Polyhedron 156 (2018) 320–331

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Polyhedron
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The induction of apoptosis in BEL-7402 cells by an iridium(III) complex

through lysosome–mitochondria pathway
Yang-Jie Wang a, Qiao-Yan Yi a, Wen-Yao Zhang a, Fan Du a, Miao He a, Yun-Jun Liu a,b,⇑
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b
Guangdong Engineering Research Center for Lead Compounds & Drug Discovery, Guangzhou 510006, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A new iridium(III) polypyridyl complex [Ir(ppy)2(DPBD)](PF6) (ppy = 2-phenylpyridine, DPBD = 4-(dipyr-
Received 4 September 2018 ido[3,2-a:20 ,30 -c]phenazin-11-yl)benzene-1,2-diamine, Ir-1) was synthesized and characterized by ele-
Accepted 19 September 2018 ment analysis, ESI-MS, IR, 1H NMR and 13C NMR. The cytotoxicity in vitro of the complex against
Available online 28 September 2018
cancer BEL-7402, A549, Eca-109 and normal LO2 cells was evaluated by 3-(4,5-dimethylthiazole)-2,5-
diphenyltetraazolium bromide (MTT) method. The IC50 values of the complex toward BEL-7402, A549
Keywords: and Eca-109 are 13.8 ± 0.7, 14.5 ± 0.6 and 15.2 ± 2.4 mM, respectively. The apoptosis was studied with
Iridium(III) complex
acridine orange (AO) and ethidium bromide (EB) staining method and comet assay. The reactive oxygen
Apoptosis
Intracellular Ca2+ levels
species including superoxide anion and NO, the location of the complex at lysosomes, lysosomal mem-
Lysosomes brane permeabilization, location of the complex at mitochondria and the change of mitochondrial mem-
Bcl-2 family proteins brane potential were assayed under a fluorescent microscope. The complex can enhance the intracellular
Ca2+ levels and induce a release of cytochrome c. In addition, the complex can cause autophagy and
upregulate the expression of LC-3 and Beclin-1 proteins. The complex inhibits the cell growth at S phase.
The effects of the complex on the expression of caspase 3 and Bcl-2 family proteins were explored. The
results show that the complex induces apoptosis in BEL-7402 cells through lysosome–mitochondrial dys-
function pathway, which was accompanied by the regulation of Bcl-2 family proteins.
Ó 2018 Elsevier Ltd. All rights reserved.

1. Introduction attention. In recent years, organometallic iridium complexes have

One of the features of cancer is the rapid generation of abnor-
mal cells that grow beyond their usual boundaries, which can then
invade adjoining parts of the body and spread to other organs. Plat-
inum based drugs are effectively used in the treatment of various
types of cancer [1–4]. Unfortunately, cisplatin exhibits some draw-
backs including severe side effects [5], drug resistance [2] and low
water-solubility [6], which seriously limit its clinical applications.
These limitations have motivated to search for other transition
metal complexes with beneficial biological properties, wide reac-
tivity ranges, and lower systemic toxicities [7–9]. Among these
metal complexes, anticancer activity of ruthenium complexes has
made great achievement [10–15]. On the other hand, cyclometa-
lated iridium(III) complexes have rich photophysical properties,
e.g., high quantum yields, large Stokes shifts, long lived lumines-
cence, good photostability and cell permeability [16], the anti-
cancer activity of iridium(III) complexes have also attracted great
⇑ Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical
University, Guangzhou 510006, PR China.
E-mail address: lyjche@gdpu.edu.cn (Y.-J. Liu).
emerged as potential candidates for new metallo-anticancer
drugs, a number of iridium(III) complexes have shown interesting
biological effect [17–23]. Complex [Ir(ppy)2(FBPIP)]+ (ppy = 2-phe-
nylpyridine, FBPIP = 2-(4-formyl)benzeno[4,5-f][1,9]phenanthro-
line) showed no cytotoxic activity against SGC-7901 cells.
However, upon irradiation, the complex displayed potent anti-pro-
liferative activity toward SGC-7901 cells, with a low IC50 value of
6.1 ± 0.6 mM, and showed selectivity between cancer and normal
cells [24]. Complex [Ir(ppy)2(BTCP)]+ (BTCP = 2-bicyclo[2.2.1]
hept-5-en-yl-1H-1,3,7,8-tetraazacyclopenta[l]phenanthrene) inhi-
bits the cell growth at G0/G1 phase in SGC-7901 cells. The complex
induces apoptosis through a ROS-mediated mitochondrial dysfunc-
tion pathway [25]. Mao et al. reported that iridium(III) complex [Ir
(dfppy)2(4,40 -bis(chloromethyl)-2,20 -bipyridine)]+ (dfppy = 2-(2,4-
difluorophenyl)pyridine) induced caspase-dependent apoptosis
through mitochondrial damage, cellular ATP depletion, mitochon-
drial respiration inhibition and reactive oxygen species (ROS)
elevation [16]. To obtain more information on anticancer activity
of iridium(III) complex, in this paper, a new iridium(III) complex
[Ir(ppy)2(DPBD)](PF6) (ppy = 2-phenylpyridine, DPBD = 4-(dipyr-
ido[3,2-a:20 ,30 -c]phenazin-11-yl)benzene-1,2-diamine, Scheme 1)

https://doi.org/10.1016/j.poly.2018.09.057
0277-5387/Ó 2018 Elsevier Ltd. All rights reserved.
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
Scheme 1. Synthetic route of complex [Ir(ppy)2(DPBD)]PF6.
was synthesized and characterized by elemental analysis, ESI-MS,
IR, 1H NMR and 13C NMR. The cytotoxicity in vitro of the complex
against BEL-7402, A549, Eca-109 and normal LO2 cells was inves-
tigated by MTT method. The apoptosis, comet assay, reactive oxy-
gen species (ROS), mitochondrial membrane potential,
intracellular Ca2+ levels, release of cytochrome c, cell cycle arrest,
cell invasion and Bcl-2 family proteins expression were studied
in detail.
2. Experimental
2.1. Materials and methods
All reagents and solvents were purchased commercially and
used without further purification unless otherwise noted. Ultra-
pure MilliQ water was used in all experiments. DMSO and RPMI
1640 were purchased from Sigma. 1,10-Phenanthroline was
obtained from the Guangzhou Chemical Reagent Factory. Cancer
cell lines of BEL-7402 (human hepatocellular carcinoma), A549
(human lung carcinoma), Eca-109 (human esophagus cancer cell
line) and normal cell LO2 (human liver cell) were purchased from
the American Type Culture Company. IrCl33H2O was purchased
from the Kunming Institution of Precious Metals.
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer
240Q elemental analyzer. Electrospray ionization mass spectra
(ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA)
using acetonitrile as mobile phase. The spray voltage, tube lens off-
set, capillary voltage and capillary temperature were set at 4.50 kv,
30.00 V, 23.00 V and 200 °C, respectively, and the quoted m/z val-
ues are for the major peaks in the isotope distribution. 1H NMR
and 13C NMR spectra were recorded on a Varian-500 spectrometer
with DMSO-d6 as solvent and tetramethylsilane (TMS) as an inter-
nal standard at 500 MHz at room temperature. UV–Vis spectra
were recorded on a Shimadzu UV-3101PC spectrophotometer
and emission spectra were recorded on a Shimadzu RF-4500 lumi-
nescence spectrometer at room temperature.
2.2. Synthesis of [Ir(ppy)2(DPBD)](PF6) (Ir-1)
A mixture of cis-[Ir(ppy)2Cl]2 (0.15 g, 0.14 mmol) [26] and DPBD
(0.11 g, 0.28 mmol) [27] in a mixture of 42 mL dichloromethane
and methanol (VCH2Cl2:VCH3OH = 2:1) was refluxed under argon for
6 h to give a clear red brown solution. Upon cooling, a brown red
precipitate was obtained by dropwise addition of saturated aque-
ous NH4PF6 solution with stirring at room temperature for 2 h.
The crude product was purified by column chromatography on
neutral alumina with a mixture of CH2Cl2–acetone (1:3, v/v) as elu-
ent. The red band was collected. The solvent was removed under
reduced pressure and a red powder was obtained. Yield: 76%. Anal.
Calc. for C46H32F6N8PIr: C, 53.37; H, 3.12; N, 10.83. Found: C, 53.48;
H, 3.34; N, 10.68%. IR (KBr, cm1): 3367m, 3043w, 2038w, 1607m,
321
1583m, 1532m, 1492s, 1479s, 1438m, 1416s, 1358s, 1316s,
1268m, 1164m, 1079m, 843s, 758s, 731m, 557s. 1H NMR (DMSO-
d6): 9.54 (d, 1Ha, J = 8.0 Hz), 9.49 (d, 1Hj, J = 8.0 Hz), 8.33–8.27
(m, 4H3,14,b,i), 8.24 (d, 2Hc,h, J = 5.0 Hz), 8.12–8.05 (m, 2H8,19),
7.97 (d, 2H1,12, J = 8.0 Hz), 7.90 (d, 3Hn,7,18, J = 7.0 Hz), 7.70 (d,
2H4,15, J = 5.5 Hz), 7.68 (d, 1Hl, J = 6.0 Hz), 7.26 (d, 1Hk, J = 5.0 Hz),
7.06 (d, 4H9,10,20,21, J = 8.0 Hz), 6.97 (t, 2H2,13, J = 6.0 Hz), 6.70 (d,
1Hp, J = 8.0 Hz), 6.30 (dd, 2Hs,t, J = 7.0, J = 7.5 Hz), 3.47 (s, 4HNH2).
13
C NMR (DMSO-d6, 125 MHz): 166.84 (Ca,j), 151.67 (C1), 151.34
(C12), 149.73 (C7), 149.61 (C18), 149.29 (Cw), 148.89 (Cx), 144.59
(C5), 144.06 (C6), 142.82 (Cq), 141.08 (Cr), 139.97 (Cm), 138.87
(Cb), 138.40 (Ci), 137.29 (Co), 134.95 (C3), 134.65 (C14), 131.52
(Cc), 131.22 (Ch), 130.69 (Cd), 130.58 (Cg), 130.36 (Ce,f), 129.37
(C4,15), 128.37 (Ck), 126.14 (Cl), 125.16 (C9,20), 123.90 (C10,21),
122.56 (C2), 121.98 (C13), 120.06 (Cn), 117.87 (Ct), 115.02 (Cs),
113.34 (Cp). ESI-MS (CH3CN): m/z 889.3 ([MPF6]+).
2.3. Cytotoxic activity assay in vitro
3-(4,5-Dimethylthiazole)-2,5-diphenyltetraazolium bromide
(MTT) method [28] was used to evaluate the cytotoxicity in vitro
of the complex against cancer cells. Cells were placed in 96-well
microassay culture plates (8  103 cells per well) and grown over-
night at 37 °C in a 5% CO2 incubator. The tested complexes were
dissolved in DMSO and then added to the wells to achieve final
concentrations ranging from 106 to 104 M (final concentration
of DMSO of 0.05%, v/v). Negative control wells were prepared by
addition of culture medium (100 lL). The plates were incubated
at 37 °C in a 5% CO2 incubator for 48 h. Upon completion of the
incubation, stock MTT dye solution (20 lL, 5 mg/mL1) was added
to each well. After 4 h, buffer (100 lL) containing dimethylfor-
mamide (50%) and sodium dodecyl sulfate (20%) was added to sol-
ubilize the MTT formazan. The optical density of each well was
measured with a microplate spectrophotometer at a wavelength
of 490 nm. The 50% inhibitory concentration (IC50) is defined as
the concentration to reduce the size of the cell population by
50%. The IC50 values were determined by the percentage of cell via-
bility versus concentration on a logarithmic graph and reading off
the concentration at which 50% of cells remain viable relative to
the control. Each experiment was repeated at least three times to
obtain the mean values.
2.4. Apoptosis assay by AO/EB staining method
BEL-7402 cells were seeded onto chamber slides in six-well
plates at a density of 2  105 cells per well and incubated for
24 h. The cells were cultured in RPMI (Roswell Park Memorial
Institute) 1640 supplemented with 10% of fetal bovine serum
(FBS) and incubated at 37 °C in 5% CO2. The medium was removed
and replaced with medium (final DMSO concentration, 0.05% v/v)
containing the complex (3.75 and 7.5 lM) for 24 h. The medium
322 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
was removed again, and the cells were washed with ice-cold phos-
phate buffer saline (PBS), and fixed with formalin (4%, w/v). Cell
nuclei were counterstained with acridine orange (AO) and ethid-
ium bromide (EB) (AO: 100 mg/mL, EB: 100 mg/mL) for 10 min.
The cells were observed and imaged under a fluorescence micro-
scope (Nikon, Yokohama, Japan) with excitation at 350 nm and
emission at 460 nm.
2.5. DNA damage assay
DNA damage was investigated by means of comet assay. BEL-
7402 cells in culture medium were incubated with 3.75 and
7.5 lM of complex for 24 h at 37 °C. The cells were harvested by
a trypsinization process at 12 and 24 h. A total of 100 lL of 0.5%
normal agarose in PBS was dropped gently onto a fully frosted
microslide, covered immediately with a coverslip, and then placed
at 4 °C for 10 min. The coverslip was removed after the gel had
been set. A mixture of 50 lL of the cell suspension (200 cells/lL)
mixed with 50 lL of 1% low melting agarose was preserved at
37 °C. A total of 100 lL of this mixture was applied quickly on
top of the gel, coated over the microslide, covered immediately
with a coverslip, and then placed at 4 °C for 10 min. The coverslip
was again removed after the gel had been set. A third coating of
50 lL of 0.5% low melting agarose was placed on the gel and
allowed to place at 4 °C for 15 min. After solidification of the agar-
ose, the coverslips were removed, and the slides were immersed in
an ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris,
90 mM sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and
10% DMSO) and placed in a refrigerator at 4 °C for 2 h. All of the
above operations were performed under low lighting conditions
to avoid additional DNA damage. The slides, after removal from
the lysis solution, were placed horizontally in an electrophoresis
chamber. The reservoirs were filled with an electrophoresis buffer
(300 mM NaOH, 1.2 mM EDTA) until the slides were just immersed
in it, and the DNA was allowed to unwind for 30 min in elec-
trophoresis solution. Then the electrophoresis was carried out at
25 V and 300 mA for 20 min. After electrophoresis, the slides were
removed, washed thrice in a neutralization buffer (400 mM Tris,
HCl, pH 7.5). Cells were stained with 20 lL of EB (20 lgmL1) in
the dark for 20 min. The slides were washed in chilled distilled
water for 10 min to neutralize the excess alkali, air-dried and
scored for comets by fluorescent microscopy.
2.6. Cell cycle arrest by flow cytometry
BEL-7402 cells were seeded into six-well plates (Costar, Corning
Corp, New York) at a density of 2  105 cells per well and incubated
for 24 h. The cells were cultured in RPMI 1640 supplemented with
10% of FBS and incubated at 37 °C in 5% CO2. The medium was
removed and replaced with medium (final DMSO concentration,
0.05% v/v) containing complex (7.5 or 15.0 lM). After incubation
for 24 h, the cell layer was trypsinized and washed with cold PBS
and fixed with 70% ethanol. Twenty mL of RNAse (0.2 mg/mL) and
20 mL of propidium iodide (0.02 mg/mL) were added to the cell sus-
pensions and they were incubated at 37 °C for 30 min. Then the
samples were analyzed with a FACSCalibur flow cytometry. The
number of cells analyzed for each sample was 10 000.
2.7. Reactive oxygen species (ROS) detection
BEL-7402 cells were seeded into six-well plates at a density of
2  105 cells per well and incubated for 24 h. The cells were cul-
tured in RPMI 1640 supplemented with 10% of FBS and incubated
at 37 °C in 5% CO2. The medium was removed and replaced with
medium (final DMSO concentration, 0.05% v/v) containing different
concentration of complex for 24 h. The medium was removed
again. The fluorescent dye 20 ,70 -dichlorodihydrofluorescein diac-
etate (DCHF-DA, 10 lM) was added to the medium to cover the
cells. The treated cells were then washed with cold PBS–EDTA
twice, collected by trypsinization and centrifugation at 1500 rpm
for 5 min, the cell pellets were suspended in PBS–EDTA and imaged
under a fluorescent microscope.
2.8. Localization assay of Ir-1 in the lysosomes
BEL-7402 cells in 12-well plates were cultured overnight. The
cells were treated with the complex (15.0 mM) for 5 h. The lyso-
somes were labeled and tracked using the lysosomotropic probe
Lyso Tracker Red (LTR) (50 nM) for 30 min in dark. After the cells
were washed with PBS twice, the stained cells were analyzed
under a fluorescence microscope.
2.9. Lysosomal membrane permeability assay
BEL-7402 cells in 12-well plates were cultured overnight. The
cells were treated with the complex (15.0 mM) for 1, 2 and 5 h.
After the cells were washed with PBS twice, the cells were stained
with acridine orange (AO, 1 mg/mL) for 30 min in dark. The stained
cells were imaged under a fluorescence microscope.
2.10. Localization assay of the complex in the mitochondria
BEL-7402 cells were placed in 12-well microassay culture plates
(4  104 cells per well) and grown overnight at 37 °C in a 5% CO2
incubator. 7.5 lM of Ir-1 was added to the wells at 37 °C in a 5%
CO2 incubator for 8 h and further co-incubated with MitoTracker
Deep Red FM (150 nM) at 37 °C for 30 min. Upon completion of
the incubation, the wells were washed three times with ice-cold
PBS. After discarding the culture medium, the cells were imaged
under a fluorescence microscope.
2.11. Mitochondrial membrane potential assay
BEL-7402 cells were treated for 24 h with different concentra-
tion of the complex in 12-well plates and were then washed three
times with cold PBS. The cells were detached with trypsin–EDTA
solution. The collected cells were incubated for 20 min with
1 lg/mL of JC-1 (5,50 ,6,60 -Tetrachloro-1,10 ,3,30 -tetraethyl-imidacar-
bocyanine iodide) in culture medium at 37 °C in the dark.
Cells were immediately centrifuged to remove the supernatant.
Cell pellets were suspended in PBS and imaged under fluorescence
microscope. The ratio of red/green fluorescence intensity was
determined by flow cytometry.
2.12. Detection of Ca2+ levels
BEL-7402 cells treated with 15.0 mM of Ir-1 for 2, 4 and 8 h were
incubated with 2.5 lM Fluo-3AM (which can cross the cell mem-
brane and be cut into Fluo-3 by intracellular esterase) for 30 min
at 37 °C, the cells were washed three times and incubated for
20 min at 37 °C to ensure that Fluo-3AM (which can specifically
bind to Ca2+ and has a strong fluorescence with an excitation wave-
length of 488 nm) has been completely transformed into Fluo-3 in
the cells. Then, the cells were washed two times with PBS and
stained with 5 lgmL1 DAPI solution. Finally, ImageXpress Micro
XLS system was used to observe fluorescence, and Multi Wave-
length Cell Scoring module was used to analyze the data. The inte-
grated intensity/cell which represented the fluorescence intensity
of each cell was used to measure the levels of Ca2+. The fluores-
cence intensity of each cell was calculated as the total fluorescence
intensity divided by the number of cells.
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
2.13. Examination release of cytochrome c by immunofluorescence
BEL-7402 cells were seeded in a 12-well plate and incubated
overnight. Then cells were treated with 15.0 lM of Ir-1 for 24 h.
Subsequently, the cells were fixed with ice-cold immunol staining
fix solution for 30 min at room temperature. After blocking cells
with immunol staining blocking buffer for 1 h, the cells were trea-
ted with the primary antibody against cytochrome c (1:50 dilution)
overnight at 4 °C. Next, the plate was washed three times with
immunol staining wash buffer and probed with Alexa Fluor 488-
Labeled Goat Anti-Mouse IgG (1:500 dilution) in the dark for 1 h
at room temperature. Finally, the cells were washed with immunol
staining wash buffer three times and the cell nuclei were stained
with DAPI. The images were obtained using ImageXpress Micro
XLS system, and Multi Wavelength Cell Scoring module was used
to analyze the data. The integrated intensity/cell which represents
the fluorescence intensity of each cell was used to measure the
release of cyto-c. The fluorescence intensity of each cell was calcu-
lated as the total fluorescence intensity divided by the number of
cells.
2.14. Autophagy induced by complex
BEL-7402 cells were seeded onto chamber slides in 12-well
plates and incubated for 24 h. The cells were cultured in RPMI
1640 supplemented with 10% of FBS and incubated at 37 °C in 5%
CO2. The medium was removed and replaced with medium (final
DMSO concentration, 0.05% v/v) containing different concentration
of complex for 24 h. The medium was removed again, and the cells
were washed with ice-cold PBS twice. Then the cells were stained
with MDC (monodansylcadaverine) solution (50 mM) for 10 min
and washed with PBS twice. The cells were observed and imaged
under fluorescence microscope. The effect of the complex on the
expression of LC3 and Beclin-1 proteins was assayed by Western
blot.
2.15. The effect of autophagy on cell viability
Cell viability was studied using the MTT method. Cells were
placed in 96-well microassay culture plates (8  104 cells per well)
and cultured overnight at 37 °C in a 5% CO2 incubator. The cells
were pretreated with or without 3-methyladenine (3-MA, 3 mM)
for 3 h, followed by different concentration of Ir-1 for 24 h. After
incubation, cells were incubated with MTT (0.5 mg/mL) for 4 h at
37 °C. Upon completion of the incubation, 100 lL DMSO was added
to solubilize the MTT formazan. The optical density of each well
was then measured with a microplate spectrophotometer at a
wavelength of 490 nm. The viability (%) of cell growth was calcu-
lated by the formula: (A490 (treatment group)/A490 (control))  100, A490
(treatment group) is the mean OD value of cells treated with the various
ruthenium complexes and A490 (control) is the mean OD value of
untreated cells. Each experiment was repeated at least three times
to obtain the mean values.
2.16. Matrigel invasion assay
The BD Matrigel invasion chamber was used to investigate the
cell invasion according to the manufacturer’s instructions. BEL-
7402 cells (4  104) in serum free media and different concentra-
tion of the complex were seeded in the top chamber of the two
chamber Matrigel system. RPMI-1640 (20% FBS) was added as
chemo-attractant into the lower chamber. Cells were allowed to
invade for 24 h. After incubation, non-invading cells were removed
from the upper surface and cells on the lower surface were fixed
with 4% paraformaldehyde and stained with 0.1% of crystal violet.
The membranes were photographed and the invading cells were
323
counted under a light microscope. The mean values from three
independent assays were calculated.
2.17. The expression of caspases 3, 8 and Bcl-2 family proteins
BEL-7402 cells were seeded in 3.5 cm dishes for 24 h and incu-
bated with 15.0 lM of the complex in the presence of 10% FBS. The
cells were harvested in lysis buffer. After sonication, the samples
were centrifuged for 20 min at 13,000 g. The protein concentration
of the supernatant was determined by BCA (bicinchoninic acid)
assay. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
was done loading equal amount of proteins per lane. Gels were
then transferred to poly (vinylidene difluoride) membranes (Milli-
pore) and blocked with 5% non-fat milk in TBST (20 mM Tris–HCl,
150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer for 1 h. Then the
membranes were incubated with primary antibodies at 1:5,000
dilutions in 5% non-fat milk overnight at 4 °C, and washed four
times with TBST for a total of 30 min. After which the secondary
antibodies conjugated with horseradish peroxidase at 1:5000 dilu-
tion for 1 h at room temperature and then washed four times with
TBST. The blots were visualized with the Amersham ECL Plus Wes-
tern blotting detection reagents according to the manufacturer’s
instructions. To assess the presence of comparable amount of pro-
teins in each lane, the membranes were stripped finally to detect
the b-actin. The gray values were calculated with BandScan.
2.18. Data analysis
All data were expressed as mean ± SD. Statistical significance
was evaluated using t-tests. Differences were considered to be sig-
nificant when the *P value was less than 0.05.
3. Results and discussion
3.1. Synthesis and characterization
Ligand DPBD was prepared according to the literature [27]. The
complex Ir-1 was synthesized by the reaction of cis-[Ir(ppy)2Cl]2
with DPBD in a mixture of dichloromethane and methanol. The
crude product was purified by column chromatography on neutral
alumina. The obtained complex was characterized by elemental
analysis, IR, ESI-MS, 1H NMR and 13C NMR. In the IR spectra, the
peaks of 3367 cm1 and 3043 cm1 for Ir-1 are assigned the
NAH and CAH stretching vibration. In the ESI-MS spectra, the peak
at a m/z value of 889.3 is assigned to the ion peak of [M-PF6]+. In
the 1H NMR spectra of complex, the single peak of 3.47 (s, 4H)
for ANH2 group was observed. The UV–Vis and luminescence of
Ir-1 (5 mM) in PBS solution is shown in Fig. 1a, the maximum
absorbance of Ir-1 appears at 204 nm, and the complex can emit
luminescence in PBS solution at ambient temperature, with a max-
imum appearing at 576 nm (Fig. 1b). The stability of the complex in
PBS solution was investigated by UV–Vis spectra at 0 and 24 h, no
obvious changes in the absorbance was observed (data no present),
indicating that the complex is stable in PBS solution.
3.2. Cytotoxic activity in vitro
The cell viability of BEL-7402, A549, Eca-109 and normal LO2
cell induced by the different concentration of complex Ir-1 and
ligand for 48 h was assayed by MTT method. Cisplatin was used
as a positive control. The IC50 values are listed in Table 1. As we
expect, ligand DPBD has no cytotoxic activity against the selected
cell lines. However, when ligand bonded metal to form complex,
the cytotoxicities in vitro are enhanced. The complex shows high
cytotoxic activity toward BEL-7402, A549 and Eca-109 with an
324 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331

Fig. 1. UV–Vis (a) and luminescence spectra (b) of 5 mM of the complex in PBS solution at room temperature.

Table 1 3.4. DNA damage studies

IC50 values of the complex Ir-1 toward selected cell lines.

Complex BEL-7402 A549 Eca-109 LO2 The apoptosis can also be confirmed by DNA damage. The DNA
QPBD >200 >200 >200 >200
damage was studied by comet assay through the single cell gel
Ir-1 13.8 ± 0.7 14.5 ± 0.6 15.2 ± 2.4 25.0 ± 2.7 electrophoresis. DNA fragmentation is a hallmark of apoptosis,
Cisplatin 11.5 ± 1.3 7.5 ± 1.3 – 9.2 ± 1.4 mitotic catastrophe or both [29,30]. As shown in Fig. 3, in the con-
trol (a), the cells fail to show any comet-like appearance. Exposure
of BEL-7402 cells to 3.75 (b) and 7.5 mM (c) of Ir-1 for 24 h caused a

IC50 value of 13.8 ± 0.7, 14.5 ± 0.6 and 15.2 ± 2.4 mM, respectively.
Interestingly, the complex exhibits relatively low cytotoxic activity
against normal LO2 cells. Comparing the IC50 values, we find that
the complex displays lower inhibitory effect against BEL-7402,
A549 and Eca-109 cell growth than cisplatin and [Ir(ppy)2(BTCP)]+
[25] under the same conditions. Because BEL-7402 cells are sensi-
tive to the complex, this cell was selected for undergoing the fol-
lowing experiments.

3.3. Apoptosis studies with AO/EB methods Fig. 3. Comet assay of BEL-7402 (a) exposure to 3.75 (b) and 7.5 (c) mM of Ir-1 for
24 h. The scale bars for all the figures are 200 .

Apoptosis known as a programmed cell death is a fundamental

and inherent biological event for maintaining the balance between
cellular proliferation and death, evasion of apoptosis constitutes a
characteristic feature of human cancers. The morphological
changes of BEL-7402 cells treated by the complex were investi-
gated with acridine orange (AO)/ethidium bromide (EB) double
staining method. It is well known that AO stained viable and apop-
totic cells and emits green fluorescence, EB stained necrotic cells
and emits red fluorescence. As shown in Fig. 2, in the control (a),
the living cells with intact cell nuclei show bright green fluores-
cence. After BEL-7402 cells were incubated with 3.75 (b) and
7.5 mM (c) Ir-1 for 24 h, the apoptotic cells with apoptotic features
such as blebbing, nuclear shrinkage and chromatin condensation
were found, simultaneously, the red necrotic cells were also
observed. The results indicate that complex Ir-1 can induce apop-
tosis in BEL-7402 cells.

Fig. 2. Apoptosis assays of BEL-7402 cells (a) exposure to 3.75 (b) and 7.5 mM (c) of Fig. 4. (A) The cell cycle distribution of BEL-7402 cells (black) exposure to 7.5 (red)
Ir-1 for 24 h and the cell nuclei were stained with AO/EB. The scale bars for all the and 15.0 mM (blue) of Ir-1 for 24 h. (B) The expression of proteins related to the cell
figures are 200 . cycle induced by different concentrations of the complex for 24 h. (Colour online.)
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331 325

considerable strand breakage on chromosomal DNA and led to the
appearance of an obscure ‘‘halo” around the nucleus of the BEL-
7402 cells. The statistically significant and well-formed comets
were observed which indicated severe DNA damage. The results
of the comet assay suggest that the DNA of a single cell underwent
degradation as a consequence of direct DNA damage or rapid apop-
tosis. Similar results were observed in other metal complexes
[13,31,32].
3.5. Cell cycle distribution detection
Cell cycle includes three phases: G0/G1, S and G2/M. The drugs
inhibit cancer cell growth through inducing cell cycle arrest at G0/
G1 or S or G2/M phase. The distribution of BEL-7402 cells in vari-
ous compartments during the cell cycle was analyzed by flow
cytometry in cells stained with propidium iodide. As shown in
Fig. 4A, in the control, the percentage in the cells at S is 11.06%.
Treatment of BEL-7402 cells with 7.5 and 15.0 mM of Ir-1 led to a
large increase of 6.95% and 12.24% in the cells at S phase, which
was accompanied by a relative reduction of 5.53% and 22.75% in
the cells at G2/M phase, respectively. These data reveal that the
complex inhibits the cell growth of BEL-7402 cells at S phase. In
our previous work [25], complex [Ir(ppy)2(BTCP)]PF6 inhibited
the cell growth at G0/G1 phase against SGC-7901 cells. Obviously,
complex [Ir(ppy)2(DPBD)]PF6 and [Ir(ppy)2(BTCP)]PF6 inhibit the
cell growth at the different phase. This may be caused by the differ-
ent structure of DBPD and BTCP and different cancer cells BEL-7402
and SGC-7901. The expression of the proteins CDK2 and Cyclin A2
related to the S phase was assayed by western blot. As shown in
Fig. 4B, treatment of BEL-7402 cells with different concentration
of Ir-1 for 24 h led to a down-regulation in the expression of
CDK2 and Cyclin A2 proteins, which further confirms the complex
induces cell cycle arrest at S phase in BEL-7402 cells.
3.6. Intracellular reactive oxygen species levels studies
Many potential anticancer and chemopreventive agents induce
apoptosis through mediated-ROS generation [33–35]. To detect
whether reactive oxygen species (ROS) are involved in apoptotic
mechanism caused by the complex, intracellular generation of
ROS was investigated using DCHF-DA as fluorescence probe.
Finally, this dye is oxidized by intracellular ROS to the highly fluo-
rescent compound, namely, 20 ,70 -dichlorofluorescein (DCF). The
fluorescent intensity of DCF is proportional to intracellular ROS
levels. As shown in Fig. 5A, in the control (a) or control + NAC (b,
5 mM), no obvious fluorescent points are observed. However, the
BEL-7402 cells were treated with 3.75 mM Ir-1 (c), 3.75 mM Ir-1
+ NAC (d), 7.5 mM Ir-1 (e) and 7.5 mM Ir-1 + NAC (f) for 24 h, a num-
ber of bright fluorescent points are found, which indicates that the
complex can increase intracellular ROS levels. In the present of
NAC, DCF fluorescent intensity decreases. This shows that NAC
inhibits the generation of ROS. To quantitatively observe the effect

Fig. 5. (A) Intracellular ROS was detected in BEL-7402 cells (a) exposure to NAC (b), Rosup (c, positive control) and different concentrations of the complex for 24 h, (d)
3.75 mM Ir-1; (e) 3.75 mM Ir-1 + NAC; (f) 7.5 mM Ir-1; (g) 7.5 mM Ir-1 + NAC. (B) The DCF fluorescent intensity was determined after BEL-7402 cells were treated with NAC and
different concentration of Ir-1 for 24 h by ImageXpress Micro XLS system. (C) The superoxide anion level was assayed after BEL-7402 cells (a) were exposed to 3.75 (b) and
7.5 mM (c) of Ir-1 for 24 h and the cells were stained with DHE. (D) DHE fluorescent intensity was determined after BEL-7402 cells were incubated with different
concentrations of the complex for 24 h. (E) The intracellular NO levels were detected after BEL-7402 cells (a) were exposed to 3.75 (b) and 7.5 mM (c) of Ir-1 for 24 h, and the
cells were stained with DAF-FMDA. (F) DAF-FMDA fluorescent intensity was determined after BEL-7402 cells were exposed to different concentrations of the complex for
24 h. *P < 0.05 represents significant differences compared with control. The scale bars for all the figures are 200 .
326 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331

of concentration of the complex on the ROS levels, the DCF fluores-
cent intensity was determined and shown in Fig. 5B, the DCF fluo-
rescent intensity increases with increasing the concentration of Ir-
1, moreover, in the present of NAC, the DCF fluorescent intensity
decreases. These data further demonstrate that the complex
enhances the intracellular ROS levels with a concentration-depen-
dent manner.
ROS include superoxide anion, hydroxyl radical and NO, etc. In
the assay of intracellular superoxide anion, dihydroethidium
(DHE) was used as fluorescent probe. After freely passing through

Fig. 6. (A) Location assay of Ir-1 (15.0 mM) in the lysosomes. (B) The lysosomal membrane permeabilization was studied after Ir-1 (15.0 mM) treated BEL-7402 cells (a) for 1
(b), 2 (c) and 5 h (d) and the cells were stained with AO. The scale bars for all the figures are 200 .

Fig. 7. (A) Location assay of Ir-1 in the mitochondria. (B) The mitochondrial membrane potential was determined under fluorescent microscope while BEL-7402 cells (a) were
treated with cccp (b, positive control), 3.75 (c) and 7.5 mM (d) of Ir-1 for 24 h. (C) The ratio of red/green fluorescence intensity was determined by flow cytometry while BEL-
7402 cells were incubated with NAC, 7.5 mM Ir-1, 7.5 mM Ir-1 + NAC, 15.0 mM Ir-1 and 15.0 mM Ir-1 + NAC for 24 h. *P < 0.05 represents significant differences compared with
control. The scale bars for all the figures are 200 .
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
plasma membrane, nonfluorescent DHE is oxidized by ROS to
ethidium, which intercalates with DNA and stains nuclei bright
red fluorescent. As shown in Fig. 5C, after treatment of BEL-7402
cells (a) with 3.75 (b) and 7.5 mM (c) of Ir-1 for 24 h, the more
bright red DHE fluorescent intensity was found, which indicated
that the complex can enhance the superoxide anion levels. To
investigate the effect of concentration of Ir-1 on the intracellular
superoxide anion levels, DHF fluorescent intensity was detected.
As shown in Fig. 5D, in the control, the DHE fluorescent intensity
is 19.40. After BEL-7402 cell was exposed to 3.75, 7.5 and
15.0 mM of Ir-1 for 24 h, the DHE fluorescent intensity increased,
which showed that the complex increases intracellular superoxide
anion in a concentration-dependent manner.
The intracellular NO levels were also determined using 3-
amino-4-aminomethyl-20 ,70 -difluorescein diacetate (DAF-FMDA)
as a fluorescent indicator of intracellular NO [36,37]. As shown in
Fig. 5E, treatment of BEL-7402 cells (a) with 3.75 (b) and 7.5 mM
(c) of Ir-1 for 24 h led to an increase of DAF-FMDA green fluores-
cence. The DAF-FMDA green fluorescent intensity was detected
using ImageXpress Micro XLS system and Multi Wavelength Cell
Scoring module. The DAF-FMDA fluorescent intensity increases
with increasing concentration of Ir-1, which suggests that the com-
plex can enhance the intracellular NO levels (Fig. 5F).
3.7. Location and lysosomal membrane permeabilization (LMP)
Lysosomes, a kind of spherical vesicles containing various
enzymes that are able to break down almost all kinds of biomole-
cules, including proteins, nucleic acids, lipids, carbohydrates, and
cellular debris, act as the waste disposal system of eukaryotes
through degradation of unwanted molecules in the cytoplasm
[38–40]. The location of the complex in the lysosomes was inves-
tigated using LysoTracker Red as fluorescent probe. It is well
known that LysoTracker Red, a fluorescent weak base, could accu-
mulate in acidic organelles and successfully stain lysosomes in liv-
ing cells [41]. After BEL-7402 cells were treated with 15.0 mM of Ir-
1 for 5 h, the cells were imaged under fluorescent microscope. As
shown in Fig. 6A, the lysosomes were stained red fluorescence,
BEL-7402 cells incubated with Ir-1 showed bright-green intracel-
Fig. 8. (A) Intracellular Ca2+ levels were assayed after BEL-7402 cells (a) were
exposed to 15.0 mM of Ir-1 for 2 (b), 4 (c) and 8 h (d). (B) The integrated fluorescent
intensity/cell was determined after BEL-7402 cells were treated 15.0 mM of Ir-1 for
2, 4 and 8 h. *P < 0.05 represents significant differences compared with control. The
scale bars for all the figures are 200 .
327
lular fluorescence, which merged very well with the red fluores-
cence emitted from the lysosomal tracker, demonstrating that
the complex locates at the lysosomes. Lysosomal membrane per-
meabilization can cause the release of lysosomal proteases, e.g.,
cathepsin B, from lysosomes to cytosol to promote apoptosis
[42]. The lysosomal membrane permeabilization induced by the
complex was studied using AO as fluorescent probe. AO is a lysoso-
motropic metachromatic fluorochrome. When excited with blue
light, AO emits red fluorescence at high concentrations (when it
is present in lysosomes) and green fluorescence at low concentra-
tions (when it is present in the cytosol and the nucleus) [43]. As
shown in Fig. 6B, after treatment of BEL-7402 cells (a) with
15.0 mM of Ir-1 for 1 (b), 2 (c) and 5 h (d), the lysosomal permeabil-
itztion can be observed, as indicated by increased numbers of ‘‘pale
cells,” i.e., cells with reduced numbers of AO-accumulating lyso-
somes. These results demonstrated that Ir-1 can induce lysosomal
membrane permeabilization in BEL-7402 cells.
3.8. Location assay of the complex and the changes of mitochondrial
membrane potential
The complex can enhance the intracellular ROS level, this
prompts us to evaluate the changes of mitochondrial membrane
potential. Mitochondria play a key role in the intrinsic apoptosis
pathway, and loss of the mitochondrial membrane potential
(DWm) appears to be a universal event during the intrinsic apop-
tosis pathway [44]. To investigate the location of the complex in
the mitochondria, Mito TrackerÒ Deep Red FM (ThermoFisher,
100 nM) was used as red fluorescent probe. As shown in
Fig. 9. (A) The release of cytochrome c was assayed after BEL-7402 cells (a) were
exposed to 7.5 mM of Ir-1 for 24 h. (B) The integrated fluorescent intensity/cell was
determined after different concentration of Ir-1 treated BEL-7402 cells for 12 and
24 h. *P < 0.05 represents significant differences compared with control. The scale
bars for all the figures are 200 .
328 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
Fig. 7A, in the control, the mitochondria are stained in bright red.
After BEL-7402 cells were exposed to 7.5 mM of Ir-1 for 8 h, the
complex emits green fluorescence. The complete merge of the
red and the green fluorescence indicates that the complex enters
into the mitochondria. The changes in the mitochondrial mem-
brane potential were also assayed under a fluorescent microscope
using JC-1 as fluorescent probe. It is well known that JC-1 emits
red fluorescence corresponding to high mitochondrial membrane
potential, and JC-1 emits green fluorescence corresponding to low
mitochondrial membrane potential. As shown in Fig. 7B, in the
control (a), JC-1 emits red fluorescence. After the treatment of
BEL-7402 cells with CCCP (b, carbonylcyanide-m-chlorophenylhy-
drazone, positive control), 3.75 mM (c) and 7.5 mM (d) for 24 h, JC-
1 emits bright green fluorescence. The changes from red to green
fluorescence suggest that the complex induces a decrease in the
mitochondrial membrane potential. To further explore the rela-
tionship between mitochondrial membrane potential and ROS,
the ratio of red/green fluorescent intensity was determined by
flow cytometry. As shown in Fig. 7C, in the control (a) or con-
trol + NAC (b), the ratios of red/green fluorescence are 2.10 and
1.97, respectively. Treatment of BEL-7402 cells with 7.5 mM Ir-1
(c), 7.5 mM Ir-1 + NAC (d), 15.0 mM Ir-1 (e) and 15.0 mM Ir-1
+ NAC (d) for 24 h, the ratios of red/green fluorescent intensity
are 0.69, 1.07, 0.33 and 0.44, respectively. The results reveal that
NAC inhibits the changes of mitochondrial membrane potential,
namely, ROS can increase the changes of mitochondrial mem-
brane potential. Furthermore, the complex shows a concentra-
tion-dependent manner to induce a decrease in the
mitochondrial membrane potential.
Fig. 10. (A) Autophagy was assayed after BEL-7402 cells (a) were treated with 3.75 (b), 7.5 (c) and 15.0 mM (d) of Ir-1 for 24 h. (B) The assay of LC3 and beclin-1 protein
expression induced by 15.0 mM of the complex by western blot. (C) Effect of 3-MA and NAC on the viability of BEL-7402 cells exposed to different concentrations of Ir-1, Ir-1
+ 3-MA and Ir-1 + NAC for 24 h. The scale bars for all the figures are 200 .
3.9. Detection of intracellular Ca2+ levels
Calcium is a prevalent signaling molecule in plentiful metabolic
processes and cellular signal responses, which can induce cell
death if maintained incessantly at high concentration [45]. In the
assay of intracellular Ca2+ levels, Fluo-3MA was used as fluorescent
probe, which combines with Ca2+ to be transferred into Fluo-3. The
intracellular Ca2+ level in BEL-7402 cells induced by the complex
was determined under ImageXpress Micro XLS system and
Multi Wavelength Cell Scoring module. As shown in Fig. 8A, in
the control (a), weak fluorescent points were found. 7.5 mM of Ir-
1 treated BEL-7402 cells at 2 (b), 4 (c) and 8 h, a number of bright
green fluorescent points were observed. The results show that the
complex can induce an increase of intracellular Ca2+ levels.
To investigate the effect of exposure time on the intracellular
Ca2+ levels, the Fluo-3 fluorescent intensity was determined. Seen
from Fig. 8B, we found that the Fluo-3 fluorescent intensity
increases with increasing the exposed time of BEL-7402 cells to
the complex.
3.10. Release of cytochrome c studies
Cytochrome c (cyt-c) can induce cell apoptosis by activating
caspases proteases, which is released from the mitochondrial
membrane space by reason of the mitochondrial dysfunction
[46]. As shown in Fig. 9A, there was no obvious green fluorescence
of cytochrome c was observed in the control cells. After BEL-7402
cells were treated with 7.5 mM of Ir-1 for 24 h, a number of green
fluorescent points were found, which indicated that the release
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331 329

of cytochrome c was occurred. The integrated intensity/cell was 3.12. Cell invasion studies
obtained by Multi Wavelength Cell Scoring module. As shown in
Fig. 9B, the integrated intensity/cell increases with increasing the Since metastasis poses a foremost threat for cancer deaths,
time of BEL-7402 exposed to Ir-1. The results show that the com- inhibiting metastasis is an urgent therapeutic need [49]. To inves-
plex can release cyt-c from the mitochondria with a time-depen- tigate the effect of the complex on inhibiting migration, the tran-
dent manner. swell invasion assay in BEL-7402 cells was treated by Matrigel
invasion assay. As shown in Fig. 11A, BEL-7402 cells (a) were
exposed to 3.75 (b), 7.5 (c) and 15.0 mM (d) for 24 h, the number
3.11. Autophagy induced by the complex of cell invasion decreases gradually. Thus, we conclude that the
complex can effectively inhibit the cell invasion. The percentage
Autophagy is a highly conserved degradation pathway for bulk of different concentration of Ir-1 inhibiting cell invasion was calcu-
cellular components; it includes macroautophagy, microautophagy lated and shown in Fig. 11B, when the concentration of Ir-1 is
and chaperone-mediated autophagy [47]. Autophagy is closely 15.0 mM, the percentage of Ir-1 inhibiting cell invasion reaches
involved in the etiology of many important human diseases,
including cancer, neurodegenerative diseases and metabolic disor-
ders [48]. To evaluate the efficiency of the complex on autophagy,
the autophagy was performed under a fluorescent microscope
using MDC as fluorescent probe. As shown in Fig. 10A, there was
no obvious fluorescent points were observed in the control (a).
Exposure of BEL-7402 cells to 3.75 (b), 7.5 (c) and 15.0 mM (d) of
Ir-1 for 24 h, the MDC labeling green fluorescent points in the cyto-
plasm were discovered, which suggests that the autophagic vac-
uoles were formed. The results show that the complex can
induce autophagy. The up-regulation of Beclin-1 and the conver-
sion of the soluble form of LC3 (LC3-I) to the lipidated and
autophagosome-associated form (LC3-II) are hallmarks of autop-
hagy. As shown in Fig. 10B, the up-regulation of Beclin-1 protein
and the conversion of LC3-1 to LC3-II compared to the control were
observed, which further shows that the complex can induce autop-
hagy. In addition, the relationship among cell viability, autophagy
and ROS was evaluated by MTT method. As shown in Fig. 10C, in
the presence of 3-MA (6 mM, an inhibitor to inhibit autophagy)
and NAC (5 mM, an inhibitor to inhibit the generation of ROS),
the cell viability decreases or increases, respectively, which indi- Fig. 12. Western blot analysis of PARP, caspase 3, Bcl-2, Bad and Bax in BEL-7402
cells treated with NAC, 15.0 mM of Ir-1 and 15.0 mM of Ir-1 + NAC for 24 h. b-actin
cates that autophagy causes less cell death and ROS induce more
was used as internal control.
cell death.

Fig. 11. (A) Microscope images of invading BEL-7402 cells (a) that have migrated through the Matrigel induced by 3.75 (b), 7.5 (c) and 15.0 mM (d) of the Ir-1 for 24 h. (B) The
percentage of inhibiting cell invasion of BEL-7402 cells induced by different concentration of Ir-1 for 24 h. *P < 0.05 represents significant differences compared with control.
The scale bars for all the figures are 200 .
330 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
Fig. 13. The molecular mechanism of the complex induced apoptosis in BEL-7402 cells.
68.1%. It is clear that the complex inhibited cell invasion with a
concentration-dependent manner.
3.13. The expression assays of the proteins related to apoptosis
PARP (poly (ADP ribose) polymerase), an enzyme involved in
DNA repair, is a preferential substrate for caspase-3 [50]. PARP
plays an active role in key biological processes, PARP cleavage
is a hallmark of apoptosis [51]. Caspases are known to mediate
the apoptotic pathway, caspase 3 mediates intrinsic pathway
[52-54]. PARP protein cleavage in the complex-treated BEL-
7402 cells was investigated. As shown in Fig. 12, BEL-7402 cells
were incubated with 15.0 mM of Ir-1 for 24 h, the complex treat-
ment caused cleavage of PARP, 116 KD into 89 KD fragment was
observed. Treatment of BEL-7402 cells with 15.0 mM of Ir-1 for
24 h resulted in a significant increase in the levels of caspase 3
expression compared to the control. On the other hand, Bcl-2
family proteins play an important role in regulating apoptosis.
Treatment of BEL-7402 cells with the complex led to down-regu-
lation in Bcl-2 protein expression, and up-regulation of the
expression of Bad and Bax proteins. In addition, in the presence
of NAC (NAC + Ir-1), the expression of PARP, caspase 3, Bcl-2,
Bad and Bax was inhibited, which exhibits that ROS enhance
apoptotic efficiency.
4. Conclusions
Complex Ir-1 was synthesized and characterized. The assays of
cytotoxic activity in vitro show that the complex displays moder-
ate cytotoxicity against the selected cancer cell lines. Morphologi-
cal changes demonstrate that Ir-1 can induce apoptosis in BEL-
7402 cells. Additionally, the complex increases intracellular ROS
and Ca2+ levels and induces cytochrome c release. Location studies
show that complex locates at the lysosomes. After inducing lysoso-
mal membrane permeabilization, the complex enters into the
mitochondria and causes a decrease in the mitochondrial mem-
brane potential. The cell cycle arrest shows that the complex inhi-
bits cell growth at S phase. The complex can effectively inhibit the
cell invasion and the percentage of Ir-1 (15.0 mM) inhibiting cell
invasion reaches 68.1%. The complex can induce autophagy and
autophagy prevents cell death, whereas ROS cause more cell death
compared to the control. The western blot reveals that the complex
up-regulates caspase 3, Bad and Bax expression, and down-regu-
lates Bcl-2 proteins expression. In summary, the complex induces
apoptosis through the following two pathways (Fig. 13): (I) the
complex induces DNA damage, then the complex induces cell cycle
arrest at S phase, finally, the complex causes apoptosis. (II) The
complex induces apoptosis through ROS-mediated lysosome–mi-
tochondrial dysfunction pathway.
Acknowledgements
This work was supported by the National Nature Science Foun-
dation of China (No 21877018) and the Natural Science Foundation
of Guangdong Province (No. 2016A030313728).
References
[1] A. Kumar, R.K. Gupta, R.P. Paitandi, K.B. Singh, S.K. Trigun, M.S. Hundal, D.S.
Pandey, J. Organomet. Chem. 801 (2016) 68.
[2] L. Kelland, Nat. Rev. Cancer 7 (2007) 573.
[3] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467.
[4] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307.
[5] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451.
[6] P.J. Loehrer, S.D. Williams, L.H. Einhorn, J. Natl. Cancer Inst. 80 (1988) 1373.
[7] F.N. Akladios, S.D. Andrew, C.J. Parkinson, J. Biol. Inorg. Chem. 21 (2016) 407.
[8] E.J. Gao, N. Sun, S.Z. Zhang, Y.Q. Ding, X. Qiu, Y. Zhang, M.C. Zhu, Eur. J. Med.
Chem. 121 (2016) 1.
[9] W.X. Chen, X.D. Song, S.F. He, J. Sun, J.X. Chen, T. Wu, Z.W. Mao, J. Inorg.
Biochem. 164 (2016) 91.
[10] A. Pastuszko, K. Majchrzak, M. Czyz, B. Kupcewicz, E. Budzisz, J. Inorg.
Biochem. 159 (2016) 133.
[11] C. Zhang, B.J. Han, C.C. Zeng, S.H. Lai, W. Li, B. Tang, D. Wan, G.B. Jiang, Y.J. Liu, J.
Inorg. Biochem. 157 (2016) 62.
[12] C.C. Zeng, S.H. Lai, J.H. Yao, C. Zhang, H. Yin, W. Li, B.J. Han, Y.J. Liu, Eur. J. Med.
Chem. 122 (2016) 118.
[13] N.A. Vyas, S.N. Ramteke, A.S. Kumbhar, P.P. Kulkarni, V. Jani, U.B. Sonawane, R.
R. Joshi, B. Joshi, A. Erxleben, Eur. J. Med. Chem. 121 (2016) 793.
[14] C. Qian, J.Q. Wang, C.L. Song, L.L. Wang, L.N. Ji, H. Chao, Metallomics 5 (2013)
844.
[15] M. Tian, J.J. Li, S.M. Zhao, L.H. Guo, X.D. He, D.L. Kong, H.R. Zhang, Z. Liu, Chem.
Commun. 53 (2017) 12810.
 Y.-J. Wang et al. / Polyhedron 156 (2018) 320–331
[16] J.J. Cao, C.P. Tan, M.H. Chen, N. Wu, D.Y. Yao, X.G. Liu, L.N. Ji, Z.W. Mao, Chem.
Sci. 8 (2017) 631.
[17] Y. Hisamatsu, N. Suzuki, A.A. Masum, A. Shibuya, R. Abe, A. Sato, S.I. Tanuma, S.
Aoki, Bioconjugate Chem. 28 (2017) 507.
[18] J.S.Y. Lau, P.K. Lee, K.H.K. Tsang, C.H.C. Ng, Y.W. Lam, S.H. Cheng, K.K.W. Lo,
Inorg. Chem. 48 (2009) 708.
[19] L. Tabrizi, H. Chiniforoshan, Dalton Trans. 46 (2017) 2339.
[20] S.K. Tripathy, U. De, N. Dehury, P. Laha, M.K. Panda, H.S. Kim, S. Patra, Dalton
Trans. 45 (2016) 15122.
[21] C.L. Wang, J.F. Liu, Z.Z. Tian, M. Tian, L.J. Tian, W.Q. Zhao, Z. Liu, Dalton Trans.
46 (2017) 6870.
[22] J.J. Li, L.H. Guo, Z.Z. Tian, M. Tian, S.M. Zhang, K. Xu, Y.C. Qian, Z. Liu, Dalton
Trans. 46 (2017) 15520.
[23] R.P. Paitandi, S. Mukhopadhyay, R.S. Singh, V. Sharma, S.M. Mobin, D.S. Pandey,
Inorg. Chem. 56 (2017) 12232.
[24] C. Zhang, S.H. Lai, H.H. Yang, D.G. Xing, C.C. Zeng, B. Tang, D. Wan, Y.J. Liu, RSC
Adv. 7 (2017) 17752.
[25] C. Zhang, S.H. Lai, C.C. Zeng, B. Tang, D. Wan, D.G. Xing, Y.J. Liu, J. Biol. Inorg.
Chem. 21 (2016) 1047.
[26] S. Sprouse, K.A. King, P.J. Spellane, R.J. Watts, J. Am. Chem. Soc. 106 (1984)
6647.
[27] A.K. Bilakhiya, B. Tyagi, P. Paul, Inorg. Chem. 41 (2002) 3830.
[28] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[29] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.
Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Environ. Mol. Mutagen. 35 (2000) 206.
[30] C. Alapetite, T. Wachter, E. Sage, E. Moustacchi, Int. J. Radiat. Biol. 69 (1996)
359.
[31] D. Wan, B. Tang, Y.J. Wang, B.H. Guo, H. Yin, Q.Y. Yi, Y.J. Liu, Eur. J. Med. Chem.
139 (2017) 180.
[32] J.F. Kou, C. Qian, J.Q. Wang, X. Chen, L.L. Wang, H. Chao, L.N. Ji, J. Biol. Inorg.
Chem. 17 (2012) 81.
331
[33] G.B. Jiang, J.H. Yao, J. Wang, W. Li, B.J. Han, Y.Y. Xie, G.J. Lin, H.L. Huang, Y.J. Liu,
New J. Chem. 38 (2014) 2554.
[34] W. Li, B.J. Han, J.H. Yao, G.B. Jiang, Y.J. Liu, RSC Adv. 5 (2015) 24534.
[35] K.E. Prosser, S.W. Chang, F. Saraci, P.H. Le, C.J. Walsby, J. Inorg. Biochem. 167
(2017) 89.
[36] H. Kojima, Y. Urano, K. Kikuchi, T. Higuchi, Y. Hirata, T. Nagano, Angew. Chem.
Int. Ed. Engl. 38 (1999) 3209.
[37] X. Zheng, G. Ning, D. Hong, M. Zhang, Mol. Biol. 279 (2004) 69.
[38] C. Settembre, A. Fraldi, D.L. Medina, A. Ballabio, Nat. Rev. Mol. Cell. Biol. 14
(2013) 283.
[39] C. Hiebel, C. Behl, Life. Sci 138 (2015) 3.
[40] D.A. Salazar, A. Rodríguez-López, A. Herren~o, H. Barbosa, J. Herrera, A. Ardila,
G.E. Barreto, J. González, C.J. Alméciga-Díaz, Mol. Genet. Metab. 117 (2016)
129.
[41] B. Sun, J. Liu, Y. Gao, H.B. Zhen, L. Li, Q.W. Hu, H.Q. Yuan, H.X. Lou, Eur. J. Med.
Chem. 136 (2017) 603.
[42] L. Galluzzi, J.M. Bravo-San Pedro, G. Kroemer, Nat. Cell Biol. 16 (2014) 728.
[43] P. Boya, G. Kroemer, Oncogene 27 (2008) 6434.
[44] C.B. Zhao, W.J. Gao, T.S. Chen, Apoptosis 19 (2014) 668.
[45] M.J. Berridge, M.D. Bootman, H.L. Roderick, Nat. Rev. Mol. Cell Biol. 4 (2003)
517.
[46] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Cell 86 (1996) 147.
[47] N. Mizushima, M. Komatsu, Cell 147 (2011) 728.
[48] A.J. Meijer, P. Codogno, Crit. Rev. Clin. Lab. Sci. 46 (2009) 210.
[49] A.F. Chambers, A.C. Groom, I.C. MacDonald, Nat. Rev. Cancer 2 (2002) 563.
[50] F. Malik, A. Kumar, S. Bhushan, S. Khan, A. Bhatia, K.A. Suri, G.N. Qazi, J. Singh,
Apoptosis 12 (2007) 2115.
[51] P.J. Duriez, G.M. Shah, Biochem. Cell Biol. 75 (1997) 337.
[52] G.S. Salvesen, V.M. Dixit, Cell 91 (1997) 443.
[53] N.A. Thornberry, Y. Lazebnik, Science 281 (1998) 1312.
[54] C.L. Hsu, Y.S. Yu, G.C. Yen, J. Agric. Food Chem. 58 (2010) 2201.