Gallium(iii)-2-benzoylpyridine-thiosemicarbazone complexes promote apoptosis through Ca2+ signaling and ROS-mediated mitochondrial pathways

Article abstract of DOI:10.1039/c8nj00697k

Ga(iii) compounds are highly promising candidates for antitumor therapy. The level of intracellular reactive oxygen species (ROS) is significantly increased after Ga(iii) complex treatment, but these complexes are redox-inactive. To investigate the effect

Full text of DOI:10.1039/c8nj00697k

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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ARTICLE
Gallium(III)-2-benzoylpyridine-thiosemicarbazone complexes
promote apoptosis through Ca2+ signaling and ROS-mediated
mitochondrial pathways
Received 00th January 20xx,
Accepted 00th January 20xx
Jinxu Qi,a Kun Qian,a Liang Tian,a Zhen Cheng b and Yihong Wang *a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Ga(III) compounds are highly promising candidates for antitumor therapy. The level of intracellular reactive oxygen species
(ROS) is significantly increased after Ga(III) complexes treatment, but these complexes are redox-inactive. To investigate
the effects of Ga(III) complexes on ROS levels, we synthesized three bis-ligated gallium(III)-2-benzoylpyridine-
thiosemicarbazone complexes and studied their antitumor mechanisms. The structures of the Ga(III) complexes were
identified by X−ray single-crystal diffraction. Cytotoxicity analysis demonstrated that ligands and gallium complexes
exerted a higher antitumor activity and lower cytotoxicity than those of normal cells. The most active complex was C3,
which exhibited a better antitumor viability than its related ligands and the anticancer agent 3−AP. Thus, Ga(III) complexes
not only transmitted the iron ions but also induced intracellular Ca2+ release. As a result, the ROS standards in redox-active
iron complexes were increased. The mechanism involved the release of Cyt C from the mitochondria which lack membrane
potential, and then activation of the Caspase family proteins stimulated cell apoptosis.
mechanism of thiosemicarbazone chelators, which are similar
15
Introduction
to
gallium(III)
nitrate.14,
3−Aminopyridine−2−carboxylaldehyde
thiosemicarbazone
Gallium(III) nitrate is a firstꢀgeneration gallium compound for
therapeutic use and shows substantial selectivity to malignant
tumors.1ꢀ3 This selectivity is probably due to the
similar chemical properties with iron that enables gallium to
interfere with the intracellular iron metabolism.4 A consequence
of galliumꢀinduced cellular iron dissipation is the inhibition of
ironꢀcontaining protein activity.5,6 Gallium nitrate, gallium
tris−maltolate and gallium tris−8−quinolinolate (KP46)
(Scheme 1) have been studied in preclinical and clinical trials
and exerted considerable therapeutic effects various solid
tumours.7,8 However, the poor bioavailability and antitumor
activity limit the development of gallium compounds.9 The
coordination of gallium(III) and ligands can improve the
(3−AP) (Scheme 1), an agent in phase II trials for treating
various kinds of cancers, is the most outstanding anticancer
agent among many thiosemicarbazone chelator derivatives.16
Further studies showed that the [C1=NNH(CS)N4] is the basic
structural unit of thiosemicarbazone compounds for biological
activity.17ꢀ19 Through their N and S atoms, thiosemicarbazone
compounds easily form stable complexes with various of metal
ions.20, 21 Many reports proposed that thiosemicarbazone−metal
complexes manifest a markedly higher antiproliferative activity
than that of their corresponding ligands.22ꢀ27 Richardson's
results showed that
the Ni(II), Zn(II), and Mn(II)
thiosemicarbazide analogy complexes display similar antitumor
activities to those of their ligands.28 Generally, the gallium(III)
complexes show increased anticancer activity relative to those
of their corresponding ligands, whereas iron(III) coordinations’
antitumor activity is substantially decreased with respect the
corresponding ligands.27, 29
The treatment of lung cancer has greatly progressed in recent
years, but numerous of patients die of this cancer every year.30
Therefore, the development of new anticancer drugs is
particularly urgent. Considering that thiosemicarbazone
compounds show significant anticancer activity and potential
medicinal value, we performed the following experiments in
this study: (1) synthesis and characterization of the crystal
structure of three 2:1 ligand/Ga (III) complexes by X−ray
(Scheme 1), (2) assay of the electrochemical properties of Ga
(III)/Fe(III) complexes by Cyclic Voltammetry, (3)
compounds’ anticancer activity by increasing their lipophilicity
10
and preventing hydrolysis.4,
Recent studies have
demonstrated that gallium complexes promote apoptosis
through the mitochondrial signaling pathway under the
regulation of the Bclꢀ2 family proteins.4, 11 In clinical practice,
Ga(NO3)3 is combined with hydroxyurea, mitoguazone or
etoposide to achieve a good therapeutic effect.12, 13
Disrupting iron ion metabolism is an antiproliferative
a.School of Chemistry and Chemical Engineering, Southeast University, Nanjing
211189, China.
b.Stanford Cancer Institute, Member of Academic Council, Stanford University,
USA.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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determination of the Ga (III) complexes’ anticancer activity in a N atoms (N1, N2, N5 and N6) and two S atoms (S1 and S2)
series of cancerous cell lines, and (4) investigation of the from two Schiff base ligands, leading to a deformation of
potential mechanism of NCIꢀH460 human lung cancer cell octahedral configuration. The C=N double bond of the
apoptosis induced by the Ga (III) complexes.
thioamide tautomer separated the ligand into 2ꢀpenzoylpyridine
and thiosemicarbazone. The bond length of Ga–N (2.036−2.107
Å) and Ga–S (2.336–2.373 Å) were comparable to analogous
4, 31, 32
distances in other known complexes.
Moreover, the
distances of C–S bond (1.731–1.737 Å) in all complexes were
similar to a single bond distance (ca. 1.81 Å) and double bond
Scheme 1. (A) Chemical structures of gallium compounds
and 3ꢀAP currently in use in the clinic; (B) 2ꢀ
benzoylpyridineꢀthiosemicarbazone
ligands;
(C)
Gallium(III)ꢀ2ꢀbenzoylpyridineꢀthiosemicarbazones
complexes.
Results and discussion
Synthesis of Ga(III) complexes.
Ligands L1ꢀL3 were prepared straightforwardly via Schiff base
condensation reactions. The synthesis reactions of the Ga(III)
complexes were similar to one another. The methyl alcohol
solution of Ga(NO3)3 was added into the solution of the ligands
in methyl alcohol with stirring. The single crystals of Ga(III)
complexes suitable for Xꢀray diffraction were crystallized from
the solution in several days.
Crystal structure description.
The structures of three Ga(III) complexes (C1, C2 and C3) have
been identified by X−ray (Fig.1). The crystal data of C1, C2
and C3 are shown in Tables S1 and Table S2. Crystal structure
showed that these Ga (III) complexes were similar in
coordination mode. The singleꢀcrystal Xꢀray diffraction
structural analysis had revealed that C1 crystallized in the
monoclinic with space group P21/c, C2 crystallized in the
triclinic with space group Pꢀ1, and C3 crystallized in a
monoclinic system with space group P21/n. The coordination
environment of Ga(III) is contented by a pyridyl ring and
thioamide tautomer form ligands. Two ligands were present in
each Ga(III) complex molecule, and a pair of coordination
atoms (N1/N2/S1 and N5/N6/S2) were both in a plane,
respectively. The Ga(III) metal center was coordinated by four
Fig. 1. The molecular structure of C1 (A), C2 (B) and C3 (C)
showing the environment about the Ga(III) atom.
(ca. 1.58 Å) within the normal range found for similar Ga(III)
complexes.33, 34 UVꢀVis spectrophotometric absorption analysis
confirmed that the ligand and complexes were stable within 48
h in 0.1% DMSO aqueous solution (Fig. S1).
Electrochemistry.
As our recent studies have shown, intracellular ROS levels are
significantly elevated when NCI−H460 cells are exposed to
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gallium complexes.
ARTICLE
Therefore,
investigating
the
These ligands and complexes showed a good activity against
electrochemistry of Ga(III) complexes is important. Cyclic these cancer cell lines, whereas Ga(NO3)3 (100 ꢁM) alone
voltammetry results indicate that the Ga(III) complex does not presented no pronounced antiproliferative effect (Table 1). The
exert any redox activity (Fig.2). A facile, reversible, single antitumor activity of L3 (IC50 = 1.08 ± 0.23 ꢁM) was
electron redox process was observed when ferric(III) nitrate significantly (p < 0.05) higher than those of L1 (IC50 = 17.54 ±
was added to the Ga(III) complex solution, which indicates that 2.2 ꢁM), L2 (IC50 = 6.88 ± 1.3 ꢁM), and 3−AP (IC50 = 4.23 ±
0.52 ꢁM) after a 48 h incubation with NCI−H460. Interestingly
the anticancer activities of C1, C2, and C3 in the MTT assay
were better than those of their corresponding ligands. In terms
of IC50 values, C3 (IC50 = 0.25 ± 0.09 ꢁM) was the most
effective among the Ga(III) complexes (compared with C1 with
IC50 = 4.89 ± 0.34 ꢁM and C2 with IC50 = 2.65± 0.22 ꢁM) for
NCI−H460 cells. This trend can be explained by the lipophilic
group (phenyl, and dimethyl) of the N4 substituents.34 Notably,
the increased activity was more strongly connected to the
different metal binding ability of the dimethylated compounds
compared to the unsubstituted ones.38,39 The increased
anticancer activity of the Ga(III) complexes relative to that of
the ligand was observed probably because the dissociation of
the complexes releases two ligands, possibly in pH 7.4 solution.
Fig 2. Cyclic voltammograms of Ga(III) complexes,
Fe(NO3)3, Ga(NO3)3 + Fe(NO3)3, C1 + Fe(NO3)3, C2 +
Fe(NO3)3 and C3 + Fe(NO3)3.
Potential anticancer mechanism of Ga(III) complexes.
Richardson et al. have demonstrated that the mechanism of
action of BpT series involves the Fe chelation and redox
cycling of its Fe complex to ROS, hydroxyl radicals (OH•): this
iron substitutes gallium to form Fe(III)–thiosemicarbazone
complexes. The cathodic shift in the FeIII/II redox potentials
reveals that oxidation–reduction reactions are prone to occur
(Fig.2).
occurrence leads to the damage of essential biomolecules
40
within cells.35,
However, the antitumor mechanism of
thiosemicarbazide Ga(III) complex has not been systematically
studied. As the gallium complexes significantly promote NCIꢀ
H460 cell apoptosis, studying their antitumor mechanisms is
important. We investigated intracellular Ca2+ and intracellular
ROS, the mitochondrial membrane potential, and the
expression of mitochondrial pathwayꢀrelated proteins to explore
the pathways by which gallium complexes promote NCIꢀH460
cell apoptosis.
Anticancer properties of Ga complexes.
The
anticancer
activity
(BpT)
of
series
the
2ꢀbenzoylpyridine
against SKꢀNꢀMC
thiosemicarbazone
neuroepithelioma cells and human DMSꢀ53 lung carcinoma
cells has been well characterized.34ꢀ37 Similar results were
obtained in our study using the NCIꢀH460 human lung cancer
cells, BELꢀ7402 human hepatoma cells and T24 human bladder
cancer cells (Table 1), which confirms the extensive
antiproliferative activity of BpT ligands. IC50 (the halfꢀmaximal
inhibitory concentration), which refers to the concentration of
an inhibitor that is required for 50% inhibition of cell growth,
was determined by MTT assay. These Ga(III) complexes were
compared with corresponding ligands and a relevant positive
control (3ꢀAP). The ligands and gallium complexes exhibited a
lower toxicity to human fetal lung fibroblast (MRCꢀ5) cells
than that of cancer cells (Table 1).
Ga(III) complexes induce intracellular Ca2+ release.
Table 1 IC50 (μM) values of ligands and Ga(III) complexes
toward the cell lines for 48h.
IC50 (ꢁM)
Fig. 3. Thiosemicarbazone ligands (L1-L3) and Ga(III)
complexes (C1-C3) induced intracellular release of Ca2+
in cancer cells. (A) Intracellular Ca2+ was detected in
NCI−H460 BELꢀ7402 T24
MRCꢀ5
3−AP
L1
4.23 ± 0.52 5.01 ± 0.43 4.18 ± 0.35 ꢀ
NCI
the flow cytometric results in (A). Results are the mean
SD (n = 6 7): *p < 0.05, **p < 0.01, ***p < 0.001.
−H460 cells by flow cytometry; (B) quantification of
17.54 ± 2.2 15.54 ± 0.98 19.06 ± 1.99 67.34 ± 4.64
6.88 ± 1.3 8.88 ± 0.56 9.32 ± 1.01 32.43 ± 2.06
1.08 ± 0.23 2.69 ± 0.37 1.23 ± 0.22 21.87 ± 1.28
4.89 ± 0.34 4.53 ± 0.54 4.98 ± 0.29 43.55 ± 2.39
2.65 ± 0.22 3.27 ± 0.28 3.52 ± 0.19 24.28 ± 2.03
0.25 ± 0.09 0.54 ± 0.07 0.37 ± 0.03 18.37 ± 1.58
L2
±
−
L3
C1
C2
C3
Ga(NO3)3 > 100
> 100
> 100
> 100
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Metalꢀbased antitumor drugs induce Ca2+ signaling by the complexes presented a green fluorescence signal, whereas the
intracellular release of the Ca2+ ion pool.8 Intracellular Ca2+ vehicleꢀtreated controls showed a red signal, which meant that
release was assayed in Ga(III) complexꢀtreated NCIꢀH460 cells Ga(III) complexes, especially C3, can lead to the collapse of the
by flow cytometry. After 30 min incubation, thiosemicarbazone mitochondrial transmembrane potential (ꢂψm).
ligands (except L1) and Ga(III) complexes significantly (p <
0.01) increased release of intracellular Ca2+ (Fig. 3).
Interestingly, Ga(III) complexes initiated intracellular Ca2+
release significantly (p < 0.05) higher than the ligand.
Fig. 5. Assay of NCI−H460 cells mitochondrial membrane
potential with JC−1 as fluorescence probe staining
method.
Fig. 4. Thiosemicarbazone ligands (L1-L3) and Ga(III)
complexes (C1-C3) increased the intracellular ROS levels
in cancer cells. (A) Intracellular ROS was detected in
NCI−H460 cells; (B) quanꢀficaꢀon of the flow cytometric
results in (A). Results are the mean ± SD (n = 6−7): *p <
0.05, **p < 0.01, ***p < 0.001.
Ga(III) complexes regulate the expression of mitochondrial
pathway-related proteins.
Intracellular calcium levels were 126%, 133% and 162%
relative to the control after C1, C2 and C3ꢀtreatment,
respectively.
Ga(III) complexes increased the intracellular ROS levels.
The release of intracellular Ca2+ induced the production of ROS
in cancer cells.41 Fluorescent DCF probe and flow cytometry
were employed to evaluate the ability of Ga(III) complexes to
affect the level of intracellular ROS in NCIꢀH460 cells (Fig. 4).
The DCF fluorescence peaks of the cells treated with Ga(III)
complexes were shifted to the right relative to those of the
control cells, indicating that these complexes increased the
intracellular ROS levels. Ga(III) complexes significantly (p <
0.001) the increased intracellular ROS levels relative to the
ligands and control (Fig. 4). The quantification of the DCF
fluorescence peaks in part A of Fig. 4 proved that C3 caused a
significant (p < 0.001) increase in H2DCF oxidation to 273% ±
10% of that of the control cells. Furthermore, C3 was found to
be 51% and 83% more effective than C2 and C1, respectively,
in inducing intracellular ROS.
Fig. 6. (A) Western blot analysis of Bcl−2, Bcl−xl, Bax, Bad
and Bim levels after Ga(III) complexes treatment. β−actin
was used as internal control. (B) Densitometric analysis of
the expression of Bcl−2, Bcl−xl, Bax, Bad and Bim. The
percentage values are those relative to the control.
Results are the mean
0.01, ***p < 0.001.
± SD (n = 6−7): *p < 0.05, **p <
The Bcl−2 family proteins play a critical role in the intrinsic
mitochondrial signal pathway of cell apoptosis.42ꢀ45 The
influence of Ga(III) complexes on the expression levels of these
proteins in NCI−H460 cells were analyzed by Western blot.
After incubation with C1, C2 and C3, the expression levels of
antiapoptotic proteins (Bcl−2 and Bcl−xl) gradually decreased,
whereas those of proapoptotic proteins (Bax, Bim and Bad)
gradually increased (Fig. 6). The ratio of Bax/Bcl−2 increased
from 1.00ꢀfold to 1.09, 1.97, and 2.44ꢀfolds after incubation
with C1, C2 and C3, respectively (Fig. 6). The increase in
Bax/Bcl−2 ratio leads to the enhanced mitochondrial membrane
permeability, which is favorable for the release of apoptogenic
factors.
Mitochondrial membrane potential assay.
The change in intracellular ROS level is an important factor
affecting the mitochondrial membrane potential.42 The change
in the mitochondrial transmembrane potential was detected by
JCꢀ1 (a kind of lipophilic fluorescent probe). After staining by
JCꢀ1, the color of the mitochondrial membrane changed from
red to green when the membrane potential decreased and
assayed by flow cytometry (Fig. 5). The mitochondrial
transmembrane of the NCIꢀH460 cells treated with Ga(III)
The consumption of mitochondrial transmembrane potential
(ꢂψm) promotes the release of apoptosis factors and causes the
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irreversible cell apoptosis.46 The release of cytochrome C (Cyt complexes has aroused our interest. NCI−H460 cells treated
C) and the activation of caspase family proteins (caspase 3 and with Ga(III) complexes (5 ꢁM) for 12 h showed increased
9) play a key role in the mitochondria mediated apoptosis apoptosis relative to that of the vehicleꢀtreated controls under
pathway.47, 48 To determine the role of the promotion of cell Annexin V−FITC/PI staining (Fig. 8). The percentages of the
apoptosis factors during Ga(III) complex−induced apoptosis in cells where C1−C3 promoted early apoptosis were 4.36%,
NCI−H460 cells, we examined the protein levels of Cyt C, 9.82% and 14.82%, respectively, relative to the control. Results
activated caspase 3 and 9, and their enzymatic activities (Fig. revealed that C3 was found to be 5% and 10% more effective
7). Incubating NCIꢀH460 cells with C1, C2 and C3 raised the than C2 and C1, respectively, in inducing cell early cell
levels of Cyt C by 1.20, 1.38 and 1.51ꢀfolds relative to that of apoptosis (Fig. 8). Our results illustrated that the intrinsic
control, respectively. After incubation with C1, C2, and C3, the mitochondrial signal pathway is involved in Ga(III) complexꢀ
activated caspase 3 protein levels increased by 1.04, 1.12, and induced apoptosis.
1.18ꢀfolds, respectively, and the activated caspase 9 protein
levels by 1.08, 1.28, and 1.48ꢀfolds, respectively, relative to
Experimental
that of the control, respectively (Fig. 7). The release of Cyt C
leads to the activation of downstream caspase family proteins
and affects the corresponding substrates; ultimately, cell
apoptosis ensues. 47
Materials.
2−Benzoylpyridine,
4,4−Dimethyl−3−thiosemicarbazide,
4−Phenyl−3−thiosemicarbazide,
N−Aminothiourea,
Ga(NO3)3 and solvents were purchased from Energy Chemical
Company (Shanghai) without further purification. Ultrapure
water was used in all experiments. Elemental analyses (C, N,
and H) were carried out on a PerkineElmer 2400 analyzer.
Primary (AntiꢀBclꢀ2 antibody, AntiꢀBcl−xl antibody, AntiꢀBax
antibody, AntiꢀBad antibody, AntiꢀBim antibody, AntiꢀCyt C
antibody, AntiꢀCaspase 9 antibody, AntiꢀCaspase 3 and Antiꢀβꢀ
Actin antibody) and secondary antibodies (Goat AntiꢀRabbit
IgG H&L) were purchased from Abcam Company (Shanghai,
China). The NCI H460, BELꢀ7402, T24 and MRCꢀ5 cell lines
−
were purchased from Chinese Academy of Sciences (Shanghai,
China).
Synthesis and characterization of Ga(III) complexes.
The ligands L1−L3 were all prepared by the published
51, 52
methods.26,
Briefly, 10 mL of 2−Benzoylpyridine (10
mmol) MeOH solution was added drop wise into 10 mL of
thiosemicarbazide or its derivatives (10 mmol) water solution
with stirring. The mixture was added with 0.05 mL of glacial
acetic acid and refluxed for 4 h. When the solution was cooled,
the thiosemicarbazone precipitate was filtered, washed with
EtOH (5 mL) and diethyl ether (5 mL), and then dried in a
vacuum desiccator.
Fig. 7. (A) Western blot analysis of Cyt C, Caspase 9 and 3
levels after Ga(III) complexes treatment. β−acꢀn was used
as internal control. (B) Densitometric analysis of the
expression of Cyt C, Caspase 9 and 3. The percentage
values are those relative to the control. Results are the
mean ± SD (n = 6−7): *p < 0.05, **p < 0.01, ***p < 0.001.
2−Benzoylpyridine thiosemicarbazone (L1): yield 81%. Anal.
Calcd for C13H12N4S: C, 60.92; H, 4.72; N, 21.86; S, 12.51.
1
Found: C, 60.90; H, 4.71; N, 21.87; S, 12.53. H NMR (400
Ga(III) complexes promote cell apoptosis.
MHz, D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ 12.43 (s, 1H), δ
9.46 (s, 1H), 9.02 (s, 1H), 8.92ꢀ8.88 (s, 1H), 8.83 (s, 2H), 8.62
(s, 1H), 8.35 (s, 1H), 8.20 (s, 1H), 8.08 (td, J=7.8, 1.5Hz, 1H),
7.87 (s, 2H). MS m/z (%) 255.07 (M − H, 100).
2−Benzoylpyridine 4−phenyl−3−thiosemicarbazone (L2): yield
79%. Anal. Calcd for C19H16N4S: C, 68.65; H, 4.85; N, 16.85;
S, 9.64. Found: C, 68.61; H, 4.87; N, 16.83; S, 9.68. 1H NMR
(400 MHz, D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ 13.15 (s, 1H),
10.34 (s, 1H), 8.89 (d, J = 4.2 Hz, 1H), 8.05 (td, J = 7.9, 1.8 Hz, 1H),
7.73 (dt, J = 8.6, 3.9 Hz, 2H), 7.61 – 7.55 (m, 3H), 7.53 – 7.45 (m,
3H), 7.40 (dd, J = 4.3, 3.2 Hz, 3H), 7.25 (dd, J = 7.3, 4.3 Hz, 1H).
MS m/z (%) 321.10 (M − H, 100).
Fig. 8. The apoptosis analysis of the NCI−H460 cells
treated by Ga(III) complexes (5 ꢁM) for 12 h, and
representative dot plots of PI and annexin V double
staining. (A) Control; (B−D) C1−C3.
MTT experiments demonstrated that the coordination of ligands
with Ga(III) ions can cause marked increases in anticancer
activity. Therefore, the antitumor activity of the three Ga(III)
2−Benzoylpyridine 4,4−dimethyl−3−thiosemicarbazone (L3):
yield 67%. Anal. Calcd for C15H16N4S: C, 63.35; H, 5.67; N,
19.70; S, 11.27. Found: C, 63.40; H, 5.64; N, 19.72; S, 11.23.
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1H NMR (400 MHz, D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ Bu4NClO4 (0.1 M), and the solutions were purged with nitrogen
12.59 (s, 1H), δ 8.93ꢀ8.84 (m, 2H), 8.45 (dd, J=12.0, 4.3 Hz, prior to measurement. The sweep rate was 100 mVsꢀ1.
1H), 8.02 (d, J=1.7 Hz, 2H), 7.69ꢀ7.67 (m, 2H), 7.58 (d, J =
Cytotoxicity assay in vitro.
7.5Hz, 1H), 3.37 (s, 6H). MS m/z (%) 283.10 (M − H, 100).
The ligands and Ga(III) complexes were dissolved in DMSO as
The synthesis method of complexes C1−C3 is as follows. A
10mM mother solutions and diluted in cell culture medium to
solution of Ga(NO3)3 (0.5 mmol) in MeOH (10 mL) was added
needed concentration. NCI−H460 cell line was cultivated at 37
drop wise into 10 mL of the relevant ligands (1 mmol) MeOH
in a humidified atmosphere of 5% CO2/95% air in an
solution with stirring, and refluxed for 30 min. The solution
incubator. Cell toxicity test is determined by 3ꢀ(4,5ꢀdimethylꢀ2ꢀ
was slowly volatilized at room temperature, and the yellow
thiazolyl)ꢀ2,5ꢀdiphenylꢀ2ꢀHꢀtetrazolium
bromide
solution
crystals formed after a few days. The crystals were filtered,
washed with EtOH (5 mL) and diethyl ether (5 mL) and dried
in a vacuum desiccator.
(MTT) assay and performed according to the published
methods. Briefly, 180 µL of 5 × 104 cells per well were seeded
in 96 well plates and cultivated for 24 h in a humidified
atmosphere of 5% CO2/95% air in an incubator. Then the
compounds at various concentrations were added into per well
and incubated for 48 h. At the end of the experimental, every
2−Benzoylpyridine thiosemicarbazone Ga(III) (C1): yield 67%.
Anal. Calcd for C26H22GaN9O3S2: C, 48.62; H, 3.45; N, 19.62;
O, 7.47; S, 9.98. Found: C, 48.66; H, 3.43; N, 19.63; O, 7.45; S,
1
9.96. MS m/z (%) 578.07 (M − H, 100). H NMR (400 MHz,
well was added with 10 µL MTT and incubated for 4 h at 37
.
D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ 9.90 (d, J = 6.8 Hz, 2H), δ
9.70 (s, 4H), 8.81ꢀ8.87 (m, 2H), 8.55 (td, J = 7.2, 1.3 Hz, 1H),
8.32 (dd, J = 8.6, 0.7 Hz,2H), 7.71ꢀ7.68 (m, 4H), 7.66 (s, 4H),
7.45 (dd, J=10, 7.2Hz, 2H).
2−Benzoylpyridine 4−phenyl−3−thiosemicarbazone Ga(III)
(C2): yield 71%. Anal. Calcd for C38H30GaN9O3S2: C, 57.44;
H, 3.81; N, 15.87; O, 6.04; S, 8.07. Found: C, 57.46; H, 3.78;
After removing the supernatant, 100 µL of DMSO were added.
The absorbance with 570/630 nm double wavelength
measurement of per well were read by enzyme labelling
instrument. IC50 values were calculated by the nonlinear
multipurpose curve−fitting program Graph Pad Prism. All of
the tests were repeated in least three measurements.
N, 15.84; O, 6.08; S, 8.05. MS m/z (%) 730.12 (M − H, 100). Intracellular Ca2+ mobilization assay.
1H NMR (400 MHz, D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ 10.44 (s, Ca2+ was measured by flow cytometry according to the
2H), 8.34 (d, J = 5.0 Hz, 2H), 8.25 (d, J = 1.2 Hz, 2H), 7.91 (d, J = specification of the Fluoꢀ3 AM (calcium fluorescence probe)
6.5 Hz, 4H), 7.81 – 7.71 (m, 10H), 7.53 (d, J = 8.0 Hz, 4H), 7.17 (t, assay kit (Beyotime, Suzhou) based on the fluorescence
J = 7.8 Hz, 4H), 7.02 (s, 2H).
method. 5 mL of 5×105 cells/mL NCI−H460 cells were seeded
2−Benzoylpyridine 4,4−dimethyl−3−thiosemicarbazone Ga(III) in 7.5 cm plates and cultivated at least 12 h before treatment.
(C3): yield 83%. Anal. Calcd for C30H30GaN9O3S2: C, 51.59; Cells were incubated with 25 ꢁM of L1ꢀL3 and C1−C3 in cell
H, 4.33; N, 18.05; O, 6.87; S, 9.18. Found: C, 51.54; H, 4.36; culture medium for 30 min at 37 . After washed twice with
N, 18.02; O, 6.89; S, 9.22. MS m/z (%) 634.13 (M − H, 100). PBS, the samples were incubated with 5 ꢁM Fluoꢀ3 AM for 30
1H NMR (400 MHz, D2O : DMSOꢀd6 = 7 : 3, pH =7.5 ): δ 8.17 (s, min at 37 . Followed by three washes with PBS. Cells were
4H), 7.89 – 7.82 (m, 4H), 7.69 (dd, J = 11.7, 4.1 Hz, 8H), 7.64 (s, suspended in Ca2+ꢀfree PBS (0.5 mL) and Fluoꢀ3 fluorescence
2H), 3.18 (d, J = 7.3 Hz, 6H), 3.09 (s, 6H).
intensity was analysed by flow cytometry.
Determination of structure of Ga(III) complexes.
Intracellular ROS measurements.
X−ray diffraction data of Ga(III) complexes were collected by a Intracellular ROS generation was measured by Flow Cytometer
Bruker SMART Apex II CCD diffract meter using according to the specification of the Reactive oxygen species
graphite−monochromatic Mo−Kα (λ = 0.71073 Å) radiation at assay kit (Beyotime, Suzhou). NCI−H460 cells were incubated
296.15 K, and then adsorption corrections with SADABS. The with 25 ꢁM of L1ꢀL3 and C1−C3 in serum−free cell culture
structures were solved and refined against F2 by full−matrix medium for 30 min at 37 . The cells were incubated with 2
least−squares methods using the Olex2 software. 53 C, N, Ga ꢁM H2DCF
−DA for 30 min at 37
℃
. After washed twice with
and O atoms were refined anisotropic ally. H atoms were added serum free medium, the samples were assayed by Flow
in theoretically ideal positions by Olex2. The Crystal Data of Cytometer with excitation wavelength at 488 nm and emission
three Ga(III) complexes are showed in Table 1. Selected bond wavelength at 525 nm. Data were analysed by FlowJo software.
lengths (Ẳ) and angles (deg) are showed in Table S1. The
The change of mitochondrial membrane potential assay.
crystal structure analysis data can get from Cambridge
Intracellular change of mitochondrial membrane potential was
Crystallographic
http://www.ccdc.cam.ac.uk/data_request/cif.
Data
Centre
via
observed by fluorescent inverted microscope according to the
specification of the JC−I assay kit (Beyotime, Suzhou). 2mL of
1 × 105 cells/mL were seeded in 6 well plates and cultivated for
Cyclic voltammetry.
Cyclic voltammetry was performed using
electrochemical workstation (Shanghai Chenhua Instruments, C1−C3 (5 ꢁM) for 30 min at 5% CO2 and 37
a
CHI660E 24 h before treatment, and then incubated with the L1ꢀL3 and
. The cells were
℃
China). Glassy carbon, aqueous Ag/AgCl and Pt wire were incubated with 1 ꢁM 1 µg/mL of 5, 5, 6, 6′−tetrachloro−1, 1', 3,
used for working, reference and auxiliary electrode, 3′−tetraethyl−imidacarbocyanineiodide iodide (JC−1) for 30
respectively. All complexes were at 1 mM concentration in min at 37
DMSO/H2O (70:30 v/v). The supporting electrolyte was
℃. After washed twice with cell culture media, the
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cells were detected by flow cytometry. Data were analysed by
FlowJo software.
Redoxꢀinactive Ga(III) complexes cannot produce ROS.32
However, substantial evidence has indicated that intracellular
ROS levels are significantly raised after gallium complex
Western blot analysis.
49, 50
treatment.8,
Electrochemical studies have shown that
5 mL of 5×105 cells/mL NCI−H460 cells were seeded in 10 cm
plates and cultivated at least 12 h before treatment. Cells were
incubated with 5 ꢁM of C1−C3 in cell culture medium for 12 h
Ga(III) complexes do not possess redox properties, whereas
Fe(III) complexes exhibit good redox activity. Because the
stability constant of the Fe(III)ꢀthiosemicarbazone complexes is
higher than that of the gallium(III) complexes, we speculated
that some gallium ions of the complexes were replaced by
intracellular iron ions to produce ROS. Ga(III) complexes
initiated the release of intracellular Ca2+, which led to the
increase in ROS levels and collapse of mitochondrial
membrane potential. In conclusion, the Ga (III) complexes
promoted apoptosis as a result of a combination of multiple
apoptotic pathways such as Ca2+ signaling triggers and ROSꢀ
mediated mitochondrial pathways. These results may contribute
to the development of novel Ga(III) anticancer agents.
at 37
in a humidified atmosphere of 5% CO2/95% air in an
incubator. NCI−H460 cells were cracked in lysis buffer.
Insoluble substance was separated by centrifuge for 30 min at
10,000g. Protein concentration was determined by the BCA
assay kit (Beyotime, Suzhou). SDS–polyacrylamide gel
electrophoresis was done by loading equal amounts of sample
total proteins in each canal. Proteins were then metastasized to
vinylidene difluoride membranes (Millipore) and blocked with
5% defatted milk in TBST buffer for 1 h. The membranes were
incubated with 1:1000 dilutions in 5% defatted milk of primary
antibodies for 12h at 4 . After being washed for two or three
times with TBST for 30 min, the membranes were incubated
with secondary antibodies at 1:1000 dilutions for 1 h at 25
and then washed for two or three times with TBST. The
immunoreactivity was visualized by Amersham ECL Plus
(Amersham) western blotting detection. The β−actin was used
for detect amount of proteins of each lane. Quantitative analysis
of electrophoretic results was performed by ImageꢀPro
software. The gray value of the target protein is equal to the
ratio of its IOD to the β−actin’s IOD value.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by National Nature Science
Foundations of China (81571812), Priority Academic Program
Development of Jiangsu Higher Education Institutions
(1107047002), the Fundamental Research Funds for the Central
Universities and Postgraduate Research& Practice Innovation
Program of Jiangsu Province (KYCX17_0135). The research
was also supported by the Scientific Research Foundation of
Graduated School of Southeast University (YBJJ1787).
Cell apoptosis assay.
The apoptotic events induced by the Ga(III) complexes
(C1−C3) were assayed by Flow Cytometer (BD, Shanghai)
according to the specification for the Annexin V−FITC
Apoptosis Detection Kit (BD, Shanghai). In this experiment,
2mL of 1 × 105 cells/mL were seeded in 6 well plates and
cultivated for 24 h, which were incubated with the C1−C3 (5
ꢁM) for 12 h at 5% CO2 and 37 . The NCI−H460 cells were
suspended in 200 µL binding buffer, then 5ꢁL of Annexin V
and PI were added to each sample. After 15 min incubation at
25 , the samples were assayed by Flow Cytometer. Cell
apoptosis rate were analysed by FlowJo software.
Notes and references
‡
CCDC 1488960ꢀ1488962 contain the supplementary
crystallographic data for this paper. Selected Bond Lengths (Ẳ)
and Angles (deg). The UVꢀVisible absorption of ligands and Ga
complexes.
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Our results revealed that the anticancer activity of these three
Ga(III) compounds are significantly higher than those of their
corresponding ligands. Modifying at the N4 position of the
ligand with lipotropic groups can also increase antitumor
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Ga (III) complexes promoted apoptosis as a result of a combination of multiple apoptotic
pathways.