1, N5–In1–S2, N5–In1–S1, N3–In1–Cl2, N3–In1–Cl1,
nisms, including the activation of mitochondrial-mediated N3–In1–S2, N3–In1–S1, N3–In1–N5, N3–In1–N4, N4–In1–Cl2,
apoptosis, MAPK-mediated apoptosis, endoplasmic reticulum N4–In1–Cl1, N4–In1–S2, N4–In1–S1, and N4–In1–N5 are
stress-mediated cell damage, and inhibition of telomerase ranging from 65.96° to 170.87°.
activity.
The structures of C2–C4 were also characterized by single-
crystal X-ray diffraction analysis and are similar to that of C1
(Fig. 1). All relative crystal data are compiled in Tables S1 and
S2.†
2 Results
2.1 Design and structure of four In(III) complexes
2.2 Stability of the In(III) complex under physiological
conditions
N-Heterocyclic thiosemicarbazones are an established impor-
tant class of N, S-donor ligands and studied for a considerable
period of time because of their variable donor properties, The In(III) complexes were analyzed by UV-vis spectroscopy and
structural diversity and biological applications.25–27 N-Donor investigated for their resistance to degradation in phosphate
coordinated to metals enhances the anticancer activities of buffer (PBS) over time. C1–C4 were dissolved in DMSO, and
thiosemicarbazide ligands, particularly affecting multiple pro- then diluted with PBS. The amount of DMSO in C1–C4 solu-
teins related to cancer and inhibiting tumor growth via mul- tion is lower than 1%. Their UV-visible spectra were recorded
tiple mechanisms.28 Specifically, 2,6-diacetylpyridine thiosemi- at different times (0–48 h). As shown in Fig. S1,† no apparent
carbazide and their N-4 position modified derivatives were red or blue shift in the absorption peaks of each group was
used to design four novel In(III) complexes, namely, C1, C2, C3 shown, and no new absorption peak appeared. These results
and C4. C1–C4 were synthesized by a stirring protocol and crys- indicated that the structure of the In(III) complexes was stable
tallized by an evaporation method. The structures of C1–C4 and was not degradable in the solution for 48 h.
Table 1 Crystal data of four In(III) complexes
Identification code
C1
C2
C3
C4
Empirical formula
Formula weight
C11H13Cl2InN7S2
494.13
C14H22Cl2InN7OS2
554.22
C46H42Cl2In2N14S4
1219.71
C16H26Cl2InN7OS2
582.28
Temperature/K
Crystal system
Space group
296.15
monoclinic
P21/n
296(2)
monoclinic
P21/c
296.15
293.15
monoclinic
C2/c
triclinic
ˉ
P1
a/Å
b/Å
c/Å
α/°
8.4977(13)
13.297(2)
15.646(2)
90.00
103.015(2)
90.00
1722.4(5)
4
1.905
10.820(7)
13.440(8)
15.215(9)
90.00
100.058(8)
90.00
2179(2)
4
1.690
1.541
8.5777(6)
11.3038(8)
13.9701(10)
94.0320(10)
102.8920(10)
106.2790(10)
1254.63(15)
1
9.7761(3)
14.2179(6)
16.7170(11)
90.00
98.698(5)
90.00
2296.9(2)
4
1.684
β/°
γ/°
Volume/Å3
Z
ρcalc (g cm−3
)
1.614
1.241
μ/mm−1
1.933
1.466
F(000)
976.0
3539/0/212
1.049
R1 = 0.0225, wR2 = 0.0515
0.46/−0.62
1961553
1112.0
3821/2/249
1.035
R1 = 0.0428, wR2 = 0.1114
1.05/−0.59
1961543
612.0
5098/2/309
1.035
R1 = 0.0235, wR2 = 0.0557
0.46/−0.26
1961545
1176.0
2683/112/145
1.171
R1 = 0.0485, wR2 = 0.0966
0.89/−0.78
1961546
Data/restraints/parameter
Goodness-of-fit on F2
Final R indexes [I ≥ 2σ (I)]
Largest diff. peak/hole (e Å−3
CCDC
)
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Fig. 1 X-ray crystallographic structures of In(III) complexes (C1–C4).
2.3 Structure–cellular uptake relationships of In(III)
complexes
suggested that the intracellular absorption of metals is struc-
ture-dependent. The modifications of the N4-position of the
thiosemicarbazide ligands differentiate the efficacy of the In
complexes. The presence of “In” measured in the mitochon-
dria was higher than nucleus, suggesting the activation of
mitochondrial-mediated apoptosis in tumor cells, which was
further supported by the cytometric and western blot results.
To conduct the cellular uptake study, we selected T24 cells.
Inductively coupled plasma (ICP)-MS was used to measure the
total intracellular indium and platinum content in T24 cells.
We observed that In(III) complexes absorbed by T-24 cells were
structure-dependent. As shown in Fig. S2,† the total intracellu-
lar contents of In and Pt ions in cells treated with C1–C4 and
cisplatin were measured with 1.02 nmol, 1.40 nmol,
1.69 nmol, 3.09 nmol, and 0.548 nmol, which were 13%, 18%,
21.8%, 39.8%, and 7.05% of the total intracellular In and Pt
ions respectively. In the cytoplasm of T-24 cells, the concen-
trations of In and Pt ions in C1–C4 and cisplatin-treated cells
were found to be 58% (0.59 nmol), 59% (0.82 nmol), 61%
(1.03 nmol), 60% (1.85 nmol), and 57% (0.31 nmol) of the
total intracellular In and Pt ions, respectively. Similarly, the
concentration of “In” in C1–C4 treated cells and “Pt” in cispla-
tin-treated cells in the mitochondria of T-24 cells was found to
be 24% (0.244 nmol), 19% (0.33 nmol), 22% (0.40 nmol), and
23% (0.74 nmol) and 20% (0.11 nmol), respectively, while the
concentration of “In” in C1–C4 treated cells and “Pt” in cispla-
tin-treated cells in the nucleus of T-24 cells was found to be
18% (18 nmol), 22% (0.31 nmol), 19% (0.32 nmol), and 17%
(0.52 nmol) and 23% (0.126 nmol), respectively. These results
2.4 Multi-functional anticancer In(III) complexes
2.4.1 Structure–activity relationships of In(III) complexes.
The cytotoxicity of four N4-modified thiosemicarbazide
ligands (L1–L4), four In(III) thiosemicarbazone complexes (C1–
C4) and cisplatin was determined separately by using MTT
assay. To conduct the cytotoxicity assay, five tumor cell lines
H460, SKOV3, MGC-803, HeLa and T24, and non-tumor cells
HL-7702 were selected. In addition, MS analysis detects that
C3 exists in the form of a single nucleus in the solution.
Therefore, the molecular weight of C3 was calculated as a
single nucleus in the cytotoxicity test. As shown in Table 2, L1–
L4 and C1–C3 have no cytotoxicity and low cytotoxicity against
five cell lines, respectively, but C4 possesses considerable cyto-
toxicity against five cell lines.
The cytotoxicity of the In(III) complexes to human urinary
bladder cells (T-24) has a relationship with N-4 substitutions.
Table 2 IC50 (μM) values of ligands (L1–L4) and In(III) complexes (C1–C4)
IC50
Compounds
H460
SKOV3
MGC-803
HeLa
T24
HL-7702
L1
L2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
L3
L4
C1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
56.78 2.10
>80
>100
>100
>100
C2
C3
C4
Cisplatin
>100
>50
20.25 1.33
14.89 1.06
43.29 0.36
30.80 1.48
19.11 1.07
15.24 1.01
>100
>50
11.77 0.88
13.36 0.61
>100
>50
15.22 1.02
15.66 1.29
52.22 2.51
46.11 1.33
8.65 0.68
19.34 1.31
>100
>50
21.09 1.01
15.38 0.56
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By adding an N4-methyl and N4-phenyl group to thiosemicar- cell spheroid protocol is used to examine the pharmacoki-
bazide in C2 and C3, their cytotoxicity (C2, IC50 = 52.22
netics and pharmacodynamics of drugs in preclinical trials.32
2.51 μM and C3, IC50 = 46.11 1.33 μM) was increased relative We developed a 3D cell culture of T24 cells and treated with
to C1 (IC50 > 80 μM), respectively. Besides, by adding C4 and cisplatin, and then analyzed the growth of the spher-
two methyl groups to thiosemicarbazide in C4, its oids for time intervals of 0–7 days. The results demonstrated
cytotoxicity against T-24 cells (IC50 = 8.65
0.68 μM) was that there is no any change for untreated spheroids, whereas
increased about 5-fold relative to C1 (52.22 2.51 μM). In the spheroids treated with C4 and cisplatin became more
conclusion, the cytotoxicity of C1–C3 is much lower than that compact in structure (Fig. 2B and S4†), which suggested that
of cisplatin against T-24 cells (IC50 = 19.34
1.31 μM). C4 can inhibit tumor growth. Besides, C4 had stronger
However, the cytotoxicity of C4 is higher about 2-fold than capacity for keeping the spheroid structure compact than
that of cisplatin against T-24 cells (19.34 1.31 μM), the cyto- cisplatin.
toxicity of C4 is lower than that of cisplatin against normal
2.4.4 In(III) complexes inhibiting metastasis of cancer cells.
cells (Table 2).
Cell migration is a process occurring in cells for tissue devel-
2.4.2 Influence of In(III) complexes on cancer cell mor- opment, wound healing, and immune responses. Cell invasion
phology. This assay was used to determine the morphological refers to the ability of cells to become motile and travel
changes of tumor cells after being treated with C4. The T-24 through the extracellular matrix within a tissue or infiltrate
cells were treated with C4 and cisplatin and analyzed for mor- into neighboring tissues. Cell migration and invasion play a
phological changes. The results showed that the shape of T-24 vital role in cancer growth and metastasis. To determine cell
cells treated with C4 was more spherical than that of the migration and metastasis in cancer cells and the effect of che-
untreated T-24 cells (Fig. 2A) and T-24 cells treated with cispla- motherapeutic agents on rapidly dividing tissues of healing
tin (Fig. S4†). The apparent change in T24 morphology could wounds and migrated cells, wound healing and transwell assay
be induced by C4, suggesting that the tumor growth is inhib- were used.33–37 We cultured T24 cells in monolayers and
ited by C4.
pseudo-wounds. The wounded cells were treated with C4 and
2.4.3 In(III) complexes inhibiting the growth of tumor cisplatin, and then analyzed for cell growth in time intervals.
spheroids. Three dimensional (3D) cell culture is a type of arti- The cells treated with C4 showed a slight increase or no
ficially created environment, in which living cells are allowed increase in migration toward the wounded side, while the cells
to grow in a three-dimensional microenvironment with intri- treated with cisplatin (Fig. S5†) and untreated cells migrated to
cate cell–cell and cell–matrix interactions and a complex trans- the wounded side, indicating the inhibition of tumor cell
port dynamics of nutrients and cells.29–31 Primarily, the 3D growth by C4 (Fig. 3A).
Fig. 2 (A) Analysis of cell morphology in T24 cells after treatment with C4. (B) 3D morphology of T24 cell tumor spheroids with vehicles, C4 with
specified concentrations for 8-days.
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2.5 Multi-targeting anticancer mechanism of In(III)
complexes
Cancer is a complicated and multiple gene/protein involved
disease, and drugs that target single proteins could
be limited with acquired resistance and adverse side
effects.11–13 Herein, we used anticancer complexes to promote
apoptotic and autophagic pathways in cancer cells via a
multi-target mechanism of actions. C4 showed higher cyto-
toxicity against T24 cells, therefore we selected C4 for further
studies.
2.5.1 Cell cycle regulation. The flow cytometry data
revealed that after treating the T24 cells with C4 (5 µM and
10 µM) and cisplatin (10 µM) the accumulation of cells in the
S-phase increased to 31.12% (5 µM) and 39.19% (10 µM)
respectively compared to that of the untreated cells that was
29.33%, suggesting that C4 arrested the cell cycle in S-phases
(Fig. 4A). Furthermore, the accumulation of T-24 cells treated
with C4 significantly increased in the S-phase compared to
that of cisplatin-treated cells (Fig. S6†). The cell cycle is regu-
lated by cyclin and cyclin-dependent kinases. Cyclin-A activates
CDK2 and promoted the cell cycle progression by inhibition of
p21.38 On the other hand, the oncogenic protein Cdc25A pro-
motes the cell cycle from G1 to the S phase. The degradation
of Cdc25A, the expression of p53 and p21, and the inhibition
of the CDK2 protein promote the cell cycle in the S-phase.39
We analyzed the expression and inhibition of these proteins by
the western blot technique. The results suggest that after treat-
ment of the T24 cells with C4 and cisplatin, the expressions of
Fig. 3 Analysis of the metastasis in T24 cells after treating with C4. (A)
Wound healing assay, during a 24-hour wound closure assay, pictures
were taken using a time-lapse microscope. (B) A diagram of the trans-
well insert apparatus used to measure cell migration and invasion cells.
Similarly, a transwell assay was used to determine the inhi-
bition of the invasion of T24. After treatment with C4 and cis-
platin, the invasion in T24 cells treated with C4 was significantly
inhibited relative to that in the untreated cells (Fig. 3B) and cis-
platin-treaded cells (Fig. S5†). These suggested that the In(III)
complex has the potential to inhibit the metastasis in T24 cells.
Fig. 4 (A) Effect of the cell cycle of T24 cells treated with C4 (5 µM and 10 µM) compared with untreated cells. (B) Analysis of the expression level
of p53, Cdc25A, Cyclin A2, CDK2, p21, and p27. (C) Percentage expression level of p53, Cdc25A, Cyclin A2, CDK2, p21, and p27; mean SD (n = 3): p
< 0.05, (**) p < 0.01.
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CDK2 and Cdc25A in T24 cells were down-regulated, and the
We examined the mitochondrial membrane permeabiliza-
expressions of p53 and p21 were up-regulated, which provides tion (Δψm) by flow cytometry using a JC-1 assay kit. The clea-
considerable indication that C4 arrests the cell cycle in vage of Cap-9 and Casp-3 and the regulation of Bax, Bcl-xl,
the S-phase through multiple protein regulations (Fig. 4B, C Bad, cytochrome C, and APAF-1 were analyzed by western blot.
and S6†).
Fig. S3 and S8† show that after the treatment of T24 cells with
2.5.2 Analysis of the mitochondrial-mediated apoptosis. C4 and cisplatin, the Δψm decreased to a varying degree com-
Apoptosis or programmed cell death is a natural process, regu- pared to the untreated T24 cells. Western blot data indicated
lated by multiple cell signaling pathways.40 An unbalance in the down-regulation of Bcl-xl and Bcl-2 and the up-regulation
apoptosis in cells or tissues results in cancer progression. of Bax, Bad, cytochrome C, and APAF-1. Similarly, the
Chemotherapy is used to promote apoptosis in tumor cells expression of Cap-9 in T24 cells sufficiently increased (Fig. 5B
without affecting healthy cells.41 We treated the T-24 cells with and C). These results indicated that the activation of mito-
C4 (5 µM and 10 µM) and cisplatin (10 µM), and then analyzed chondrial apoptosis by the regulations of multiple proteins
apoptosis by using Annexin-V/Propidium Iodide double stain- induce apoptosis in tumor cells.
ing assay by flow cytometry. Fig. 5A shows that the occurrence
2.5.3 Endoplasmic reticulum-mediated stress pathways
of apoptosis was increased by 34% (5 µM) and 47% (10 µM) and ROS generation. The endoplasmic reticulum has a flat
when compared with untreated cells. This experimental data structure containing a network of tubules. It functions as the
suggested that C4 induces apoptosis in T24 cells. In addition, folding and processing of secretory proteins.47 The function of
C4 induced more apoptosis in T-24 cells than cisplatin the ER is calcium (Ca2+) dependent. A high concentration of
(Fig. S7†).
Ca2+ requires regulating the homeostatic condition of the
Chemotherapeutics aims to activate apoptotic cascades and ER.48,49 The imbalance in ionic or redox state results in ER-
induces mitochondrial-mediated apoptosis in various cancer stress and accumulation of unfolded proteins.50 Under the stress
cells.42 The Bcl-2 family proteins play a critical role in mito- condition, the ER-transmembrane receptors, such as protein-
chondrial apoptosis regulations. The Bax inhibited the kinase-RNA-like ER-kinase (PERK), phosphorylates the eIF2α
expression of Bcl-2 and Bcl-xl and activated the expression of protein, resulting in the activation of unfolding protein response
the Bad protein, resulting in the release of cytochrome-c from (UPR).51,52 On the other hand, phosphorylation of eIF2α by
the intermembrane space of the mitochondria into the cyto- PERK activates the transcriptional factor CCAATEnhancer-
plasm. Cytochrome-c then makes a complex with procaspase-9 binding protein homologous protein (CHOP).53,54 Under stress
called apoptotic proteases activator factor 1 (APAF-1), which conditions, CHOP is overexpressed and activates the pro-apopto-
activates cap-3 and leads to cell death.43–46
tic proteins and leads to mitochondrial-mediated apoptosis.55
Fig. 5 (A) Effect of cell apoptosis of T24 cells treated with C4 (5 µM and 10 µM) compared with untreated cells. (B) Western blot analysis of Apaf-1,
Bad, Bax, Bcl-2, Bcl-xl, and Cytochrome C. (C) Percentage expression level of Apaf-1, Bad, Bax, Bcl-2, Bcl-xl, Cytochrome C, Casp-9 and Casp-3
relative to control; mean SD (n = 3): p < 0.05, (**) p < 0.01.
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Fig. 6B indicates that compared with untreated T24 cells, the
treated cells with C4 showed more deviated peaks, indicating the
imbalance of Ca2+ concentration in T24 cells. Moreover, the
western blot results signified the activation of PERK and eIF2α
and the up-regulation of CHOP in T24 cells, which suggested
that the ER-stress-mediated apoptosis is activated in T24 cells by
C4 (Fig. 6C and D).
Reactive oxygen species are generated in the mitochondria
in a balanced manner. The imbalance in the induction of the
mitochondrial ROS leads to dysfunction in cell physiology.56
Anticancer chemotherapeutics have the ability to generate ROS
and induce ER-stress, which could be the potential anticancer
agents.57 After treatment with C4, we analyzed the ROS gene-
ration in T24 cells using 2′,7′-dichlorodihydrofluorescein diace-
tate (H2DCFDA) which was oxidized by cellular ROS to 2′,7′-
dichlorofluorescein (DCF), while ER-tracker red was used to
analyze the ER-stress, which was bound to sulfonylurea recep-
tors in the ER. The T24 cells were incubated with C4 and clear
colonization was observed (Fig. 7). In time intervals, the ROS
were found to be more diffused and less colonized, which may
indicate the more permeabilization of the ER membrane and
the induction of ER-mediated apoptosis.
Fig. 7 Generation of ROS by using 2’,7’-dichlorofluorescein (DCF) and
analysis of ER-tracker red by binding to sulfonylurea receptor in the ER.
Besides, the T24 cells were analyzed for ROS generation by
flow cytometry. The cells were observed to make deviated
peaks from the untreated cells, suggesting the activation of expression of its catalytic subunit, telomerase reverse transcrip-
ROS in cancer cells treated with C4 (Fig. 6A). Furthermore, the tase (TERT).59 Human telomerase reverse transcriptase (hTERT)
C4-treated cells significantly increased the ROS and ER-stress is upregulated in most cancer cells but do not express in normal
than cisplatin-treated cells (Fig. S9 and S10†).
cells.60 Myc is thus viewed as a promising target for anti-cancer
2.5.4 Regulation of the telomerase reverse transcriptase by drugs.61 We analyzed the expressions of hTERT and c-Myc by
C4. The MYC proto-oncogene encodes a transcription factor the western blotting technique. After the T24 cells were treated
(c-Myc) that plays a key role in the control of cell proliferation with C4 (5 μM and 10 μM) and cisplatin (10 μM), the expression
and differentiation.58 c-Myc activates telomerase by the of these proteins was remarkably inhibited (Fig. 8A–C and S11†),
which suggested that C4 and cisplatin inhibit the telomerase
activities in T-24 cells. However, C4 had stronger capacity for
inhibiting telomerase activity than cisplatin.
2.5.5 Induction of the MAPK apoptotic pathway by C4.
Mitogen-activated protein kinase (MAPK) also called the extra-
cellular signal-regulated kinases (ERK) pathway is a chain of
proteins in the cell that communicates a signal from a receptor
on the surface of the cell to the DNA in the nucleus of the
cell,62,63 that regulated apoptotic and autophagic pathways in
cells.64 An extracellular mitogen is phosphorylated, which helps
the activation of MAPKs. MAPKs further activate the transcrip-
tional factors such as cyclins and cyclin-dependent kinases.65
P38MAPKs, ERKs, and JNKs are the isoforms of MAPK family
proteins.66–68 In response to cellular stress, these proteins are
activated by phosphorylation and their cell functions, including
apoptosis, are regulated.69 On the other hand, protein kinase B
(AKT) is a cell survival protein that inhibits apoptosis and pro-
motes growth factor-mediated cell survival. AKT phosphorylates
the BAD-protein, isolates from the Bcl-2/Bcl-xl complex and
inhibits its pro-apoptotic function. Numerous compounds that
Fig. 6 T24 cells treated with C4 and analysis of (A) ROS concentration,
targeted the MAP/ERK pathway are considered to be the poten-
(B) Ca2+ concentration in T24 cells, (C) western blot analysis of PERK,
tial anticancer chemotherapeutics.70,71 T24 cells were treated
eIF2α and CHOP in T24 cells, (D) graphical presentation of the
expression level of PERK, eIF2α, and CHOP; mean SD (n = 3): p < 0.05,
(**) p < 0.01, (***) p < 0.001.
with C4 (5 µM and 10 µM) and then analyzed by western blot
for the expressions of p-P38, p-JNKs, p-ERK, and p-Akt-1 pro-
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Fig. 8 (A) Western blot analysis of c-myc and hTERT in T24 cells treated with C4 for 24 h, (B) densitometric analysis of hTERT and c-myc proteins,
(C) the influence on the telomerase activity in the T24 cells treated with C4; mean SD (n = 3): p < 0.05, (**) p < 0.01.
teins. As shown in Fig. 9A and B, the expressions of p-P38, II) and induce cell autophagy.72 Furthermore, an autophagic
p-JNK, and p-ERK were upregulated, while that of p-Akt-1 was receptor, sequestosome 1 (SQSTM1/p62), interacting with LC3,
down-regulated, indicating the activation of MAPK-mediated is constantly degraded during autophagy and eliminates
apoptosis in T24 cells. These results indicated that both C4 and autophagic-dependent substances.73 Besides, beclin-1, an
cisplatin induce apoptosis and autophagy in T24 cells and are autophagic-associated protein, interacts with Bcl-2 and Bcl-xl
associated with MAPK and Akt signaling pathways. However, C4 proteins and regulates the cell death and survival and acts as a
possessed higher activity than cisplatin (Fig. S12†).
crosslink between apoptosis and autophagy.74 The treated T24
2.5.6 Induction of the autophagic pathway by C4. The cyto- cells were analyzed by western blot for LC3-II, p62, and beclin-
solic protein, namely, the microtubule-associated tubule 1A/1B 1 expression. The western blot data showed that the LC3-II and
light chain-3 protein (LC3) is associated with autophagy. The beclin-1 were up-regulated, while the p62 was down-regulated
LC3 is phosphorylated in the cytosol to form a complex (LC3- (Fig. 10A–C). These results not only confirmed the activation
Fig. 9 (A) Western blot analysis of Akt1/2 and P38, (B) analysis of the expression percentage level of Akt1/2 and P38; mean SD (n = 3): p < 0.05,
(**) p < 0.01.
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Fig. 10 (A) Morphological analysis of the induction of autophagy after treated with In(III) complex C4, (B) western blot analysis of the expression
level of LC3-II, Beclin-1 and P62, relative to the control, (C) graphical presentation of the percentage expression level of LC3-II, Beclin-1 and P62;
mean SD (n = 3): p < 0.05, (**) p < 0.01, and (***) p < 0.001.
of autophagy in T24 cells but also revealed the crosstalk of To overcome the deficiency of Pt-based anticancer agents, on
autophagy with apoptosis. In addition, C4 had stronger the one hand, inorganic medicine researchers attempted to
capacity for autophagy modulation than cisplatin (Fig. S13†).
design multi-target metal agents; on the other hand, they
designed metal agents based on non-DNA targets or other anti-
cancer mechanisms. As a matter of fact, In bis(thiosemicarba-
zide) complexes acted against cancer cells by multiple
approaches, including working on proteins related to cancer
and other anticancer mechanisms. It is known that the main
cause of cancer death is the fact that cancer cells can spread to
other organs and tissues in the advanced stage of cancer. It is
highly challenging for designing a metal agent that inhibits
tumor metastasis. Excitingly, the In bis(thiosemicarbazide)
complex can be effective in inhibiting metastasis of cancer
cells. Obviously, the In bis(thiosemicarbazide) complex has
shown promise for the development as a multi-functional and
multi-targeting anticancer lead metal drug.
3 Discussion
Over the past several decades, the characterization of novel In
(III) thiosemicarbazide complexes has been extensively studied
and investigated for their biological applications,22,23,75 but
only a few anticancer In thiosemicarbazide complexes have
been investigated.76,77 To develop potential non-Pt anticancer
agents, In complexes are worthwhile to be deeply studied.
Herein, we have been successful to synthesize four novel In bis
(thiosemicarbazide) complexes for the first time. Interestingly,
previous studies revealed that the anticancer activity of metal
thiosemicarbazide complexes is directly related to the anti-
cancer activity of thiosemicarbazides. Indeed, the results of In
bis(thiosemicarbazide) complexes confirmed the rule again.
The lipophilic groups attached to the thiosemicarbazide
played an important role in increasing the anti-cancer activity
4 Experiments
4.1 Materials
of the In complexes. When the H atom(s) in the N4 location of All the chemicals used were in a highly pure form. H2O used in
the thiosemicarbazide was replaced by more lipophilic groups, all reactions was distilled prior to use. 2,6-Diacetylpyridine and
the IC50 value of C4 is lower than that of C1–C3 (Table 1). thiosemicarbazide were provided by Energy Chemical Company
Therefore, to regulate the anticancer properties of In com- Shanghai, (Shanghai, China). Single crystals were analyzed
plexes, we can modify the lipophilic group(s) of the using a Bruker APEX-II CCD diffractometer. UV-Vis spectra were
thiosemicarbazide.
recorded using
a
UV-Vis spectrophotometer (Shimadzu,
Cisplatin is commonly used to treat various solid cancers, UV-2600). All the cell lines (H460, SKOV3, 803, T24 and HeLa
but it produced severe side effects because it damaged DNA. and non-tumor cells HL-7702) were provided by the Shanghai
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Institute of Biological Sciences. All the antibodies used in under solvothermal conditions at 80 °C for 2 days. C1–C4 were
western blot were provided by Abcam (Cambridge, UK).
characterized by X-ray crystallography, infrared spectroscopy
and electrospray ionization mass spectrometry (ESI-MS) (ESI†).
[In(L1)Cl2] (C1): yield: 62.5%, Anal. calcd for
4.2 Synthesis of ligands (L1–L4)
Ligands L1–L4 were synthesized by the reflux method. As C11H14Cl2InN7S2: C, 26.13; H, 2.85; N, 19.84; S, 12.97. Found:
shown in Scheme S1,† 5 mM 2,6-diacetylpyridine and thiose- C, 26.73; H, 2.86; N, 19.85; S, 12.95. IR, cm−1: IR, cm−1: 3348
micarbazides (L1), 5 mM 2,6-diacetylpyridine and 4-methyl-3- (s, amide), 3311 (s, NH), 3384, 3193, 2345 (s, aromatic), 1622
thiosemicarbazide (L2),
5 mM 2,6-diacetylpyridine and (s, CvN), 1586, 1543, 1399, 1363 (s, thioamide), 1296, 1207,
4-phenyl-3-thiosemicarbazide (L3), and 5 mM 2,6-diacetylpyri- 1011, 986 (m, C–H), 804, (m, CvS), 787, 646, 574. ESI + m/z:
dine 4,4-dimethyl-3-thiosemicarbazide (L4) were stirred in C11H14Cl2InN7S2, 421.97 [M − 2Cl]+.
C2H5OH for 3 h at 65 °C. The ligands were precipitated and fil-
[In(L2)Cl2] (C2): yield: 59.1%, Anal. calcd for
tered. L1–L4 were characterized by infrared spectroscopy, elec- C13H18Cl2InN7S2: C, 30.00; H, 3.28; N, 18.81; S, 12.30. Found:
1
trospray ionization mass spectrometry (ESI-MS), and H NMR C, 29.95; H, 3.29; N, 18.82; S, 12.28. IR, cm−1: IR, cm−1: 3398
(ESI†).
(s, amide), 3334 (s, NH), 2928, 2345, (s, aromatic), 1637, 1563,
L1: 2,6-diacetylpyridine thiosemicarbazide; yield: 60.6%. 1512 (s, CvN), 1326 (s, thioamide), 1392, 1366, 1327, 1274,
Anal. calcd1 for C11H15N7S2: 309.414, C, 42.72; H, 4.88; N, 1200, 1045 (m, C–H), 804, 740, 708, (m, CvS), 651. ESI + m/z:
31.68; S, 20.72. Found: C, 42.71; H, 4.89; N, 31.71; S, 20.69. IR, C13H18Cl2InN7S2, 450.00 [M − 2Cl]+.
cm−1: 3388 (s, amide), 3261 (s, NH), 3122 (m, aromatic hydro-
[In(L3)Cl]2 (C3): yield: 54.7%, Anal. calcd for
gen), 1663 (s, aromatic), 1570, 1503, 1454, 1379 (m, CvN), C46H42Cl2In2N14OS4: C, 45.22; H, 3.88; N, 16.05; S, 10.49.
1280, 1204, 1169, 1102 (s, thioamide), 860, 818, (m, C–H), 559, Found: C, 45.22; H, 3.88; N, 16.06; S, 10.48. IR, cm−1: IR, cm−1
:
509 (m, CvS). 1H NMR (400 MHz, DMSO) δ 10.33 (s, 2H), 3413 (s, amide), 3304 (s, NH), 1677, 1593, 1481 (s, CvN), 1454
8.49–8.36 (m, 4H), 8.17 (s, 2H), 7.79 (t, J = 7.9 Hz, 1H) and 2.45 (s, thioamide), 1367, 1322, 1165, 1189, 1074 (m, C–H), 807 (m,
(s, 6H). ESI + MS: m/z = 310.09 [M + H]+.
L2: 2,6-diacetylpyridine 4-methyl-3-thiosemicarbazide;
CvS), 621, 508. ESI + m/z: C23H21ClInN7S2, 574.03 [M − Cl]+.
[In(L4)Cl2] (C4): yield: 49.2%, anal. calcd for
yield: 56.7%. Anal. calcd1 for C13H19N7S2: 337.46, C, 46.26; H, C16H25Cl3InN7S2: C, 32.68; H, 4.20; N, 17.78; S, 11.63. Found:
5.67; N, 29.05; S, 19.00. Found: C, 46.28; H, 5.68; N, 29.08; S, C, 32.68; H, 4.21; N, 17.79; S, 11.61. IR, cm−1: IR, cm−1: 3417
18.97. IR, cm−1: 3409, 3299 (s, amide), 3299 (s, NH), 2936 (m, (s, amide), 2927, 2351, (s, NH), 1619, 1567, 1499 (s, aromatic),
aromatic hydrogen), 1692 (s, aromatic), 1549, 1513, 1534 (m, 1464 (s, CvN), 1377 (s, thioamide), 1316, 1297, 1249, 1167,
CvN), 1494, 1447, 1392, 1256, 1175 (s, thioamide), 851, 811, 1113 (m, C–H), 809 (m, CvS), 761, 437. ESI
754 (m, C–H), 693, 576 (m, CvS). 1H NMR (400 MHz, DMSO) δ C15H23Cl2InN7S2, 478.03 [M − 2Cl]+.
10.35 (s, 2H), 8.64 (q, J = 4.6 Hz, 2H), 8.41 (d, J = 7.9 Hz, 2H),
+ m/z:
4.4 X-Ray crystallography of In(III) complexes
7.84 (t, J = 7.9 Hz, 1H), 3.06 (d, J = 4.6 Hz, 6H) and 2.44 (s, 6H).
ESI + MS: m/z = 360.10 [M + Na]+.
A single crystal each of C1–C4 was selected to analyze using a
4-phenyl-3-thiosemicarbazide; Bruker APEX-II CCD diffractometer. The crystals were kept at
L3:
2,6-diacetylpyridine
yield: 49.8%. Anal. calcd1 for C23H23N7S2: 461.61, C, 59.84; H, 296.15 K during the data collection. Using Olex2, the structure
5.02; N, 21.33; S, 13.89. Found: C, 59.85; H, 5.03; N, 21.26; S, was solved with the XS structure solution program using direct
13.87. IR, cm−1: 3544 (s, amide), 3299 (s, NH), 1596, 1534 (m, methods and refined with the XL refinement package using
aromatic hydrogen), 1494, 1447 (s, aromatic), 1320, 1256 (m, Least Squares minimization. The data in the CIF format were
CvN), 1175 (s, thioamide), 851, 811 (m, C–H), 754, 693, 576 input at the Cambridge crystallographic data center under the
(m, CvS). 1H NMR (400 MHz, DMSO) δ 10.22 (s, 2H), 8.05 (q, J CCDC number 1961553 for C1, 1961543 for C2, 1961545 for
= 7.8 Hz, 2H), 7.92–7.85 (m, 3H), 2.45 (s, 6H) and 1.56 (s, 18H). C3, and 1961546 for C4.†
ESI + MS: m/z = 484.13 [M + Na]+.
4.5 MTT assay
L4: 2,6-diacetylpyridine 4,4-dimethyl-3-thiosemicarbazide;
yield: 45.9%. Anal. calcd1 for C15H23N7S2: 365.520, C, 49.28; H, H460, SKOV3, MGC-803, HeLa, T24 and non-tumor cell
6.34; N, 26.82; S, 17.54. Found: C, 49.30; H, 6.35; N, 26.84; S, HL-7702 were analyzed to determine the cytotoxicity of C1–C4.
17.51. IR, cm−1: 3462, 3269 (s, amide), 2971 (s, NH), 1696, Each of the cells was grown in Dulbecco’s modified Eagle’s
1574 (m, aromatic hydrogen), 1504, 1448 (s, aromatic), 1358, medium (DMEM) at 37 °C and under 5% CO2 conditions sep-
1242 (m, CvN), 1113, 1017 (s, thioamide), 817 (m, C–H), 698, arately. C1–C4 were dissolved into DMSO and then diluted
1
642, 596 (m, CvS), H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1 with PBS. The amount of DMSO in C1–C4 solution is lower
H), 8.52 (dd, J = 15.5, 2.4 Hz, 2 H), 3.77–3.63 (m, 4 H), 3.22 (q, J than 1%. The stability of In(III) complexes in solution was
= 7.4 Hz, 2 H), 2.36 (s, 3 H), 1.91 (s, 4 H), 1.22 (t, J = 7.4 Hz, 3 checked by using UV. Cisplatin was directly dissolved into PBS.
H). ESI + MS: m/z = 366.42 [M + H]+.
To determine the cytotoxicity of C1–C4, we used 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. To initiate this assay, 96-well plates were used. 180 µL of
4.3 Synthesis of In(III) complexes (C1–C4)
C1–C4 were prepared by treating the ligands L1–L4 each of the cell solution was added to each well and incubated
(0.10 mmol) with InCl3 (0.10 mmol) in methanol (2.5 mL) for 24 h. The cells were then treated with different concen-
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trations of C1–C4, and cisplatin for 48 h. 10 µL of MTT was ml−1) in serum-free chambers, and then placed the transwell
added to each well and incubating for 4 h. The media were into lower chambers and incubated at 37 °C for 15 h. The
removed, 100 µL of DMSO was added to each well and ana- media were removed and washed with PBS. The cells were
lyzed using enzyme labeling instruments to determine the IC50 fixed with 3.7% formaldehyde, washed and permeabilized
values of In(III) complexes against tumor cells.
with 100% methanol. The cells were stained with Giemsa for
15 min, washed and analyzed under a bright field microscope.
4.6 Cellular uptake assay
Inductively coupled plasma (ICP-MS) was used to determine
the distribution of “In” in cancer cells. T24 cells were cultured
and treated with In complexes C1, C2, C3, and C4, and cispla-
tin for 24 h. The mitochondrial membrane fraction, nuclear
fraction and cytoplasmic fraction were separated and extracted
using the NE-PER assay protocol. The cell extract each for
mitochondrial fraction, nuclear fraction and the cytoplasmic
fraction was stored at −80 until analyzed using ICP-MS.
4.11 Cell cycle analysis
T24 cells with a concentration of 1 × 105 cell per mL were cul-
tured in DMEM and incubated at 37 °C and under 5% CO2
conditions for 12 h. The cells were treated with C4 and cispla-
tin for the next 24 h, collected by centrifugation, washed with
bovine phosphate serum (PBS) and fixed with cold ethanol fol-
lowed by overnight storage at −20 °C. The cells were washed
with PBS and incubated with DNA-free RNase-A for 30 min.
10 µL of 1 mg mL−1 propidium iodide solution was added to
each cell’s solution and analyzed by flow cytometry.
4.7 Cell morphology analysis
Tumor cells T24 with concentrations of 1 × 105 cells per mL
were cultured in six-well plates and incubated at 37 °C and
under 5% CO2 environments. The cells were then treated with
4.12 Cell apoptosis assay
C4 and cisplatin for 24 h and were observed under a bright- We cultured the T24 cell in DMEM and treated it with C4 and
field microscope.
cisplatin. The double staining (Annexin-V and PI) protocol was
used to determine apoptosis in T-24 cells. The cells were col-
lected by centrifugation, incubated in 200 µL of Annexin-V
4.8 Tumor spheroid growth inhibition assay
The three-dimensional (3D) spheroid assay was used to binding buffer, and 5 µL of Annexin-V, added to each cell’s
analyze the physiological characteristics of cells. To perform tube, excepting the PI control for 20 min and 37 °C in the
this protocol, 96-well microtiter plates were used. Agarose- dark. After 20 min, 300 µL of 1×-Annexin-V binding buffer was
DMEM solution (1.5% (wt/v)) was sterilized and heated to added to each tube, followed by 4 µL of PI to each cell’s tube,
80 °C. Agarose solution (50 µL) was added to each well and except the Annexin-V control and incubated for 20 min in the
allowed to solidify. T24 cells with a concentration of 1 × 103 dark at 37 °C. The cells were then analyzed for apoptosis using
cells per well were grown on the upper lips of cell culture flow cytometry.
plates and incubated at 37 °C, and under a 5% CO2 atmo-
sphere. After 72 h, one of each T24 cell spheroid was trans-
ferred into each agarose coated well and allowed to settle.
Each T24 cell spheroid except the control was treated with C4
and cisplatin and then analyzed for spheroid growth every
48 h for up to seven days.
4.13 Measurements of mitochondrial membrane potential
Δψm
To determine the mitochondrial membrane potential (Δψm),
we used a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazo-
lylcarbocyanine iodide (JC-1) assay kit. T-24 cells were seeded
in 6-well cell plates, treated with C4 and cisplatin then incu-
bated for 15 h at 37 °C. The cells were then collected by cen-
trifugation, washed with PBS and incubated with 500 µL of
JC-1 solution in the dark. The cells were then analyzed for
Δψm using flow cytometry.
4.9 Wound healing assay
Wound healing assay was made to determine the efficacy of
the In(III) complex C4 against T-24. The cells were seeded in
6-well plates and allowed to grow in monolayers. The mono-
layered cells were wounded using a sterile 200 µL pipette tip,
generated a clean wound area and washed with PBS. The cells
were incubated C4 and cisplatin for 24 h and analyzed under a
bright field microscope.
4.14 Imagining of the ROS in the endoplasmic reticulum
2 × 106 cells per well T24 cells were cultured in six-well plates
on poly-L-lysine-coated coverslips in heat deactivated complete
RPMI, treated with C4 and cisplatin for 6 hours and collected
4.10 Cell invasion assay
To measure the capacity of cell motility and invasiveness by centrifugation. The cells were washed with pre-warmed PBS,
toward chemotherapeutics, we used cell invasion assay. T-24 followed by incubation with 25 µM pre-warmed 2′,7′-dichloro-
cells were cultured in 24-well plates and placed in the dihydro-fluorescein diacetate (H2DCF-DA) for 20 min at 37 °C.
Transwell chamber in 24-well plates. 100 µL of Matrigel (a 1 µM pre-warmed ER-Tracker Red was added to the cells and
serum-free media) with and without treated with C4 and cis- incubated for 15 min and washed with PBS. The cells were
platin was added to the upper chambers and incubated at mounted onto slides and analyzed using confocal microscopy.
37 °C for 24 h. We added 750 µL of culture media to the lower A 20× objective lens was acquired to take images and pro-
chambers and placed 200 µL of T24 cell solution (2.5 × 105 cessed using the Zeiss FLUOVIEW Viewer software.
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4.15 Analysis of the intracellular Ca2+ ion
AKT
Protein kinase-B
PERK
ROS
MAPK
LC3
Protein-kinase-RNA-like
Reactive oxygen species
Mitogen-activated protein kinase
Microtubule-associated protein 1A/B-light
chain 3
The Fluo-3 AM assay protocol was used to determine the intra-
cellular Ca2+. T24 cells were cultured in six-well cell plates and
incubated at 37 °C for 24 h. The cells were then incubated
with C4 and cisplatin for another 24 h. The cells were then
incubated with fluo-3 AM for Ca2+ staining and analyzed using
flow cytometry.
4.16 Western blot analysis
Conflicts of interest
T-24 cells were cultured and treated with C4 and cisplatin for
24 h. The cells were collected by centrifugation and washed
thrice with cold PBS. Radioimmunoprecipitation (RIPA) assay
buffer was used to lyse the treated T24 cells. To determine the
protein concentration in the T-24 cell’s lysate, we used the
bicinchoninic acid (BCA) assay protocol. 10% SDS-polyacryl-
amide gel electrophoresis was used to separate the equal
amount of total proteins. The proteins were then transferred
to poly(vinylidene difluoride) membranes (Millipore, MA, USA)
and blocked in 5% fat-free milk for 30 min and washed with
TBST (20 mM Tris, 150 mM NaCl, pH 8.0 and 0.05% tween 20)
three times. The membrane was then incubated overnight in
primary antibodies at 4 °C, washed thrice with TBST and incu-
bated in secondary antibodies for two hours at room tempera-
ture. Amersham ECL plus (western blotting detecting reagent)
was used to detect the immunoreactivity of the proteins.
There are no conflicts of interest to declare.
Acknowledgements
This work received financial support from the Natural
Science Foundation of Guangxi (2017GXNSFEA198002
and AD17129007), the Guangxi “Bagui” Scholar Program
to HB Sun, and the High-Level Innovation Team
and Distinguished Scholar Program of Guangxi Universities
to F Yang.
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