Lysosome-Targeted Chemotherapeutics: Half-Sandwich Ruthenium(II) Complexes That Are Selectively Toxic to Cancer Cells

Article abstract of DOI:10.1021/acs.inorgchem.8b01944

Poor selectivity between cancer cells and normal cells is one of the major limitations of cancer chemotherapy. Lysosome-targeted ruthenium-based complexes target tumor cells selectively, only displaying rather weak cytotoxicity or inactivity toward normal cells. Confocal microscopy was employed for the first time to determine the cellular localization of the half-sandwich Ru complex.

Full text of DOI:10.1021/acs.inorgchem.8b01944

Communication
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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Lysosome-Targeted Chemotherapeutics: Half-Sandwich
Ruthenium(II) Complexes That Are Selectively Toxic to Cancer Cells
Zhenzhen Tian,†,§ Juanjuan Li,†,§ Shumiao Zhang,† Zhishan Xu,†,‡ Yuliang Yang,† Deliang Kong,†
Hairong Zhang,† Xingxing Ge,† Junming Zhang,† and Zhe Liu*,†
†Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key
Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Department of Chemistry and Chemical
Engineering, Qufu Normal University, Qufu 273165, China
‡Department of Chemistry and Chemical Engineering, Shandong Normal University, Jinan 250014, China
S
* Supporting Information
this complex has very poor water solubility, which is a
ABSTRACT: Poor selectivity between cancer cells and
normal cells is one of the major limitations of cancer
chemotherapy. Lysosome-targeted ruthenium-based com-
plexes target tumor cells selectively, only displaying rather
weak cytotoxicity or inactivity toward normal cells.
Confocal microscopy was employed for the first time to
determine the cellular localization of the half-sandwich Ru
complex.
disadvantage for further application as an anticancer agent.
Lysosomes are organelles containing a variety of hydrolases
to break down endogenous and exogenous macromolecules.
Increased permeability of the membrane in lysosomes and
release of the enzyme may cause cell apoptosis. Moreover,
lysosomes are involved in many aspects of apoptosis and are
becoming an attractive anticancer target.22,23 Cyclometalated
Ru complexes containing three bidentate ligands have shown
lysosome targeting and other organelle targeting.24,25 The
inherent fluorescence of these complexes helps people to track
and monitor them successfully in cells. However, intracellular
observation for half-sandwich Ru complexes is poorly
investigated due to the absence of or very poor fluorescence.
Confocal microscopy is a powerful technique that can provide
clear cell images for cyclometalated complexes and shine a light
on their mechanisms of action (MoAs).
Inspired by these findings, herein we design and synthesize a
series of Ru complexes containing ethanol or butanol
substituted arene and N^N-chelating imino-pyridyl ligands,
Chart 1, to improve anticancer activity, selectivity, and water
solubility. Confocal microscopy and flow cytometry helped us
to provide new insights into the MoAs. The opposite
intracellular performances of the complexes in cancer and
normal cells were compared.
iomedical applications of metal-based compounds have
Bprovided an essential contribution to the development of
diagnostic and therapeutic agents.1 Platinum (Pt)-based
anticancer drugs are widely used in clinical treatment, whereas
they have encountered much resistance, undesirable non-
cancer-cell toxicity, and limited activity.2,3 These unsatisfactory
outcomes of treatment thus have spurred scientists to search
for non-platinum-metal-based anticancer complexes such as
gold, ruthenium, rhodium, osmium, and iridium complexes in
order to find better selectivity between cancer cells and normal
cells and to overcome platinum resistances.4−9 A large number
of non-platinum-metal-based complexes have shown more
potent in vitro antiproliferative activity against a wide range of
cancer cells than Pt drugs. However, they are hindered by the
absence of clear modes of action and reasonable cytotoxic
selectivity.10 In the design of novel anticancer drugs, ruthenium
(Ru) complexes have attracted a great deal of attention due to
high kinetic stability and extremely beneficial biological
properties.11−17 Moreover, three Ru complexes have entered
clinical trials.18
Chart 1. Ru Complexes 1−5 Studied in This Work
Metal compounds containing imino-pyridyl ligands, due to
their redox properties and good stability, play a significant role
in the research of catalytic processes.19 We have reported a
series of half-sandwich iridium compounds containing imino-
pyridyl ligands that displayed highly potent antiproliferative
activity against cancer cells, but poor cytotoxic selectivity.20
Substitution of the cyclopentadienyl iridium moiety with
ruthenium benzene led to a decrease of potency but better
cytotoxic selectivity.21 However, only one complex possessed
potent anticancer activity and promising selectivity simulta-
neously. In addition, it’s cellular uptake, distribution, local-
ization, and targeting organelles are still unknown. Moreover,
Received: July 12, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b01944
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Table 1. IC50 Values of Tested Complexes Towards Different Cell Lines
a
IC50 (μM)
complex
A549
HeLa
BEAS-2B
16HBE
R1
R2
1
2
3
4
81.5 0.8
33.8 0.8
53.8 0.2
20.8 2.4
19.5 2.1
21.3 1.7
98.2 3.6
35.4 1.2
53.1 0.8
23.8 0.8
18.1 0.1
7.5 0.2
109.2 17.5
68.1 2.4
71.0 13.8
84.0 15.7
93.2 9.6
42.0 2.3
144.8 12.1
199.5 13.9
152.3 17.0
147.4 7.6
144.1 5.2
18.6 2.0
1.3
2.0
1.3
4.0
4.8
2.0
1.8
5.9
2.8
7.1
7.4
0.9
5
cisplatin
a
The drug treatment period was 24 h. R1: IC50 ratio of BEAS-2B cells to A549 cells. R2: IC50 ratio of 16HBE cells to A549 cells.
Complexes 1−5 were newly prepared and characterized by
(0.47) > 2 (0.28) > 3 (0.25) > 1 (−0.58), which is consistent
with their toxicity and selectivity.
1H NMR and 13C NMR spectra, elemental analysis, and mass
spectroscopy. The X-ray crystal structure of complex 4 can be
seen in Figure S1, and the crystallographic data are listed in
Tables S1 and S2. Complex 4 adopts the expected half-
sandwich piano-stool geometry. A significant difference
between Ru−N1 (2.142(5) Å) and Ru−N2 (2.088(5) Å)
bond lengths were observed.
Complex 5 as the best candidate was selected for further
investigation of the underlying mechanisms. Complex 5 is
stable in aqueous environment, Figures S3−S6.
Coenzyme NADH and NAD+ play a vital role in numerous
biocatalyzed processes. Sadler and co-workers have shown that
NADH can donate a hydride to aqua IrIII cyclopentadienyl
complexes and produce ROS H2O2, thus providing an oxidant
MoA.32−34 We found the Ru complex 5 had strong catalytic
ability in the oxidation of NADH with a turnover number of
40.5, Figure S7, probably providing a potential pathway for
generation of ROS in cells.
Complexes 1−5 were tested for their in vitro antiproliferative
activity against human cervical carcinoma (HeLa) and human
lung cancer cells (A549). Their selectivity was also determined
by comparing the toxicity between cancer cells and normal
cells (human bronchial epithelial cells 16HBE and BEAS-2B),
Table 1 and Figure S2. Excitingly, all five complexes displayed
cytotoxicity (IC50 < 100 μM) against both cancer cell lines.
More importantly, all of them showed lower cytotoxicity
toward normal cells over cancer cells. Especially, complexes 4
and 5 possess not only as potent anticancer activity as cisplatin
but also high cytotoxic selectivity, approximately 7 times more
active toward cancer cells. The two complexes displayed great
potential as new chemotherapeutic agents as they can
effectively kill cancer cells while not causing or only causing
very weak damage to normal cells. Same antiproliferative
tendency was observed after 72 h (Table S3).
Contrary to our anticipation, the introduction of a
hydrophilic chain on the benzene ring did not decrease
anticancer activity. Dyson and co-workers also have reported
that the change in the hydrophilic chain on the benzene ring of
the RuII complexes did not have much effect on the anticancer
activity.26 Increasing the length of the carbon chain on the
arene ring may not have a significant impact on the anticancer
activity as complexes 4 and 5 showed similar antiproliferative
activities. Compared to 2, introduction of a methyl group on
the pyridine ring ligand in complex 3 reduced anticancer
activity. Excitingly, we found that complex 5 with the quinolyl
group in the ligand showed much higher anticancer efficacy
than complex 2 that contains a pyridyl group. The introduction
of the quinolyl group not only increased antiproliferative
activity but also improved the selectivity for cancer cells,
suggesting that the quinolyl group played a crucial role in
contribution to anticancer activity. Moreover, the iridium
analogues synthesized by our group were almost nonselective,
indicating that these ligands and metal ruthenium may
constitute a more specific structure with selectivity.20 Previous
results indicate that Ru could mimic Fe in the interaction with
proteins, such as transferrin and albumin. As more transferrin
receptors were located on the surface of cancer cells, Ru
complexes usually show better selectivity.27−29
The intracellular Ru contents of 5 in the nucleus, cytosol,
nuclear chromatin, and cytoskeleton fractions isolated from
A549 and BEAS-2B cells were determined,35 Table S4 and
Figure S8. Complex 5 is mainly accumulated in the cytosol as
compared with the nucleus, suggesting a cytosol-targeting
pathway. Very interestingly, the Ru content in A549 cells is
about 2.5 times higher than that in BEAS-2B normal cells,
indicating that 5 is more easily absorbed by cancer cells.
Although the cyclometalated Ru complexes have been
successfully observed with help of their luminescent prop-
erty,36 for a long time, however, half-sandwich Ru metal
complexes have been generally thought to be invisible in cells
due to a lack of effective conjugate structure and therefore a
lack of fluorescent property. This becomes a major obstacle to
understanding their anticancer mechanisms. Very excitingly,
we successfully observed the intracellular images of the half-
sandwich complex 5 and determined the specific location in
both A549 and BEAS-2B cells with help of confocal laser
scanning microscopy. As shown in Figure 1, complex 5
specifically localized in the lysosome in A549 cells with a high
Pearson’s colocalization coefficient (0.81−0.93) and showed
no different organelle-targeting behavior after 6 h.23 On the
contrary, very low overlap with mitochondria (0.05−0.09), the
Golgi apparatus (0.00), and nucleus (0.00) was observed for 5,
Figures S9−S11.37 Similarly, complex 5 also mainly targeted
the lysosome in BEAS-2B cells (Figures S12−S15). However,
due to low uptake of the complex in BEAS-2B cells, only rather
weak luminescence intensity was observed. These results
indicate that 5 could specifically label lysosomes.
Next, we tested the lysosomal integrity of A549 cells in the
presence of 5 by confocal microscopy with Acridine orange
(AO) staining. The red fluorescence intensity decreased with
the increase of 5, indicating that the integrity of lysosomes was
destroyed, Figure 2. It has been reported that drugs targeting
lysosomes may result in an increase of intracellular ROS, which
in turn leads to increased lysosomal permeability.38,39 The
increase of lysosomal permeability may be one of the pathways
inducing cell apoptosis.
Cellular uptake efficiency often correlates with their log Po/w
values.30,31 We compared the log P values of 5 (0.59) > 4
B
DOI: 10.1021/acs.inorgchem.8b01944
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We investigated the interaction of complex 5 with BSA,
indicating that 5 has a certain binding ability to BSA.
Therefore, the compound may also bind to other proteins.
More details of interaction can be seen in the Supporting
Information. Lysosome contains a wide range of membrane
proteins, such as acid sphingomyelinase (ASM), which are
responsible for the lysosomal integrity.43,44 Probably, complex
5 also binds to these proteins that are responsible for
specifically targeting lysosomes, and resulting in the
destruction of lysosomal integrity. In addition, the OH−
groups in the long tail of the benzene ring make the complexes
stable in an acid environment, probably also contributing to
the selectivity toward acidic cancer cells and lysosome.
In summary, five novel half-sandwich Ru compounds
containing N^N-chelating imino-pyridyl ligands were synthe-
sized. The complexes showed significant selectivity toward
cancer cells over normal cells. In addition, the complexes
selectively targeted lysosome and caused injury toward the
integrity of lysosome. The complexes displayed catalytic
activity for conversion of NADH to NAD+ and possibly
contributed to the production of ROS. Moreover, the
complexes promotes apoptosis through production of ROS,
mitochondrial dysfunction, and lysosomal destruction in
cancer cells, while the same events do not happen in normal
cells. This class of complexes has great potential to be
developed as novel anticancer agents with fewer side effects.
Figure 1. Determination of intercellular localization of complex 5 in
A549 cells by confocal microscopy. The A549 cells were labeled with
LysoTracker Deep Red (LTDR) and then exposed to complex 5 (5
μM) for various periods of time. Lysosomes and Ru complex were
visualized by red and green fluorescence, respectively. The Ru
complex was excited at 488 nm, and the emission was collected at 550
30 nm. LTDR was excited at 594 nm, and the emission was
collected at 630 30 nm. Scale bar: 10 μm.
ASSOCIATED CONTENT
* Supporting Information
■
Figure 2. Observation of lysosomal disruption in A549 cells caused by
complex 5 with AO (5 μM) staining.
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b01944.
Small molecules penetrate the cell membrane via energy-
dependent and non-energy-dependent pathways.40,41 Complex
5 enters both cancer cells and normal cells through a non-
energy-dependent pathway, Figures S16−S17.
Experimental details, characterization data, supplemen-
tary tables and figures (PDF)
Flow cytometry results suggested that 5 induced clear
apopotosis in A549 cells with a concentration-dependent
increasing manner; however, no obvious changes were
observed in BEAS-2B cells with the increase of concentration
of 5, Figures S18−S19, and Tables S5 and S6.
Accession Codes
CCDC 1828898 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
We further determined the level of ROS in A549 cells and
BEAS-2B cells induced by complex 5 measured by flow
cytometry, Figure S20. In comparison with the untreated cells,
0.25 × IC50 treatment in A549 cells resulted in an obvious
increase in mean fluorescence intensity, while there were no
significant changes in BEAS-2B cells. The dramatic increase of
ROS in A549 cells may be an important pathway of cell death.
Mitochondria play an important role in cell survival.42 To
further confirm the effect of complex 5 on cell apoptosis, the
changes of mitochondrial membrane potential (ΔΨm) in A549
cells and BEAS-2B cells after exposure to complex 5 were
determined using flow cytometry. The impairment in ΔΨm
induced by 5 was clearly concentration-dependent in A549
cells, and no significant changes were observed in BEAS-2B
cells (Figure S21 and Tables S7 and S8). These results are
consistent with the data from the ROS assays.
The cytotoxicity of many anticancer drugs is often linked to
genomic DNA damage and cell cycle disturbance. However, no
obvious cell cycle arrest were observed for 5 for both A549 and
BEAS-2B cells (Figures S22 and S23 and Tables S9 and S10).
The antimetastatic property of complex 5 was evaluated by a
wound-healing assay using A549 cells, Figure S24. Complex 5
has weak antimetastatic properties (12.37%).
AUTHOR INFORMATION
Corresponding Author
*E-mail: liuzheqd@163.com.
■
ORCID
Zhe Liu: 0000-0001-5796-4335
Author Contributions
§The first two authors are equal first authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China
(Grant No. 21671118) and the Taishan Scholars Program for
support.
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DOI: 10.1021/acs.inorgchem.8b01944
Inorg. Chem. XXXX, XXX, XXX−XXX