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Photoinduced ROS regulation of apoptosis and mechanism studies of iridium(iii) complex against SGC-7901 cells
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PAPER
Cite this: RSC Adv., 2017, 7, 17752
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Photoinduced ROS regulation of apoptosis and
mechanism studies of iridium(III) complex against
SGC-7901 cells
Cheng Zhang,a Shang-Hai Lai,a Hui-Hui Yang,a De-Gang Xing,*b
Chuan-Chuan Zeng,a Bing Tang,a Dan Wana and Yun-Jun Liu*ac
A new iridium(III) complex, [Ir(ppy)2(FBPIP)]PF6 (Ir-1), was synthesized and characterized. The cytotoxic
activity of this complex against SGC-7901, PC-12, SiHa, HepG2, BEL-7402, A549, HeLa and normal LO2
cells was investigated using the MTT method. The complex showed no cytotoxic activity against the
selected cell lines. However, upon irradiation, the complex displayed potent anti-proliferative activity
toward SGC-7901 cells, with a low IC50 value of 6.1 0.6 mM, and showed selectivity between cancer
and normal cells. Apoptosis was assayed using the AO/EB staining method. The level of reactive oxygen
species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under
a fluorescence microscope. The cell cycle distribution was studied by flow cytometry. The expression of
caspase and Bcl-2 family proteins was investigated by western blot. Complex Ir-1 was shown to
Received 17th January 2017
Accepted 27th February 2017
accumulate preferentially in the mitochondria of SGC-7901 cells and induced apoptosis via the
DOI: 10.1039/c7ra00732a
mitochondrial pathway, which involved ROS generation, mitochondrial membrane potential
depolarization, and Bcl-2 and caspase family member activation. These results demonstrate that the
rsc.li/rsc-advances
complex induces cancer cell apoptosis by acting on mitochondrial pathways.
1. Introduction
Bioinorganic chemistry is a fast-growing area of fascinating
opportunities and creative power.1 A great number of transition
metal ions play essential roles in biological processes and have
been implicated in many diseases, including microbial infections, cancer, and neurodegenerative and autoimmune disorders.2,3 Due to the disadvantage of the dose-dependent side
effects of cisplatin as well as the resistance of some carcinomas,
it is understandable that a wide range of novel transition metal
complexes have been screened for their use as therapeutic
agents.4 Among all the transition metal complexes, ruthenium
complexes appear to be particularly interesting compounds.
Presently, one ruthenium(III) complex, namely KP1019 ([IndH]
[trans-RuCl4(Ind)2], where Ind ¼ indazole), has successfully
entered clinical trials.5 On the other hand, iridium-based
compounds show a wide range of biological activities.6–18 Espinosa reported that iridium complexes inhibit cell growth
through DNA interaction.19 Tris-cyclometalated iridium(III)
complexes contain cationic peptides as inducers and detectors
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, P.
R. China. E-mail: lyjche@gdpu.edu.cn
b
School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510006,
P. R. China. E-mail: degangxing@126.com
c
Guangdong Cosmetics Engineering & Technology Research Center, Guangzhou,
510006, P. R. China
17752 | RSC Adv., 2017, 7, 17752–17762
of cell death via a calcium-dependent pathway.20 Chao and Mao
reported that Ir(III) complexes induce apoptosis through mitochondrial targets.14,15 In particular, induction of mitochondrial
dysfunction has attracted great attention because the major
function of mitochondria in human cells is to provide ATP by
oxidative phosphorylation, and the mitochondrial respiratory
chain is a major source of damaging free radicals. Therefore, the
mitochondria are an important target for iridium complexes. To
obtain more insight into the anticancer activity and further
understanding of the anticancer mechanism of iridium
complexes, in this article, a new cyclometalated iridium(III)
complex, [Ir(ppy)2(FBPIP)]PF6 (Ir-1, ppy ¼ 2-phenylpyridine, FBPIP
¼ 2-(4-formyl)benzeno[4,5-f][1,10]phenanthroline, Scheme 1), was
synthesized and characterized by elemental analysis, IR, ESI-MS,
1
H NMR and 13C NMR. The in vitro cytotoxic activity of the
complex against SGC-7901, PC-12, SiHa, HepG2, BEL-7402, A549,
HeLa and normal LO2 cells was investigated using the MTT (3(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method.
The results obtained demonstrated that the complex induced
SGC-7901 cell apoptosis by acting on mitochondrial apoptosis
pathways.
2.
Results and discussion
2.1. Synthesis and characterization
The ligand FBPIP was prepared according to the method
described in the literature.21 The complex Ir-1 was synthesized
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Scheme 1
The synthetic route of the ligand and complex Ir-1.
by the direct reaction of [Ir(ppy)2Cl2]2 with FBPIP in a mixture of
dichloromethane and methanol. The crude product was puried by column chromatography on neutral alumina. The obtained complex was characterized by elemental analysis, ESIMS, IR, 1H NMR and 13C NMR. In the ESI-MS spectra, the
peak at an m/z value of 824.5 corresponds to the ion peak of [M
PF6]+. In the IR spectra for Ir-1, the peak at 1696 cm1 is
attributed to C–H (formyl group) stretch vibration. In the 1H
NMR spectrum, the peak at a chemical shi of 10.08 ppm is
characteristic of the hydrogen of a formyl group, whereas the
peak at 210.21 ppm in the 13C NMR spectrum is assigned to the
carbon atom in the formyl group. The UV-Vis and luminescence
spectra of the complex in PBS solution are shown in Fig. 1. The
maximum absorbance of Ir-1 appears at 430 nm, and the
complex can emit luminescence in PBS solution at ambient
temperature, with a maximum appearing at 563 nm.
2.2. The in vitro cytotoxicity assay using the MTT method
The in vitro cytotoxicity of Ir-1 against SGC-7901, PC-12, SiHa,
BEL-7402, A549, HeLa, HepG2 and normal LO2 cells was
determined using the MTT method. The 50% inhibitory
concentrations (IC50), dened as the concentration required to
reduce the size of the cell population by 50%, of the ligand and
Ir-1 complex are listed in Table 1. As expected, the ligand FBPIP
shows certain cytotoxic activity against the selected cell lines. It
is unexpected to nd that the Ir-1 complex displays no cytotoxicity in vitro without irradiation. However, when the cells are
treated with Ir-1 and irradiation (7.03 J cm3) for 45 min, Ir-1
exhibits high cytotoxic activity against SGC-7901, SiHa, A549
Fig. 1
and BEL-7402 cells. In particular, Ir-1 exhibits the highest
cytotoxicity against SGC-7901 cells, with an IC50 value of 6.1
0.6 mM, among the selected cancer cells. Interestingly, the
complex exhibits no or very weak cytotoxicity toward normal
LO2 cells with or without irradiation. Comparing the IC50 values
with those of cisplatin, the cytotoxic activity of Ir-1 is lower than
that of cisplatin and [Ir(ppy)2(BTCP)]+ (IC50 ¼ 3.9 0.5 mM)3
toward SGC-7901 cells. Because SGC-7901 cells are sensitive to
Ir-1 upon irradiation, this cell line was selected for the following
experiments.
2.3. Apoptosis assay using the AO/EB staining method
It is well known that acridine orange (AO) can stain living and
apoptotic cells green, while ethidium bromide (EB) stains
necrotic cells red. Apoptosis is primarily a physiological process
necessary to remove individual cells that are no longer needed
or that function abnormally.22 As shown in Fig. 2A, in the
control ((a–c): (a) bright; (b) green; (c) merge), the living cells
exhibit bright green uorescence and retain their integrated
morphology. Aer the SGC-7901 cells were exposed to 6.25 mM
of Ir-1 for 24 h ((d–f): (d) bright; (e) green; (f) merge), apoptotic
cells also displayed bright green uorescence with apoptotic
characteristics, such as nuclear shrinkage and chromatin
condensation. These characteristics indicated that the Ir-1
complex can induce apoptosis in SGC-7901 cells. N-Acetylcysteine (NAC, 5 mM) is an inhibitor that inhibits the production of
reactive oxygen species (ROS). To investigate the relation
between apoptosis and ROS, apoptosis was also studied by ow
cytometry. As shown in Fig. 2B, upon irradiation, in the control
The UV-Vis (a) and luminescence (b) spectra of complex Ir-1 (10 mM) at room temperature.
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Table 1
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The IC50 (mM) values of Ir-1 toward selected cell linesa
Complex
SGC-7901
PC-12
SiHa
HepG2
BEL-7402
A549
HeLa
LO2
FBPIP
Ir-1
Ir-1 (light)
Cisplatin
30.5 3.4
>200
6.1 0.6
3.6 0.5
44.6 2.3
113.7 4.2
92.1 2.4
11.2 0.7
nd
>200
39.4 2.6
13.6 2.2
nd
>200
>200
25.4 3.3
15.5 1.6
>200
35.5 1.8
11.1 1.2
>200
>200
22.9 1.2
6.3 1.1
nd
>200
>200
7.4 1.3
>200
>200
53.7 4.2
nd
nd: not determined.
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a
Fig. 2 (A) SGC-7901 cells stained with AO/EB and imaged under a fluorescence microscope. SGC-7901 cells incubated without (a–c) and with
exposure to 6.25 mM of the complex (d–f) for 24 h. (B) The percentage of apoptotic cells determined by flow cytometry. SGC-7901 cells alone (a),
SGC-7901 cells + NAC (b), and SGC-7901 cells exposed to 12.5 mM of Ir-1 (c), Ir-1 + NAC (d), Ir-1 + light (e) and Ir-1 + NAC + light (f) for 24 h.
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(a), the percentage of early apoptotic cells was 0.32%. In the
presence of NAC (b), the percentage of early apoptotic cells was
0.25%. Aer SGC-7901 cells were treated with 6.25 mM of Ir-1 for
24 h, the percentage in the apoptotic cells was 0.82% without
irradiation (c) and 5.92% upon irradiation (e). However, in the
presence of NAC, the percentage of apoptotic cells was 1.06%
((d) without irradiation) and 2.78% ((f) with irradiation). Obviously, in the presence of NAC, the complex induces less
apoptosis, i.e. low ROS levels cause the complex to produce less
apoptosis. These results indicate that the apoptotic effect
induced by the complex is closely related to ROS levels.
2.4. Assay of intracellular ROS levels
The apoptosis assays demonstrated that ROS can regulate the
apoptotic effect. To quantitatively investigate the intracellular
generation of ROS induced by different concentrations of
complex Ir-1, ROS levels were detected using DCFH–DA dye as
a uorescent probe. In the presence of ROS, this dye is changed
into a highly uorescent compound (20 ,70 -dichloro uorescein,
DCF). As shown in Fig. 3A, in the control (a), no obvious uorescent points were observed. Aer the treatment of SGC-7901
cells with Rosup (b, positive control) and 6.25 mM of Ir-1 (c) for
24 h, the bright green uorescent points were found. The
uorescence intensity of DCF is proportional to the amount of
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peroxide (ROS) produced by the cells.23 As shown in Fig. 3B, in
the control ((a) with irradiation), the DCF uorescence intensity
is 20. Aer treating the SGC-7901 cells with 6.25 (b) and 12.5 mM
(d) Ir-1 without irradiation for 24 h, the DCF uorescence
intensity is 12.9 and 25.6, respectively. However, upon irradiation for 45 min, the SGC-7901 cells that were exposed to 6.25 (c)
and 12.5 (e) mM Ir-1 for 24 h displayed a DCF uorescence
intensity of 85.7 and 158.0, which is a DCF uorescence intensity increase of 4.29 and 7.90 times relative to the original. Thus,
we can conclude that it is difficult for Ir-1 to produce ROS
without light, but upon irradiation, the complex can increase
the intracellular ROS levels in a concentration-dependent
manner. To further investigate the relationship between ROS
levels and cell viability, SGC-7901 cells were treated with
different concentrations of Ir-1 in the absence or presence of
NAC (5 mM) upon irradiation for 24 h. As shown in Fig. 3C, Ir-1
can cause more cell death than Ir-1 + NAC. This demonstrates
that ROS levels can regulate cell growth.
2.5. Evaluation of the location of Ir-1 and mitochondrial
membrane potential
The location of the complex in the mitochondria was inspected
using MitoTracker® Deep Red FM (ThermoFisher, 100 nM) as
a uorescent dye. As shown in Fig. 4A, in the control, the
Fig. 3 (A) Intracellular ROS detected in untreated SGC-7901 cells (a) and in SGC-7901 cells exposed to Rosup ((b) positive control) or 6.25 mM of
the complex (c) for 24 h. (B) The DCF fluorescence intensity determined in untreated SGC-7901 cells exposed to light (a), and in SGC-7901 cells
exposed to 6.25 (b) and 12.5 mM (d) of the complex without light and 6.25 (c) and 12.5 mM (e) of the complex with light for 24 h. (C) The viability of
cells incubated with the complex for 24 h assayed in the absence or presence of NAC.
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(A) The location of the complex in the mitochondria. (B) The mitochondrial membrane potential was assayed using JC-1 as fluorescent
probe. SGC-7901 cells (a) were exposed to cccp (b, positive control), 6.25 (c) and 12.5 mM (d) of Ir-1 for 24 h. (C) The ratio of red/green fluorescence was determined by flow cytometry.
Fig. 4
mitochondria were stained red. SGC-7901 cells were treated with
6.25 mM of Ir-1 for 4 h, and the complex emits bright green
uorescence. The merge of the red and green uorescence
images suggests that the complex can enter into the mitochondria. To further investigate whether or not cell apoptosis is
accompanied by mitochondrial dysfunction, the mitochondrial
membrane potential was detected using JC-1 as a uorescent
17756 | RSC Adv., 2017, 7, 17752–17762
probe. It is well known that JC-1 forms aggregates and emits red
uorescence when the mitochondrial membrane potential is
high. On the other hand, JC-1 exists as monomer and emits green
uorescence when the mitochondrial membrane potential is low.
As shown in Fig. 4B, in the control (a), JC-1 emits red uorescence
corresponding to the high mitochondrial membrane potential.
Aer SGC-7901 cells were exposed to cccp (b, positive control)
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and 6.25 (c) and 12.5 mM (d) for 24 h, JC-1 emits green
uorescence corresponding to low mitochondrial membrane
potential. The ratio of the red/green uorescence suggests the
change of mitochondrial membrane potential. As shown in
Fig. 4C, in the control (top and bottom), the ratio of red/green
uorescence is 1.24. Aer the SGC-7901 cells were incubated
with 6.25 mM of Ir-1 (top and bottom) for 24 h, the ratio was 1.02.
Upon irradiation for 30 min, in the control (light), the ratio was
1.06, while aer treatment of the SGC-7901 cells with 6.25 mM Ir-1
(top and bottom) or Ir-1 + NAC (top and bottom), the ratios were
0.41 and 0.49, respectively. Comparing the ratios with the
control, the ratios decrease, which indicates that the red uorescence decreases and the green uorescence increases. These
results show that the complex can induce a decrease in the
mitochondrial membrane potential. Moreover, in the presence of
NAC, the ratio of red/green uorescence decreases. This also
demonstrates that the changes in mitochondrial membrane
potential are closely related to ROS levels.
2.6. Anti-metastatic studies
As a conrmatory efficacy test, the anti-invasive potential of
different concentrations of Ir-1 toward invasive SGC-7901 cells
was evaluated using a Matrigel assay. As shown in Fig. 5A and B,
the translocated cells that migrated from the upper to the lower
side of the lter were labeled with crystal violet dye. Aer SGC7901 cells were exposed to 6.25 (b), 12.5 (c) and 25 mM (d) of Ir1, a signicant reduction in the number of invasive cells that
penetrated the Matrigel was observed compared with the control
(a). The percentage inhibition of cell invasion is shown in Fig. 5C.
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When the concentration of Ir-1 reaches 50 mM, the percentage
inhibition of cell invasion reaches 73.1%. This concentration of
Ir-1 can effectively inhibit cell migration. Furthermore, the antimetastatic effect of Ir-1 is concentration-dependent.
2.7. Autophagy induced by the Ir-1 complex
Autophagy is the process of sequestering cytoplasmic proteins
into lytic components and is characterized by the formation and
promotion of AVOs (acidic vesicular organelles).24,25 The autophagy induced by the complex was investigated using monodansylcadaverine (MDC) as a uorescent probe. MDC is
a specic in vivo marker for autophagic vacuoles.26 As shown in
Fig. 6A, in the control (a), no obvious green spots were observed.
Aer treatment of the SGC-7901 cells with Ir-1 (6.25 mM) with (d)
or without (b) irradiation, MDC emits a number of green spots,
which indicates that the complex induces autophagy with the
formation of autophagic vacuoles. However, in the presence of
NAC (5 mM), which is an inhibitor of ROS, the mixture of Ir-1 +
NAC without (c) and with (e) irradiation can also induce SGC7901 cell autophagy, but autophagy occurs at the autophagosome stage. One of the hallmarks of autophagy is the conversion of the soluble form of LC3 (LC3-I) to the lipidated and
autophagosome-associated form (LC3-II).27 As shown in
Fig. 6B, the conversion of LC3-I into LC3-II and an increase in
the amount of LC3-II were observed. Moreover, upon irradiation, the Ir-1 complex can induce more effective autophagy than
without light. Comparing the amount of LC3-II induced by Ir-1
and Ir-1 + NAC, Ir-1 induces a higher expression than Ir-1 + NAC
under identical conditions. Thus, NAC can inhibit SGC-7901
Fig. 5 (A) Microscope images of invading SGC-7901 cells that have migrated through the Matrigel: untreated cells (a) and cells exposed to 6.25
(b), 12.5 (c) and 25 mM (d) of the complex for 24 h. (B) The number of invading SGC-7901 cells induced by different concentrations of the
complex. (C) The percentage inhibition of cell invasion induced by different concentrations of the complex. *P < 0.05 represents significant
differences compared with the control.
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(A) Autophagy assayed with MDC staining in untreated SGC-7901 cells (a) and cells exposed to 6.25 mM Ir-1 (b) and Ir-1 + NAC (c) without
light, and Ir-1 (d) and Ir-1 + NAC (e) with light for 24 h. (B) The assay of LC3 and beclin-1 protein expression induced by 6.25 mM of the complex by
western blot. (C) Effect of an autophagy inhibitor on the viability of cells exposed to different concentrations of Ir-1 and Ir-1 + 3-MA.
Fig. 6
cell autophagy. Similar results were found for the expression of
beclin-1. Autophagy can prevent or induce cell death. To
investigate whether the complex prevents or induces cell death,
the cell viability was determined using the MTT method in the
presence of 3-MA. SGC-7901 cells were exposed to different
concentrations of Ir-1 or Ir-1 + 3-MA upon irradiation for 24 h.
As shown in Fig. 6C, the cell viability with Ir-1 is higher than
that with Ir-1 + 3-MA, which suggests that the complex protects
the cell and does not induce cell death.
2.8. Cell cycle distribution studies
Inhibition of cancer cell proliferation by cytotoxic drugs could
be the result of induction of apoptosis or cell cycle arrest, or
a combination of these. Cell cycle distribution was investigated
by ow cytometry in propidium iodide (PI) stained cells. It is
well known that among the cell cycle regulating mechanisms,
arrest in the S phase is related to cellular damage.28 As shown in
17758 | RSC Adv., 2017, 7, 17752–17762
Fig. 7 Cell cycle distribution of SGC-7901 cells exposed to 6.25 mM of
the complex for 24 h.
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Fig. 8 Western blot analysis of caspase 3, Bcl-2, Bak and Bax in SGC7901 cells treated with 6.25 mM of Ir-1 in the absence and presence of
NAC for 24 h. b-Actin was used as an internal control.
demonstrate that the complex can increase ROS levels. In the
presence of NAC, the cell viability of SGC-7901 cells decreases.
The complex can induce a decrease in the mitochondrial
membrane potential and inhibit cell growth at the S phase. In
addition, Ir-1 can cause autophagy at different stages in the
presence of 3-MA with or without irradiation. Furthermore,
autophagy enhances the cell viability. Western blot analysis
indicates that the complex can down-regulate the expression of
Bcl-2 and up-regulate the expression of pro-apoptotic proteins
Bak and Bax. In summary, the anticancer activity of the Ir-1
complex is closely related to the ROS levels. The complex
induces apoptosis through a ROS-mediated mitochondrial
dysfunction pathway. This work will be helpful for designing
and synthesizing iridium complexes as potential anticancer
drugs.
4. Experimental
4.1. Materials and methods
Fig. 7, in the control, the percentage of cells in the S phase is
27.97%. Aer treatment of the SGC-7901 cells with Ir-1 for 24 h,
cell cycle distribution analysis reveals that the percentage of
cells in the S phase is 32.90%. An increase of 4.93% in the
percentage of cells in the S phase was observed, which is
accompanied by a concomitant decrease in the fraction of G0/
G1 and G2/M cells. The data show that the complex inhibits
SGC-7901 cell growth during the S phase.
2.9. The expression of Bcl-2 family proteins induced by Ir-1
The activation of caspase 3 as an “effector caspase” serves as an
important event in the apoptotic pathway. To study the effect of
Ir-1 on the expression of caspase 3, SGC-7901 cells were incubated with 6.25 mM of Ir-1 in the absence or presence of NAC
with or without irradiation for 24 h. As shown in Fig. 8, upon
irradiation, Ir-1 can increase the expression levels of caspase 3,
and Ir-1 induces more expression than Ir-1 + NAC. In apoptotic
cells, Bcl-2 and Bax are two members of the Bcl-2 protein family
that regulate the balance between cell proliferation and
apoptosis. The anti-apoptotic protein Bcl-2 can block the release
of cytochrome c from the mitochondria, whereas pro-apoptotic
protein Bax induces the release of cytochrome c and other proapoptotic factors from the mitochondria.29 Upon irradiation,
Fig. 8 shows that Ir-1 can down-regulate the expression of the
Bcl-2 protein, and up-regulate the expression of the Bak and Bax
proteins. In the presence of NAC, Ir-1 induces less expression of
Bcl-2 family proteins compared with cells induced by Ir-1 alone.
The results indicate that the expression levels of Bcl-2 family
proteins induced by the Ir-1 complex can be regulated by ROS
levels.
3.
Conclusions
A new iridium(III) polypyridyl complex, [Ir(ppy)2(FBPIP)]PF6, has
been synthesized and characterized. Upon irradiation, this
complex shows high cytotoxicity. In particular, the complex can
effectively inhibit cell growth in SGC-7901 cells. ROS assays
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All reagents and solvents were purchased commercially and
used without further purication unless otherwise noted.
Ultrapure 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.
The cancer cell lines SGC-7901 (human gastric adenocarcinoma), HeLa (human cervical cancer), PC-12 (pheochromocytoma), BEL-7402 (human hepatocellular carcinoma), SiHa
(human cervical carcinone), A549 (human lung carcinoma) and
HepG2 (human hepatocellular carcinoma), and normal LO2
cells (human liver cells) were purchased from the American
Type Culture Collection. IrCl3$3H2O was purchased from the
Kunming Institution of Precious Metals.
Microanalysis (C, H, and N) was carried out with a PerkinElmer 240Q elemental analyzer. Electrospray ionization mass
spectra (ESI-MS) were recorded on a LCQ system (Finnigan
MAT, USA) using acetonitrile as the mobile phase. The spray
voltage, tube lens offset, 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 values 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
a solvent and tetramethylsilane (TMS) as an internal standard at
500 MHz at room temperature.
4.2. Synthesis of complex [Ir(ppy)2(FBPIP)]PF6 (Ir-1)
A mixture of cis-[Ir(ppy)2Cl2]2 (0.15 g, 0.14 mmol)30 and FBPIP21
(0.091 g, 0.28 mmol) in a mixture of 21 mL of dichloromethane
and methanol (vCH2Cl2 : vCH3OH ¼ 2 : 1) was reuxed under
argon for 6 h to give a clear yellow solution. Upon cooling, a yellow
precipitate was obtained by dropwise addition of saturated
aqueous NH4PF6 solution with stirring at room temperature for
2 h. The crude product was puried by column chromatography
on neutral alumina with a mixture of CH2Cl2/acetone (1 : 3, v/v) as
eluent. The yellow band was collected. The solvent was removed
under reduced pressure and a yellow powder was obtained. Yield:
70%. Anal. calc. for C42H28N6OIrPF6: C, 52.01; H, 2.91; N, 8.66%.
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Found: C, 52.23; H, 3.02; N, 8.53%. IR (KBr, cm1): 3044m, 1696s,
1607s, 1549m, 1479s, 1452w, 1424w, 1363m, 1309m, 1212m,
1193m, 1172m, 1071w, 1032w, 1015m, 952s, 838m, 805m, 738m,
703m, 645s, 623s. 1H NMR (DMSO-d6): d 10.08 (s, 1H), 9.23 (d, 1H,
J ¼ 8.0 Hz), 9.16 (d, 2H, J ¼ 8.5 Hz), 8.57 (s, 1H), 8.52 (d, 2H, J ¼ 8.5
Hz), 8.28 (d, 2H, J ¼ 6.0 Hz), 8.15 (t, 2H, J ¼ 5.0 Hz), 8.08 (dd, 2H, J
¼ 5.0, J ¼ 5.0 Hz), 7.98 (d, 4H, J ¼ 6.5 Hz), 7.89 (d, 2H, J ¼ 7.5 Hz),
7.53 (d, 4H, J ¼ 8.0 Hz), 7.08 (t, 2H, J ¼ 6.5 Hz), 7.00 (d, 2H, J ¼ 5.5
Hz). 13C NMR: (DMSO-d6, 125 MHz, ppm): 210.21, 166.91, 150.46,
149.12, 148.20, 144.22, 144.03, 138.68, 136.56, 132.19, 131.22,
130.30, 129.91, 126.93, 125.06, 123.83, 122.80, 122.35, 119.97. ESIMS (DMSO): m/z 824.5 ([M PF6]+).
4.3. In vitro cytotoxicity assay
MTT assay procedures were used.31 Cells were placed in 96-well
microassay culture plates (8 103 cells per well) and grown
overnight at 37 C in a 5% CO2 incubator. The tested complex
was dissolved in DMSO and then added to the wells to achieve
nal concentrations ranging from 106 to 104 M. Control wells
were prepared by addition of culture medium (100 mL). 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
mL, 5 mg mL1) was added to each well. Aer 4 h, buffer (100 mL)
containing dimethylformamide (50%) and sodium dodecyl
sulfate (20%) was added to solubilize the MTT formazan. The
optical density of each well was measured with a microplate
spectrophotometer at a wavelength of 490 nm. The IC50 values
were determined by plotting the percentage of viable cells 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 mean values.
4.4. Apoptosis assay using AO/EB staining methods
SGC-7901 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 medium supplemented with 10% fetal bovine
serum (FBS) and incubated at 37 C in 5% CO2. The medium
was removed and replaced with medium (nal DMSO concentration, 0.05% v/v) containing the complex (6.25 mM) for 24 h.
The medium was removed again, and the cells were washed
with ice-cold phosphate buffered saline (PBS), and xed with
formalin (4%, w/v). The cell nuclei were counterstained with
acridine orange (AO) and ethidium bromide (EB) (AO: 100 mg
mL1, EB: 100 mg mL1) for 10 min. The cells were then
observed and imaged under a uorescence microscope (Nikon,
Yokohama, Japan) with excitation at 350 nm and emission at
460 nm.
4.5. Reactive oxygen species (ROS) detection
SGC-7901 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
medium supplemented with 10% FBS and incubated at 37 C in
5% CO2. The medium was removed and replaced with medium
17760 | RSC Adv., 2017, 7, 17752–17762
Paper
(nal DMSO concentration, 0.05% v/v) containing different
concentrations of the complex for 24 h. The medium was
removed again. The uorescent dye 20 ,70 -dichlorodihydrouorescein diacetate (DCHF-DA, 10 mM) was added to the
medium to cover the cells. The treated cells were then washed
with cold PBS–EDTA twice, and collected by trypsinization and
centrifugation at 1500 rpm for 5 min, and the cell pellets were
suspended in PBS–EDTA. The DCF uorescence intensity was
determined by ow cytometry.
4.6. Location assay of the complex in the mitochondria
SGC-7901 cells were placed in 24-well microassay culture plates (4
104 cells per well) and grown overnight at 37 C in a 5% CO2
incubator. 6.25 mM of the complex was added to the wells at 37 C
in a 5% CO2 incubator for 4 h and further co-incubated with
MitoTracker Deep Red FM (150 nM) at 37 C for 0.5 h. Upon
completion of the incubation, the wells were washed three times
with ice-cold PBS. Aer discarding the culture medium, the cells
were imaged under a uorescence microscope.
4.7. Mitochondrial membrane potential assay
SGC-7901 cells were treated for 24 h with different concentrations 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 mg mL1 of JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylimidacarbocyanine iodide) in culture medium at 37 C. The
cells were then immediately centrifuged to remove the supernatant. The cell pellets were suspended in PBS. The ratio of red/
green uorescence intensity was determined by ow cytometry.
4.8. Cell cycle arrest by ow cytometry
SGC-7901 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
medium supplemented with 10% FBS and incubated at 37 C in
5% CO2. The medium was removed and replaced with medium
(nal DMSO concentration, 0.05% v/v) containing the complex
(6.25 mM). Aer incubation for 24 h, the cell layer was trypsinized and washed with cold PBS and xed with 70% ethanol. 20
mL of RNAse (0.2 mg mL1) and 20 mL of propidium iodide
(0.02 mg mL1) were added to the cell suspensions and they
were incubated at 37 C for 30 min. Then the samples were
analyzed with a FACSCalibur ow cytometer. The number of
cells analyzed for each sample was 10 000.32
4.9. Autophagy induced by the complex
SGC-7901 cells were seeded onto chamber slides in 12-well plates
and incubated for 24 h. The cells were cultured in RPMI 1640
medium supplemented with 10% FBS and incubated at 37 C in
5% CO2. The medium was removed and replaced with medium
(nal DMSO concentration, 0.05% v/v) containing different
concentrations of the 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
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RSC Advances
mM) for 10 min and washed with PBS twice. The cells were
observed and imaged under a uorescence microscope. The effect
of the complex on the expression of LC3 and beclin-1 proteins was
assayed by western blot.
reagents according to the manufacturer's instructions. To
assess the presence of comparable amounts of protein in each
lane, the membranes were stripped nally to detect the b-actin.
The gray values were calculated using BandScan.
4.10.
5.
The effect of autophagy on cell viability
Cell viability was determined 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 (3MA, 3 mM) for 3 h, followed by different concentrations of Ir-1
for 24 h. Aer incubation, the cells were incubated with MTT
(0.5 mg mL1) for 4 h at 37 C. Upon completion of the incubation, 100 mL of 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 calculated using the formula:
(A490 (treatment group)/A490 (control)) 100, where A490 (treatment group)
is the mean OD value of cells treated with the complex and A490
(control) is the mean OD value of untreated cells. Each experiment
was repeated at least three times to obtain mean values.
4.11.
Matrigel invasion assay
A BD Matrigel invasion chamber was used to investigate cell
invasion according to the manufacturer's instructions. SGC7901 cells (4 104) in serum free medium containing
different concentrations of the complex were seeded into the
top chamber of the two-chamber Matrigel system. RPMI 1640
medium (20% FBS) was added into the lower chamber. The cells
were allowed to invade for 24 h. Aer incubation, non-invading
cells were removed from the upper surface and cells on the
lower surface were xed with 4% paraformaldehyde and stained
with 0.1% crystal violet. The membranes were photographed
and the invading cells were counted under a light microscope.
The mean values from three independent assays were
calculated.
4.12.
The expression of caspases and Bcl-2 family proteins
SGC-7901 cells were seeded into 3.5 cm dishes for 24 h and
incubated with 6.25 mM of the complex in the presence of 10%
FBS. The cells were harvested in lysis buffer. Aer sonication,
the samples were centrifuged for 20 min at 13 000g. The protein
concentration of the supernatant was determined by BCA
(bicinchoninic acid) assay. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed by loading equal
amounts of protein per lane. The gels were then transferred to
poly(vinylidene diuoride) membranes (Millipore) 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 : 5000 dilutions in
5% non-fat milk overnight at 4 C, and washed four times with
TBST for a total of 30 min. Aer this, the membranes were
incubated with secondary antibodies conjugated with horseradish peroxidase at 1 : 5000 dilution for 1 h at room temperature and then washed four times with TBST. The blots were
visualized using Amersham ECL Plus western blotting detection
This journal is © The Royal Society of Chemistry 2017
Data analysis
All data were expressed as mean SD. Statistical signicance
was evaluated using t-tests. Differences were considered to be
signicant when the *P value was less than 0.05.
Acknowledgements
This work was supported by the Natural Science foundation of
Guangdong Province (No. 2016A030313728) and the Project
of Innovation for Enhancing Guangdong Pharmaceutical
University, Provincial Experimental Teaching Demonstration
Center of Chemistry & Chemical Engineering.
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