← Back
Anticancer mechanism studies of iridium(III) complexes inhibiting osteosarcoma HOS cells proliferation.
Journal of Inorganic Biochemistry 237 (2022) 112011
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Anticancer mechanism studies of iridium(III) complexes inhibiting
osteosarcoma HOS cells proliferation
Fu-Li Xie a, c, 1, Yan Wang a, c, 1, Jian-Wei Zhu a, c, 1, Hui-Hua Xu a, c, Qi-Feng Guo a, c, *,
Yong Wu b, c, *, Si-Hong Liu a, c, *
a
b
c
Department of Orthopaedics, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510180, PR China
Department of Oncology, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong 510180, PR China
Guangzhou First People’s Hospital, Guangzhou, Guangdong 510180, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Osteosarcoma
Iridium (III) complexes
Autophagy
Mitochondria
Antitumor
HOS cells
Three iridium (III) polypyridine complexes [Ir(bzq)2(maip)](PF6) (Ir1，
，bzq = benzo[h]quinoline, maip = 3aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(bzq)2(apip)](PF6) (Ir2, apip = 2-aminophenyl-1Himidazo[4,5-f][1,10]phenanthroline) and [Ir(bzq)2(paip)](PF6) (Ir3, paip = 4-aminophenyl-1H-imidazo[4,5-f]
[1,10]phenanthroline) were synthesized and characterized. The cytotoxic activities of the three complexes
against human osteosarcoma HOS, U2OS, MG63 and normal LO2 cells were evaluated by MTT (3-(4,5-dime
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results showed that Ir1–3 exhibited moderate
antitumor activity against HOS with IC50 of 21.8 ± 0. 4 μM,10.5 ± 1.8 μM and 7.4 ± 0.4 μM, respectively. We
found that Ir1–3 can effectively inhibit HOS cells growth and blocked the cell cycle at the G0/G1 phase. Further
studies revealed that complexes can increase intracellular reactive oxygen species (ROS) and Ca2+, which
accompanied by mitochondria-mediated intrinsic apoptosis pathway. In addition, autophagy was also investi
gated. Taken together, the complexes induce HOS apoptosis through a ROS-mediated mitochondrial dysfunction
pathway and inhibition of the PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR (mammalian
target of rapamycin) signaling pathway. This study provides useful help for understanding the anticancer
mechanism of iridium (III) complexes toward osteosarcoma treatment.
1. Introduction
Osteosarcoma (OS) is the most common primary malignant bone
cancer in children and young adults, with an annual incidence of be
tween 1 and 3 cases per million worldwide [1]. The treatment of newly
diagnosed osteosarcoma is a combination of preoperative and post
operative chemotherapy and surgery [2]. There is no specific chemo
therapy for osteosarcoma at present. Despite the continuous
improvement of new drugs, methotrexate, doxorubicin and cisplatin are
still the most commonly used combination chemotherapy schemes for
children and young adult patients [3,4]. Although neoadjuvant therapy
and extensive tumor resection have improved survival, clinical out
comes and 5-year survival in patients with OS remain unsatisfactory due
to early lung metastasis and drug resistance of the tumor [5–7]. As the
first generation of anticancer drugs, cisplatin has severe toxic side effects
and drug resistance which limit long-term use and clinical efficacy
[8–10]. In recent years, iridium, ruthenium, rhodium, titanium and
other non‑platinum metal complexes have developed rapidly as sub
stitutes for platinum organometallic anticancer drugs [11–14]. Iridium
complexes are widely used as cell probes, organelle-targeted imaging
reagents and tumor chemotherapy drugs because of their excellent
photophysical properties, low toxicity and strong anti-tumor activity
[15–22]. Zhang et al. reported that the iridium(III) complexes contain
ing maip (3-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline),
apip (2-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline) and paip
(4-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline) show high
anticancer efficacy against B16 and A549 cells [18] This stimulates us to
choose these ligands to synthesize iridium(III) complexes, owing to
benzo[h]quinoline (bzq) possessing good planarity and hydrophobicity,
we chose bzq as an ancillary. At present, the cancer cells including A549,
* Corresponding authors at: Department of Orthopaedics, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou,
Guangdong, 510180, PR China
E-mail addresses: eyguoqf@scut.edu.cn (Q.-F. Guo), eywuyong@scut.edu.cn (Y. Wu), eyliush@scut.edu.cn (S.-H. Liu).
1
These authors contribute equally.
https://doi.org/10.1016/j.jinorgbio.2022.112011
Received 25 March 2022; Received in revised form 14 September 2022; Accepted 14 September 2022
Available online 20 September 2022
0162-0134/© 2022 Elsevier Inc. All rights reserved.
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
HeLa, HepG2, BEL-7402 and SGC-7901 cells are chosen for many
iridium(III) complexes, very few literatures reported the studies on os
teosarcoma cells of iridium(III) complexes. Therefore, in this paper, we
choose HOS, U2OS, MG63 osteosarcoma cells and investigate the anti
tumor effect of the iridium(III) complexes. To obtain much anticancer
information and further understanding the anticancer mechanism of
iridium(III) complexes, herein, three new iridium(III) complexes [Ir
(bzq)2(maip)](PF6) (Ir1), [Ir(bzq)2(apip)](PF6) (Ir2) and [Ir
(bzq)2(paip)](PF6) (Ir3, Scheme 1) were designed and synthesized. The
complexes were characterized by HRMS,1H NMR and 13C NMR. The
cytotoxic activity of the complexes against osteosarcoma HOS, U2OS,
MG63 and normal LO2 cells was investigated by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT). The anticancer efficacy
was evaluated by endocytosis, apoptosis, cell cycle arrest, reactive ox
ygen species, mitochondrial membrane potential. The expression of Bcell lymphoma-2 family proteins was explored by western blot.
2.2. Synthesis of complexes
2.2.1. Synthesis of [Ir(bzq)2(maip]PF6 (Ir1)
Maip (0.16 g, 0.5 mmol) [23] and cis-[Ir(bzq)2Cl]2 (0.292 g, 0.25
mmol) [24] was dissolved in a mixture of dichloromethane and meth
anol (45 mL, VCH3OH:VCH2Cl2 = 1:2, v/v) and refluxed under argon for 6
h. After cooling, NH4PF6 (1.5 g) was added and stirred for 30 min. The
dark red precipitate was obtained. With a mixture of CH2Cl2/acetone (v/
v, 1:3) as eluent, neutral alumina column was used to purify the crude
product and finally a yellow-brown powder was obtained. Yield: 69%.
Anal. Cacld for C45H29N7IrPF6: C, 53.78; H, 2.91; N, 9.76%. Found:
53.95; H, 2.83; N, 9.61%. HRMS (CH3CN): Calcd. for C45H29N7IrPF6: m/
z = 860.2116 ([M-PF6]+), found: m/z = 860.2117 ([M-PF6]+) (Error
0.12 ppm). 1H NMR (DMSO‑d6, 500 MHz): δ 9.18 (d, 2H, J = 8.0 Hz),
8.51 (d, 2H, J = 7.5 Hz), 8.09 (d, 2H, J = 5.5 Hz), 7.99–7.92 (m, 6H),
7.87 (d, 2H, J = 8.5 Hz), 7.58 (d, 3H, J = 8.5 Hz), 7.48–7.42 (m, 3H),
7.26–7.20 (m, 3H), 6.73 (d, 1H, J = 8.5 Hz), 6.32 (d, 2H, J = 7.5 Hz),
5.40 (s, 2H). 13C NMR (DMSO‑d6, 125 MHz): 158.46, 151.28, 150.82,
150.42, 149.34, 146.32, 142.39, 139.52, 135.77, 134.21, 131.74,
131.51, 130.56, 128.81, 128.71, 126.22, 124.76, 122.34, 117.76,
116.30, 113.84.
2. Experimental
2.1. Materials and methods
All reagents purchased are analytically pure. The experimental water
was Milli-Q ultra-pure water. The fluorescent dye kit was obtained from
Beyotime Biotechnology. The human osteosarcoma cell lines HOS, U2OS
and MG63 and normal human hepatocytes LO2 were obtained from the
experimental center of Sun Yat-Sen university (Guangzhou, China).
HOS, U2OS, MG63 and LO2 cells were cultured in Dulbecco’s Modified
Eagle Medium (DMEM) at 37 ◦ C and 5% CO2. All culture mediums were
added with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/
streptomycin.
2.2.2. Synthesis of [Ir(bzq)2(apip)]PF6 (Ir2)
Ir2 was synthesized in the same method described as Ir1, using apip
[25] in place of maip. Yield: 64%. Anal. Cacld for C45H29N7IrPF6: C,
53.78; H, 2.91; N, 9.76%. Found: C, 53.61; 3.03; N, 9.88%. HRMS
(CH3CN): Calcd. for C45H29N7IrPF6: m/z = 860.2116 ([M-PF6]+), found:
m/z = 860.2120 ([M-PF6]+) (Error 0.47 ppm). 1H NMR (DMSO‑d6, 500
MHz): δ 9.13 (d, 2H, J = 8.0 Hz), 8.51 (d, 2H, J = 8.0 Hz), 8.09 (d, 2H, J
= 4.5 Hz), 8.01–7.93 (m, 8H), 7.88 (d, 2H, J = 8.0 Hz), 7.58 (d, 2H, J =
8.0 Hz), 7.44 (t, 2H, J = 6.0 Hz), 7.22 (t, 2H, J = 7.5 Hz), 6.76 (d, 2H, J
= 8.5 Hz), 6.32 (d, 2H, J = 7.0 Hz), 5.77 (s, 2H). 13C NMR (DMSO‑d6,
125 MHz): 164.39, 158.45, 156.49, 153.14, 150.81, 150.39, 149.31,
Scheme 1. Synthetic route for the complexes Ir1, Ir2 and Ir3.
2
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
146.04, 142.38, 139.53, 135.76, 134.11, 131.75, 131.52, 130.56,
130.06, 128.76, 128.71, 126.23, 124.76, 122.36, 118.53, 115.69.
2.6. Cell cycle analysis
HOS cells (1 × 105 cells per well) were seeded at in 6-well plates
overnight. Then the cells were exposed to IC50 concentrations of Ir1, Ir2
and Ir3 for 24 h. The cells were fixed with 70% ethanol at 4 ◦ C over
night, and subsequently stained with propidium iodide (PI) for 15 min in
the dark. The cell cycle distribution was detected by flow cytometry.
2.2.3. Synthesis of [Ir(bzq)2(paip]PF6 (Ir3)
Ir3 was synthesized in the same method described as Ir1, using paip
[23] in place of maip. Yield: 61%. Anal. Cacld for C45H29N7IrPF6: C,
53.78; H, 2.91; N, 9.76%. Found: 53.92; H, 2.78; N, 9.68%. HRMS
(CH3CN): Calcd. for C45H29N7IrPF6: m/z = 860.2116 ([M-PF6]+), found:
m/z = 860.2120 ([M-PF6]+) (Error 0.47 ppm). 1H NMR (DMSO‑d6, 500
MHz): δ 9.11 (d, 2H, J = 8.5 Hz), 8.50 (d, 2H, J = 8.0 Hz), 8.07 (d, 2H, J
= 5.0 Hz), 8.01–7.91 (m, 8H), 7.87 (d, 2H, J = 9.0 Hz), 7.57 (d, 2H, J =
8.0 Hz), 7.45–7.42 (m, 2H), 7.22 (t, 2H, J = 7.5 Hz), 6.73 (d, 2H, J = 8.5
Hz), 6.31 (d, 2H, J = 7.0 Hz), 5.71 (s, 2H). 13C NMR (DMSO‑d6, 125
MHz): 158.48, 152.86, 150.74, 150.10, 149.40, 145.87, 142.40, 139.51,
135.77, 134.03, 131.73, 131.52, 130.56, 130.01, 128.71, 128.60,
126.21, 124.75, 122.32, 115.69.
2.7. Apoptosis assay
HOS cells were grown in six-well plates and then treated with IC50
concentrations of Ir1, Ir2 and Ir3 for 24 h. Afterwards, the cells were
washed three times with phosphate buffer saline (PBS) and resuspended
in 195 μL of buffer. The cells were dyed with 5 μL Annexin V-FITC
(fluorescein isothiocyanate) and 10 μL propidium iodide (PI) for 20 min
in the dark at room temperature. The apoptotic percentage in the cells
was measured by flow cytometry.
2.3. Cell viability assay
2.8. Intracellular ROS level assays
The osteosarcoma cells HOS, U2OS, MG63 and normal cells LO2
were seeded into 96-well plates at 2 × 103 cells/well. After 24 h, the cells
were added with various concentrations (3.125, 6.25, 12.5, 25, 50, 100
μM) of iridium (III) complexes for 48 h. Upon completion of the incu
bation, DMEM (90 μL) and MTT (10 μL) was added in and the cells were
incubated for 4 h at 37 ◦ C. After the media were removed and replaced
with 100 μL DMSO (final concentration of 0.05%). The absorbance was
determined at 490 nm using a microplate reader. The experiment was
repeated three times independently and the mean value was calculated.
Intracellular reactive oxygen species (ROS) generation was measured
using a dichlorofluorescein diacetate (DCFH-DA) assay kit according to
the manufacturer’s instructions. Briefly, after HOS cells were treated
with IC50 concentrations of Ir1, Ir2 and Ir3 for 24 h, the cells were
harvested and then stained with 200 μL DCFH-DA solution for 30 min at
37 ◦ C. After rinsed twice, the cells were imaged under a fluorescence
microscope and the fluorescence intensity of dichlorofluorescein (DCF)
was examined to assess intracellular ROS level.
2.4. Cell uptake analysis
HOS cells (6 × 104 cells per well) were cultured on 12-well plates
overnight. After 4 h incubation with IC50 concentrations of Ir1, Ir2 and
Ir3, the cells were stained with LysoTracker Red at 37 ◦ C for 30 min.
Then the cells were observed under a fluorescence Microscope.
2.9. Lysosomal localization assay
HOS cells were inoculated into 12-well plates and incubated over
night in an incubator. When the density of each well reaches 40–50%,
IC50 concentrations of Ir1, Ir2 and Ir3 were added into the cells and
incubated for 24 h. The cells were washed twice with PBS and incubated
with 75% ethanol (300 μL/well) for 20 min. The samples were observed
under fluorescence microscope.
The cell uptake was quantitatively determined by inductively
coupled plasma-mass spectrometry (ICP-MS, Thermo Fisher Scientific
iCAP Qc) according to the literature [26]. HOS cells (5.0 × 104 cells/
well) were seeded in 6-well plate and incubated with 20.0 μM of Ir1, Ir2
and Ir3 for 8 h when the cells arrived logarithmic phase. Then, washing
the adherent cells twice with PBS containing 5 mM EDTA. After tryp
sinization and centrifugation of the suspension at 800 rpm for 5 min,
repeating the above processes till the residual complexes were
completely removed. The cells were digested with 60% HNO3 at 60 ◦ C to
completely release the endocytosed iridium(III) complexes from the
cells and then a 5 mL solution was obtained by adding Milli-Q water. The
endocytosed mounts were calculated through the following procedures:
(I) determining the intensity (193Ir) of different concentrations of
iridium standard solution, through linear fitting to obtain a fitting
equation (x-axis: concentration of sample; y-axis: intensity). (II) deter
mining the intensity (193Ir) in the sample, then calculate the uptake
amount according to the fitting equation.
2.10. Autophagy
After the HOS cells were treated with IC50 concentrations of Ir1, Ir2
and Ir3 for 24 h, the cells were washed with PBS twice, and stained with
50 μM dansylcadaverine (MDC) at 37 ◦ C for 25 min. Then the cells were
washed with PBS twice and the cells was photographed under a fluo
rescence microscope.
2.11. Measurement of intracellular Ca2+
The levels of intracellular Ca2+ were measured using the Fluo-3pentaacetoxymethyl ester (Fluo-3 AM) fluorescent dye (Beyotime,
Guangzhou) according to the manufacture’s instruction. HOS cells were
treated with IC50 concentrations of Ir1, Ir2 and Ir3 for 24 h, and the cells
were dyed with Fluo-3 AM at 37 ◦ C for 25 min in darkness. Then the cells
were washed twice times with PBS, the cells were observed under
fluorescence microscopy.
2.12. Localization of the complexes at the mitochondria
HOS cells were incubated in a six-well plate overnight, the cells were
exposed to IC50 concentrations of Ir1, Ir2 and Ir3 for 5 h. After incu
bation, the cells were washed twice with PBS and dyed at 37 ◦ C for 30
min. The residual dye was then washed with PBS, and the cells were
observed under a fluorescence microscope.
2.5. Cell cloning experiment
HOS cells (700 cells/well) were cultured into 6-well plates overnight.
The cells were treated with IC50 concentrations of Ir1, Ir2 and Ir3 and
incubated for 48 h. The culture medium was removed every two days
and continuously cultured for one week. After washing with PBS, the
cells were stained with 0.1% (w/v) crystal violet and observed under a
fluorescence microscope.
2.13. Detection of mitochondrial membrane potential (MMP)
HOS cells (6 × 104 cells per well) were plated in 12-well plate
overnight, and then the cells were treated with IC50 concentrations of
3
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
Ir1, Ir2 and Ir3 for 24 h. Next, the cells were harvested and dyed with 1
μg/mL of JC-1 (5,5′ ,6,6′ -tetrachloro-1,1′ ,3,3′ -tetraethyl-imidacarbo
cyanineiodide) for 30 min at room temperature. After washed twice with
PBS, the cells were observed under a fluorescence microscope.
and Ru(II) complex [Ru(dmp)2(dcdppz)](ClO4)2 (IC50 = 22.51 ± 1.35
μM, dmp = 2,9-dimethyl-1,10-phenanthroline, dcdppz = 7,8-dichlor
odipyrido[3,2-a:2′ ,3′ -c]phenazine) [29] against MG63 cells. Although
the complexes show markedly higher activity than cisplatin toward
MG63 cells, we found that many literatures reported that osteosarcoma
MG63 cells were used as research subject [30–32], few literatures re
ported the anticancer effect on the compound on osteosarcoma HOS
cells, and the IC50 values of complexes Ir1, Ir2 and Ir3 toward HOS cells
are lower than toward MG63 cells, therefore, in this paper, we chose
HOS cell to undergo the following cell experiments.
2.14. Western blotting analysis
HOS cells were harvested after the treatment with IC50 concentra
tions of Ir1, Ir2 and Ir3 for 24 h and total proteins from the cells were
extracted. Protein concentrations were determined using the bicincho
ninic acid (BCA) method. Proteins separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto
polyvinylidene difluoride (PVDF) membranes, which were blocked with
5% non-fat milk for 3 h. The membranes were incubated with primary
antibodies overnight at 4 ◦ C and then were probed with secondary
antibody for 70 min at room temperature. Immunoblot signals were
visualized with enhanced chemiluminescence.
3.3. Assay of cellular uptake of complexes
After uptake by the cell, the complexes can accumulate in the
different subcellular structures of the cell. The intracellular uptake level
was detected by fluorescence technique to investigate the relationship
between uptake level and antitumor activity. As shown in Fig. 1a, we
found that the complexes can enter to the cells and emit green fluores
cence compared to the blank group. To compare the amount of the
complexes entering the cells, the green fluorescence intensity was
quantified by flow cytometry. As shown in Fig. 1b, after HOS cells were
exposed to IC50 concentrations of Ir1, Ir2 and Ir3 for 24 h, the fluo
rescence intensity increases by 6.76, 1.57 and 1.46 times for Ir1, Ir2 and
Ir3 compared to that in the control. These results indicate that the
complexes were successfully endocytosed. In addition, we use induc
tively coupled plasma-mass spectrometry (ICP-MS) to quantitatively
determine the amounts of the complexes entering the cells. After 8 h of
exposure of HOS cells with 20.0 μM of the complexes, the amounts of the
complexes entering the cells are 103.94 ± 6.11, 4.26 ± 0.14 and 5.95 ±
0.45 ng/106 cells for Ir1, Ir2 and Ir3, respectively. The amounts of the
complexes entering the cells are not consistent with the cytotoxic ac
tivity of the complexes against HOS cells.
3. Results and discussion
3.1. Synthesis and characterization
The ligands maip, apip and paip were prepared according to the
literature. The complexes Ir1, Ir2 and Ir3 were synthesized by the direct
reaction with ligands and [Ir(bzq)2Cl]2 in dichloromethane and meth
anol. The complexes were purified by neutral alumina column using
CH2Cl2/acetone as eluent. In the HRMS spectra, the determined mo
lecular weights are consistent with the expected values. In the 1H NMR
spectra, the peaks of 5.40 for Ir1, 5.77 for Ir2 and 5.71 ppm for Ir3 are
assigned to the hydrogen atoms in the -NH2. The complexes can be
dissolved in CH3OH, CH3CH2OH, CH2Cl2 and DMSO. The UV–Vis and
luminescence spectra of the complexes in PBS solution was determined.
As shown in Fig. S1a and S1b (supporting information), complexes
Ir1, Ir2 and Ir3 show one distinct band at 261 (ε = 20,300), 259 (ε =
32,500), 258 nm (ε = 30,950), respectively. The range of the emission
spectra of the complexes is 580–610 nm, and the emission maxima of
complexes Ir1-Ir3 appeared at 598 nm (λex = 295 nm), 595 nm (λex =
295 nm) and 596 nm (λex = 295 nm), respectively.
3.4. Colony formation assays
Excessive proliferation and invasion of tumor cells are hallmarks of
cancer [33]. To evaluate the effect of complexes on osteosarcoma cell
proliferation, the colony formation assay was investigated. As shown in
Fig. S2 (supporting information), the cells in the control grew rapidly,
and large colony distribution was observed under the microscope after
crystal violet staining. However, after HOS cells were treated with IC50
concentrations of Ir1, Ir2 and Ir3, the number of colonies of cells was
significantly reduced. The results show that complexes Ir1, Ir2 and Ir3
can significantly inhibit the proliferation of HOS cells.
3.2. Cytotoxic activity in vitro studies
The in vitro cytotoxicity of the complexes Ir1, Ir2 and Ir3 against
HOS, U2OS, MG63 and LO2 was investigated by MTT assay (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) [27]. The re
sults of the antitumor activity of these complexes against selected tumor
cell lines compared to cisplatin are shown in Table 1. The complexes
exhibited moderate cytotoxic activity against the selected cancer cells.
In addition, the complex [Ir(bzq)2(paip)](PF6) (Ir3) show lower cyto
toxic activity than the complex [Ir(piq)2(paip)](PF6) (piq = 1-phenyl
isoquinoline, IC50 = 4.7 ± 0.2 μM) [18] against HepG2 cells. Comparing
complexes [Ir(bzq)2(paip)](PF6) and [Ir(piq)2(paip)](PF6), the two
complexes containing the same main ligand paip and different ancillary
ligands (bzq or piq), the difference in the cytotoxic activity toward
HepG2 cells may be caused by different ancillary ligands. The cytotoxic
activity of Ir1, Ir2 and Ir3 is higher than that of complex VO(od)phen
(IC50 = 58 μM, od = oxodiacetate, phen = 1,10-phenanthroline) [28]
3.5. Effect of complexes on cell cycle distribution
Cell proliferation inhibition or death is the result of apoptosis, cell
cycle arrest, or both [34–36]. To explore the mechanism of the com
plexes inhibiting the cell proliferation, the cell cycle arrest was studied
by flow cytometry. As shown in Fig. 2, in the control, the percentage in
the cell at G0/G1 phase is 35.19%. After a 24 h treatment of HOS cells
(a) with IC50 concentrations of Ir1 (b), Ir2 (c) and Ir3 (d), the percentage
in the cells at G0/G1 phase increased by 5.68%, 16.85% and 20.67%
compared to that in the control. Obviously, complexes Ir2 and Ir3 show
higher efficacy on the cell cycle arrest than Ir1 under the same condi
tions. These results indicated that Ir1–3 inhibit the cell growth at G0/G1
phase.
Table 1
IC50 values (μM) of the cisplatin and complexes toward the selected cancer cells
for 48 h.
Complexes
HOS
U2OS
MG63
HepG2
LO2
Ir1
Ir2
Ir3
cisplatin
21.8 ± 0.4
10.5 ± 1.8
7.4 ± 0.4
7.8 ± 0.5
27.8 ± 1.3
28.3 ± 2.9
9.5 ± 0.7
8.8 ± 0.1
31.1 ± 1.3
19.5 ± 0.3
9.5 ± 0.7
48.7 ± 2.0
95.5 ± 6.6
> 200
15.1 ± 1.0
12.2 ± 1.4
> 100
> 100
32.5 ± 1.5
18.7 ± 0.7
3.6. Ir1–3 promote cell apoptosis
Apoptosis, known as programmed death, exhibits early phosphati
dylserine externalization and can be detected by staining with the
phospholipid-binding protein Annexin V and PI [37,38]. To evaluate the
effect of the complexes on apoptosis, the apoptosis was investigated by
flow cytometry. As shown in Fig. 3, Q2, Q3 and Q4 stand for late
4
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
Fig. 1. (a) The cell uptake was observed under a fluorescence microscope after HOS cells were exposed to IC50 concentrations of Ir1, Ir2 and Ir3 for 24 h. (b) After
incubation with IC50 concentrations of Ir1 (II), Ir2 (III) and Ir3 (IV) for 24 h, the fluorescence intensity in HOS cells (I) was measured by flow cytometry.
Fig. 2. Cell cycle distribution assay of HOS cells (a) treated with IC50 concentrations of Ir1 (b), Ir2 (c) and Ir3 (d) for 24 h.
apoptosis, early apoptosis and living cells, respectively. The apoptosis
rate was quantified by the sum of early and late apoptosis. In the control,
the apoptotic rate is 4.66%. After HOS cells (a) were exposed to IC50
concentrations of cisplatin (b), Ir1 (c), Ir2 (d) and Ir3 (e) for 24 h, the
apoptotic rates increased by 7.73, 4.67, 6.90 and 9.15%, respectively.
The apoptotic efficacy follows the order of Ir3 > cisplatin > Ir2 > Ir1.
This is line with those of cytotoxic activity of the complexes against HOS
cells, Ir3 exhibits higher apoptotic effect than cisplatin. These results
demonstrated that the complexes can cause apoptosis in HOS cells.
3.7. Reactive oxygen species (ROS) determination
Intracellular reactive oxygen species (ROS) are mainly produced in
mitochondria, and oxidative stress caused by ROS accumulation triggers
physiological disorders and cellular damage and plays a key role in
tumor cell apoptosis [39–41]. The changes of intracellular ROS induced
by the complexes were assessed using 2′ ,7′ -dichlorodihydrofluorescein
diacetate (DCFH-DA) as fluorescence probe. As seen from Fig. S3a
(supporting information), after a 24 h treatment of HOS cells (I) with
5
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
Fig. 3. Apoptosis analysis of HOS cells (a) exposure to cisplatin (b) and IC50 concentrations of Ir1 (c), Ir2 (d) and Ir3 (e) for 24 h.
IC50 concentrations of Ir1 (II), Ir2 (III) and Ir3 (IV) for 24 h, the green
fluorescence obviously increases compared with that in the control,
indicating that Ir1–3 enhance the intracellular ROS levels. The quanti
fication of intracellular fluorescence intensity was performed by flow
cytometry. As shown in Fig. S3b (supporting information), the green
fluorescence increased by 1.43 times for Rosup (positive control, II),
4.47 times for Ir1 (III), 2.99 times for Ir2 (IV) and 2.29 times for Ir3 (V)
compared with that in the control (I). This further confirms that the
complexes can increase intracellular ROS levels.
3.8. Location of the complexes at the lysosomes and autophagy
Lysosomes are degrading organelles containing acidic hydrolases
whose primary role is the degradation and recycling of extracellular
substances through endocytosis and phagocytosis [42,43]. Autophagy is
the process in which cells are stimulated to engulf their cytoplasm or
organelles and finally degrade the engulfed cells in lysosomes [44,45].
As shown in Fig. S4a (supporting information), after 4 h of HOS cells
exposure to IC50 concentrations of Ir1, Ir2 and Ir3, the lysosomes were
stained red, the complexes emit green fluorescence. The overlap of red
and green fluorescence indicates that the complexes enter the
Fig. 4. (a) Colocalization of HOS cells incubated with Ir1, Ir2 and Ir3 for 5 h. (b) Assay of the change of mitochondrial membrane potential after HOS cells were
treated with IC50 concentration of Ir1, Ir2 and Ir3 for 24 h.
6
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
lysosomes. On the other hand, MDC is commonly used as a fluorescent
probe to detect autophagy. As shown in Fig. S4b (supporting infor
mation), HOS cells (I) were treated with IC50 concentrations of Ir1 (II),
Ir2 (III) and Ir3 (IV) for 24 h, a number of autophagic vacuoles were
observed, which suggests that the complexes can cause autophagy.
3.9. Determination of intracellular Ca2+ concentration
Mitochondria are one of the subcellular organelles involved in the
regulation of intracellular calcium ion signaling. When large amounts of
Ca2+ accumulate in mitochondria, the pro-apoptotic factor caspase-3
and the death-related factor poly ADP-ribose polymerase (PARP-1) are
activated, which in turn induce apoptosis [46–48]. The calcium fluo
rescent probe Fluo-3 AM was used to detect intracellular
Ca2+concentration. As shown in Fig. S5a (supporting information),
comparing with the control, the treatment of HOS cells with IC50 con
centrations of Ir1, Ir2 and Ir3 leads to an increase of green fluorescence
intensity. The green fluorescence intensity was quantitatively deter
mined by flow cytometry. As shown in Fig. S5b (supporting informa
tion), the green fluorescence intensity increases by 1.34 times for Ir1,
1.63 times for Ir2 and 1.09 times for Ir3, respectively. The efficacy of the
complexes on intracellular Ca2+ level follows the order of Ir2 > Ir1 >
Ir3. These results indicates that the complexes can increase intracellular
Ca2+ concentration.
Fig. 5. The expression of Bcl-2 family protein was assayed after an exposure of
HOS cells to IC50 concentration of Ir1, Ir2 and Ir3 for 24 h. β-actin was used as
internal control.
[56–58]. The expression of PARP and apoptotic execution factor
caspase-3 was significantly up-regulated in the Ir1–3-treated cells. The
PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR
(mammalian target of rapamycin) pathway is an important intracellular
signaling pathway. It plays an integral role in abnormal cell growth,
tumor invasion and chemoresistance, and is considered an attractive
therapeutic target for osteosarcoma [59,60]. The PI3K/AKT/mTOR
pathway is involved in regulating mitochondrial activity and ROS pro
duction, it also is an important pathway for autophagy [61–63]. The
results from the western blotting indicate that complexes Ir1, Ir2 and Ir3
inhibit the expression of PI3K, AKT and mTOR. Hence, we consider that
Ir1–3 induce apoptosis through inhibition of PI3K/AKT/mTOR
pathway.
3.10. Subcellular localization and membrane potential detection
Mitochondria play an important role as organelles in individual life
activities and are involved in the regulation of normal cell signaling
[49]. To detect whether the complexes inhibit tumor cell proliferation
and induce apoptosis through mitochondrial dysfunction pathway, the
location of the complexes at the mitochondria was studied. As seen in
Fig. 4a, mitochondria are stained red, the complexes emit green fluo
rescence, the overlap of red and green fluorescence indicates that the
complexes locate at the mitochondria and can damage mitochondrial
dysfunction. Hence, the complexes locate at both lysosomes and mito
chondria, moreover, we used ICP-MS to determine the distribution of the
Ir1 at the lysosomes and mitochondria. After the HOS cells were exposed
to 20.0 μM of Ir1 for 5 h, the distribution of Ir1 at the lysosomes and
mitochondria is 12.65 ± 2.31 and 8.57 ± 1.34 ng/106 cells, respec
tively. The results demonstrate that the complexes prefer to accumulate
at the lysosomes. The increase of intracellular ROS levels induces a rapid
depolarization of the inner mitochondrial membrane potential (MMP).
The decrease in mitochondrial membrane potential is a hallmark event
in the early stages of apoptosis [50–53]. The mitochondrial membrane
potential was assessed using 5,5′ ,6,6′ -Tetrachloro-1,1′ ,3,3′ -tetraethylimidacarbocyanine iodide (JC-1) as a fluorescent probe. As shown in
Fig. 4b, JC-1 in the control group emitted bright red fluorescence cor
responding to a high MMP. In contrast, HOS cells were exposed to the
carbonylcyanide-m-chlorophenylhydrazone (CCCP, positive control)
and IC50 concentration of Ir1–3, JC-1 exhibit a lot of green fluorescence
and a little of red fluorescence points corresponding to a low MMP. The
results demonstrate that the complexes cause a decrease of MMP. In
summary, complexes-triggered oxidative stress causes Ca2+ release and
induced apoptosis through mitochondrial membrane depolarization.
4. Conclusions
In this study, three iridium (III) complexes were synthesized and
characterized. Their antitumor properties were studied. we observed
that Ir1–3 can inhibit HOS cells proliferation, induce cell cycle arrest at
G0/G1 phase. At the same time, Ir1–3 locate at the mitochondria and
cause a decrease of mitochondrial membrane potential. The complexes
also locate at the lysosomes and induce autophagy. Additionally, the
complexes can increase intracellular ROS and Ca2+ levels, downregulate the expression of Bcl-2 protein, simultaneously, up-regulate
the expression of PARP and caspase 3, inhibit the expression of PI3K,
AKT and mTOR (Fig. 6). In conclusion, the complexes induce apoptosis
via a ROS-mediated mitochondrial dysfunction pathway and inhibition
of PI3K/AKT/mTOR signaling pathway. This work is helpful for design
and synthesis of new iridium (III) complexes as potent antiosteosarcoma drugs.
3.11. Effects of complexes on the expression of Bcl-2 family proteins
Author statement
To investigate the mechanism of Ir1–3 inducing cancer cell death,
we treated HOS cells with IC50 concentration of Ir1, Ir2 and Ir3 for 24 h
and examined the expression levels of apoptosis-related proteins. The Bcell lymphoma-2 (Bcl-2) family proteins play a critical dual regulatory
role between autophagy and mitochondrial apoptosis [54,55]. As shown
in Fig. 5, the results showed that the expression of anti-apoptotic protein
Bcl-2 was decreased compared to the control. Caspase-3 is an important
pro-apoptotic regulator and PARP acts as a substrate for caspase-3
We declare that this manuscript has been finished by all authors
listed in this manuscript, and all data are original and real. We agree to
be accountable for all aspects of the work.
All authors have read this manuscript and approved the manuscript
to be submitted to JIB.
7
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
Fig. 6. The mechanism of the complexes inducing apoptosis in HOS cells.
Declaration of Competing Interest
[9] M. Chovanec, M. Abu Zaid, N. Hanna, N. El-Kouri, L.H. Einhorn, C. Albany, Ann.
Oncol. 28 (2017) 2670–2679.
[10] V. Brabec, J. Kasparkova, Drug Resist. Updat. 8 (2005) 131–146.
[11] Q. Sun, Y. Wang, Q. Fu, A. Ouyang, S. Liu, Z. Wang, Z. Su, J. Song, Q. Zhang,
P. Zhang, D. Lu, Angew. Chem. Int. Ed. Eng. 60 (2021) 4841–4848.
[12] Z.Y. Liu, J. Zhang, Y.M. Sun, C.F. Zhu, Y.N. Lu, J.Z. Wu, J. Li, H.Y. Liu, Y. Ye,
J. Mater. Chem. B 8 (2020) 438–446.
[13] J. Wang, J.J. Nie, P. Guo, Z. Yan, B. Yu, W. Bu, J. Am. Chem. Soc. 142 (2020)
2709–2714.
[14] G. Lan, K. Ni, S.S. Veroneau, X. Feng, G.T. Nash, T. Luo, Z. Xu, W. Lin, J. Am. Chem.
Soc. 141 (2019) 4204–4208.
[15] S.M. King, S. Claire, R.I. Teixeira, A.N. Dosumu, A.J. Carrod, H. Dehghani, M.
J. Hannon, A.D. Ward, R. Bicknell, S.W. Botchway, N.J. Hodges, Z. Pikramenou,
J. Am. Chem. Soc. 140 (2018) 10242–10249.
[16] Q. Chen, C. Jin, X. Shao, R. Guan, Z. Tian, C. Wang, F. Liu, P. Ling, J.L. Guan, L. Ji,
F. Wang, H. Chao, J. Diao, Small 14 (2018), e1802166.
[17] S. Kuang, X. Liao, X. Zhang, T.W. Rees, R. Guan, K. Xiong, Y. Chen, L. Ji, H. Chao,
Angew. Chem. Int. Ed. Eng. 59 (2020) 3315–3321.
[18] Y.Y. Zhang, Y. Zhou, H.W. Zhang, L. Tian, J. Hao, Y.H. Yuan, W.L. Li, Y.J. Liu,
J. Inorg. Biochem. 224 (2021), 111580.
[19] Y. Zhou, L. Bai, L. Tian, L.L. Yang, H.W. Zhang, Y.Y. Zhang, J. Hao, Y.Y. Gu, Y.
J. Liu, J. Inorg. Biochem. 223 (2021), 111550.
[20] F.L. Xie, Z.Z. Huang, L. Bai, J.W. Zhu, H.H. Xu, Q.Q. Long, Q.F. Guo, Y. Wu, S.
H. Liu, J. Inorg. Biochem. 225 (2021), 111603.
[21] I. Echevarra, E. Zafon, S. Barrabés, M.A. Martíne, S. Romas-Gómez, N. Ortega, B.
R. Manzano, E.A. Jalón, R. Quesada, G. Espino, A. Massaguer, J. Inorg. Biochem.
231 (2022), 111790.
[22] Y.H. Yuan, C.L. Shi, X.Y. Wu, W.L. Li, C.X. Huang, L.J. Liang, J. Chen, Y. Wang, Y.
J. Liu, J. Inorg. Biochem. 232 (2022), 111820.
[23] H.L. Huang, Y.J. Liu, C.H. Zeng, L.X. He, F.H. Wu, DNA Cell Biol. 29 (2010)
261–270.
[24] S. Sprouse, K.A. King, P.J. Spellane, R.J. Watts, J. Am. Chem. Soc. 106 (1984)
6647–6653.
[25] Z.H. Liang, Z.Z. Li, H.L. Huang, Y.J. Liu, J. Coord. Chem. 64 (2011) 3342–3352.
[26] W.L. Li, C.L. Shi, X.Y. Wu, Y.Y. Zhang, H.M. Liu, X.Z. Wang, C.X. Huang, L.J. Liang,
Y.J. Liu, J. Inorg. Biochem. 236 (2022), 111977.
[27] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.
[28] I.E. León, N. Butenko, A.L.D. Virgilio, C.I. Muglia, E.J. Baran, I. Cavaco, S.
B. Etcheverry, J. Inorg. Biochem. 134 (2014) 106–117.
[29] H.L. Huang, Z.Z. Li, Z.H. Liang, Y.J. Liu, Eur. J. Inorg. Chem. 2011 (2011)
5538–5547.
[30] E. Blanchet, M. Vansteelandt, R. Le Bot, M. Egorow, Y. Guitton, Y.F. Pouchus,
O. Grovel, Eur. J. Med. Chem. 79 (2014) 244–250.
[31] S.Q. Zhang, H. Ren, H.T. Sun, S.H. Cao, Saudi, J. Biol. Sci. 28 (2021) 4908–4915.
[32] Y.D. Qin, F.M. Fang, J.J. Zhou, R.B. Wang, S.X. Xu, L.H. Li, H.L. Zhang, Fitoterapia
146 (2020), 104727.
[33] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646–674.
The authors have declared that no competing interest exists.
Data availability
Data will be made available on request.
Acknowledgements
The present study was supported by National Natural Science
Foundation of China (31601031, 31971164), Guangdong Special Fund
Project of Fundamental and Applied Research (2022A1515012332),
Guangzhou Planned Project of Science and Technology
(202102010032), and the Science Foundation of Guangzhou First Peo
ple’s Hospital (Q2019019).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2022.112011.
References
[1] M. Kansara, M.W. Teng, M.J. Smyth, D.M. Thomas, Nat. Rev. Cancer 14 (2014)
722–735.
[2] A.J. Chou, D.S. Geller, R. Gorlick, Paediatr. Drugs 10 (2008) 315–327.
[3] M.S. Isakoff, S.S. Bielack, P. Meltzer, R. Gorlick, J. Clin. Oncol. 33 (2015)
3029–3035.
[4] S.S. Bielack, S. Hecker-Nolting, C. Blattmann, L. Kager, F1000Res 5 (2016) 2767.
[5] S.S. Bielack, B. Kempf-Bielack, G. Delling, G.U. Exner, S. Flege, K. Helmke, R. Kotz,
M. Salzer-Kuntschik, M. Werner, W. Winkelmann, A. Zoubek, H. Jürgens,
K. Winkler, J. Clin. Oncol. 20 (2002) 776–790.
[6] E. Ward, C. DeSantis, A. Robbins, B. Kohler, A. Jemal, CA Cancer J. Clin. 64 (2014)
83–103.
[7] B. Kempf-Bielack, S.S. Bielack, H. Jürgens, D. Branscheid, W.E. Berdel, G.U. Exner,
U. Göbel, K. Helmke, G. Jundt, H. Kabisch, M. Kevric, T. Klingebiel, R. Kotz,
R. Maas, R. Schwarz, M. Semik, J. Treuner, A. Zoubek, K. Winkler, J. Clin. Oncol.
23 (2005) 559–568.
[8] E. Wiltshaw, Platin. Met. Rev. 23 (1979) 90–98.
8
F.-L. Xie et al.
Journal of Inorganic Biochemistry 237 (2022) 112011
[52] M. Redza-Dutordoir, D.A. Averill-Bates, Biochim. Biophys. Acta 1863 (2016)
2977–2992.
[53] Y.X. Zhang, P.F. Yu, Z.M. Gao, J. Yuan, Z. Zhang, Eur. Rev. Med. Pharmacol. Sci. 21
(2017) 1665–1671.
[54] J. Kale, E.J. Osterlund, D.W. Andrews, Cell Death Differ. 25 (2018) 65–80.
[55] C. He, M.C. Bassik, V. Moresi, K. Sun, Y. Wei, Z. Zou, Z. An, J. Loh, J. Fisher,
Q. Sun, S. Korsmeyer, M. Packer, H.I. May, J.A. Hill, H.W. Virgin, C. Gilpin,
G. Xiao, R. Bassel-Duby, P.E. Scherer, B. Levine, Nature 481 (2012) 511–515.
[56] I. Tamm, Y. Wang, E. Sausville, D.A. Scudiero, N. Vigna, T. Oltersdorf, J.C. Reed,
Cancer Res. 58 (1998) 5315–5320.
[57] K. Blomgren, C. Zhu, X. Wang, J.O. Karlsson, A.L. Leverin, B.A. Bahr, C. Mallard,
H. Hagberg, J. Biol. Chem. 276 (2001) 10191–10198.
[58] A. Bressenot, S. Marchal, L. Bezdetnaya, J. Garrier, F. Guillemin, F. Plénat,
J. Histochem. Cytochem. 57 (2009) 289–300.
[59] K. Zhang, S. Wu, H. Wu, L. Liu, J. Zhou, Aging (Albany NY) 13 (2021)
21090–21101.
[60] C.Y. Meng, Z.Q. Zhao, R. Bai, W. Zhao, Y.X. Wang, H.Q. Xue, L. Sun, C. Sun,
W. Feng, S.B. Guo, Oncol. Rep. 43 (2020) 1169–1186.
[61] S.Y. Kim, H. Hwangbo, M.Y. Kim, S.Y. Ji, H. Lee, G.Y. Kim, C.Y. Kwon, S.H. Leem,
S.H. Hong, J. Cheong, Y.H. Choi, Arch. Biochem. Biophys. 697 (2021), 108688.
[62] W. Yao, Z. Lin, P. Shi, B. Chen, G. Wang, J. Huang, Y. Sui, Q. Liu, S. Li, X. Lin,
Q. Liu, H. Yao, Biochem. Pharmacol. 171 (2020), 113680.
[63] Z. Xu, X. Han, D. Ou, T. Liu, Z. Li, G. Jiang, J. Liu, J. Zhang, Appl. Microbiol.
Biotechnol. 104 (2020) 575–587.
[34] S.M. Wang, J. Pang, K.J. Zhang, Z.Y. Zhou, F.Y. Chen, Mol. Ther. Oncolytics 21
(2021) 62–73.
[35] T. Liang, X. Ye, Y. Liu, X. Qiu, Z. Li, B. Tian, D. Yan, Exp. Mol. Med. 50 (2018)
1–12.
[36] M. Malumbres, M. Barbacid, Nat. Rev. Cancer 9 (2009) 153–166.
[37] S. Jitkaew, A. Trebinska, E. Grzybowska, G. Carlsson, A. Nordström, J. Lehtiö, A.
S. Fröjmark, N. Dahl, B. Fadeel, J. Biol. Chem. 284 (2009) 27827–27837.
[38] S. Kesavardhana, R.K.S. Malireddi, T.D. Kanneganti, Annu. Rev. Immunol. 38
(2020) 567–595.
[39] R.P. Chakrabarty, N.S. Chandel, Cell Stem Cell 28 (2021) 394–408.
[40] H. Sies, D.P. Jones, Nat. Rev. Mol. Cell Biol. 21 (2020) 363–383.
[41] V.N. Kotiadis, M.R. Duchen, L.D. Osellame, Biochim. Biophys. Acta Gen. Subj. 1840
(2014) 1254–1265.
[42] F. Wang, R. Gómez-Sintes, P. Boya, Traffic 19 (2018) 918–931.
[43] S.R. Bonam, F. Wang, S. Muller, Nat. Rev. Drug Discov. 18 (2019) 923–948.
[44] N. Mizushima, Genes Dev. 21 (2007) 2861–2873.
[45] J.M.M. Levy, C.G. Towers, A. Thorburn, Nat. Rev. Cancer 17 (2017) 528–542.
[46] T. Pathak, M. Trebak, Pharmacol. Ther. 192 (2018) 112–123.
[47] Z. Xia, C. Wang, X. Wang, H. Yu, H. Yao, H. Shen, X. Lan, X. Wu, G. Zhang, Toxicol.
in Vitro 72 (2021), 105104.
[48] H. Wang, N. Zhai, Y. Chen, H. Xu, K. Huang, J. Inorg. Biochem. 172 (2017) 16–22.
[49] F.J. Bock, S.W.G. Tait, Nat. Rev. Mol. Cell Biol. 21 (2020) 85–100.
[50] H.S. Wong, B. Benoit, M.D. Brand, Free Radic. Biol. Med. 130 (2019) 140–150.
[51] M.D. Brand, Free Radic. Biol. Med. 100 (2016) 14–31.
9