Photodamaging of Mitochondrial DNA to Overcome Cisplatin Resistance by a RuII–PtII Bimetallic Complex

Article abstract of DOI:10.1002/chem.201803630

Emerging studies have shown that mitochondrial DNA (mtDNA) is an attractive target for anticancer therapeutics. Herein, a heterobimetallic complex [Ru(dip)₂(μ-bpm)PtCl₂]Cl₂ (RuPt; dip=4,7-diphenyl-1,10-phenanthroline; bpm=2,2′-bipyrimidine) and the corresponding mononuclear complex [Ru(dip)₂(bpm)]Cl₂ (Ru) have been designed and synthesized. RuPt can bind to mtDNA and damage it both in the dark and upon visible light irradiation. By using a variety of methods, it was demonstrated that RuPt can interfere with the function of mtDNA by decreasing the amplification and copy number of mtDNA, and affecting the transcriptional level of mitochondria-encoded genes. Furthermore, RuPt can disturb the physiological processes of mitochondria and induce caspase-dependent apoptosis in the presence of light. In addition, RuPt shows low systemic toxicity and potent in vivo anticancer potency upon light irradiation. This study provides strong evidence that mtDNA is an important molecular target of RuPt, and photodamaging mtDNA is an effective strategy to overcome cisplatin resistance.

Full text of DOI:10.1002/chem.201803630

A Journal of
Accepted Article
Title: Photodamaging Mitochondrial DNA to Overcome Cisplatin
Resistance by a Ru(II)-Pt(II) Bimetallic Complex
Authors: Yue Zheng, Dong-Yang Zhang, Hang Zhang, Jian-Jun Cao,
Cai-Ping Tan, Liang-Nian Ji, and Zong-Wan Mao
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To be cited as: Chem. Eur. J. 10.1002/chem.201803630
Link to VoR: http://dx.doi.org/10.1002/chem.201803630
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Photodamaging Mitochondrial DNA to Overcome Cisplatin
Resistance by a Ru(II)-Pt(II) Bimetallic Complex
Yue Zheng, Dong-Yang Zhang, Hang Zhang, Jian-Jun Cao, Cai-Ping Tan*, Liang-Nian Ji and Zong-
Wan Mao*
Abstract: Emerging studies have shown that mitochondrial DNA
(mtDNA) is an attractive target for anticancer therapeutics. Herein, a
heterobimetallic complex [Ru(dip)2(μ-bpm)PtCl2]Cl2 (RuPt; dip = 4,7-
et al. designed a mtDNA-targeted cisplatin derivative with
mitochondria-penetrating peptide that could induce apoptosis
without damaging nuclear DNA (nucDNA).[7] Rerouting an active
nucDNA alkylation agent to mitochondria can effectively combat
drug deactivation and resistance.[8]
diphenyl-1,10-phenanthroline; bpm
= 2,2'-bipyrimidine) and the
corresponding mononuclear complex [Ru(dip)2(bpm)]Cl2 (Ru) have
been designed and synthesized. RuPt can bind to mtDNA and
damage it both in dark and upon visible light irradiation. By using a
variety of methods, we show that RuPt can interfere with the
function of mtDNA by decreasing the amplification and copy number
of mtDNA, and affecting the transcriptional level of mitochondria-
encoded genes. Furthermore, RuPt can disturb the physiological
processes of mitochondria and induce caspase-dependent apoptosis
in the presence of light. In addition, RuPt shows low systemic
toxicity and potent in vivo anticancer potency upon light irradiation.
Our study provides strong evidence that mtDNA is an important
molecular target of RuPt, and photodamaging mtDNA is an effective
strategy to overcome cisplatin resistance.
Photodynamic therapy (PDT) can damage neoplastic cells
by producing massive cytotoxic reactive oxygen species
(ROS).[9] Light-activated PDT allows accurate targeting of tumor
sites and can reduce the off-site effects associated with
systemic chemotherapies.[10] Ruthenium complexes have been
proved to be potent photosensitizes.[11-12] For example, TLD-
1433, a visible light-activated ruthenium complex, is recently
infused into clinical trials in Canada for bladder cancer.[13]
The interaction of octahedral ruthenium polypyridyl
complexes with different DNA structures has been intensively
explored.[14] The coordination-saturated ruthenium complexes
can bind to DNA mainly via non-covalent interactions, e.g.,
intercalation, non-specific electrostatic association and groove
binding.[14-15] DNA is also generally considered as the major
biological target of cisplatin, and cisplatin can damage DNA by
forming intrastrand or interstrand cross-links through covalently
binding to the N7 position of purine bases.[16] Interestingly, a
recent genome-wide profiling study showed that mtDNA was an
important target of cisplatin.[17]
Introduction
Mitochondria are essential cellular organelles that play central
roles in energy production and cell death, and they are
considered to be potential anticancer drug targets.[1] Human
mitochondrial genome is a tiny 16.6 kb circle containing 37
genes coding for 2 rRNAs, 22 tRNAs and 13 essential subunits
of the oxidative phosphorylation (OXPHOS) system.[2] Individual
mitochondrion contains multiple copies of mitochondrial DNA
(mtDNA) that are packaged into structures known as
nucleoids.[3] The genome multiplicities, high mutation rates and
limited repair capacities of mtDNA are related to several
diseases, such as cancer, cardiac failure and degenerative
diseases.[4] MtDNA is devoid of protective histone and nucleic
excision repair (NER), and it is more susceptible to damage by
reactive oxygen species (ROS), chemical carcinogens, and
Due to their high lipophilicity, the positively charged
ruthenium complexes may specially target mitochondria, similar
to other lipophilic cations such as triphenylphosphonium and
rhodamine 123.[18] Herein, we design a heterobimetallic complex
[Ru(dip)2(μ-bpm)PtCl2]Cl2 (RuPt; dip
=
4,7-diphenyl-1,10-
phenanthroline; bpm = 2,2'-bipyrimidine) as a PDT agent that
can damage mtDNA (Figure 1a and 1b). RuPt contains a
ruthenium polypyridyl moiety that can target mitochondria and
be photo-activated to generate singlet oxygen (1O2). As ROS,
particularly singlet oxygen, generated during PDT have
extremely short half-life and small radius of action, the platinum
moiety is expected to rivet the complex near mtDNA by covalent
interactions. The in vitro DNA-binding properties and induction of
mtDNA damage at the cellular level were investigated. The
induction of mitochondrial dysfunction and apoptotic cell death
by RuPt in the presence of light were demonstrated. Finally, we
investigated the in vivo effects of RuPt-mediated PDT in a
human tumor xenograft model. In all cases, the mononuclear
complex [Ru(dip)2(bpm)]Cl2 (Ru) was kept as the control. As far
as we know, this is the first detailed investigation that shows
mtDNA is a valid molecular target for metal-based photodynamic
therapeutic agents. In all, our work provides a new insight into
the design of novel metal-based anticancer drugs with high
potential to overcome cisplatin resistance.
ionizing radiation as compared with nucDNA.[5]
A higher
percentage of damaged/mutated mtDNA can disrupt
mitochondrial function and implicate in many biological
processes related to tumorigenesis and cancer progression.[6]
Although mtDNA is emerging as an important target for cancer
therapy, the reports on the action mechanisms of anticancer
compounds targeting mitochondria are still very limited. Lippard
[a]
Y. Zheng, Dr. D. Zhang, H.Zhang, J. Cao, Dr. C. Tan, Prof. L. Ji,
Prof. Z. Mao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry, Sun Yat-Sen University
Guangzhou 510275 (P.R. China)
Email: cesmzw@mail.sysu.edu.cn; tancaip@mail.sysu.edu.cn
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
Results and Discussion
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Synthesis, characterization and photophysical properties
Figure 2. Photooxidation of DPBF (50 μM) by Ru and RuPt in aerated DMSO.
Changes in absorbance of DPBF at 417 nm upon irradiation at 450 nm (17
mW cm‒2) in the presence of Ru and RuPt were monitored. MB was used as
the standard.
RuPt to generate photosensitized 1O2 was determined by
monitoring the photooxidation of 1,3-diphenylisobenzofuran
(DPBF). The 1O2 quantum yield (Ф△) was calculated using
methylene blue (MB, Ф△ = 0.52) as the standard (Figure 2).
These results show that the Ф△ values of Ru and RuPt are 0.22
and 0.16, respectively. These values fall in the range of Ф△
values reported for other ruthenium complexes with similar
structures.[22]
Figure 1. a) Chemical structures of complexes Ru and RuPt. b) Schematic
illustration of the action mechanisms of RuPt.
Complex Ru was synthesized by refluxing the precursor cis-
Ru(dip)2Cl2 and the ligand bpm in an ethanol-water solution.
RuPt was synthesized by reacting cis-Pt(DMSO)2Cl2 (DMSO =
dimethyl sulfoxide) with Ru in ethanol. The synthetic route is
showing in Scheme S1† (Supporting Information). Pure products
1
of Ru and RuPt were characterized by H NMR spectroscopy,
DNA interaction and damage in vitro
ESI-MS and CHN elemental analysis (Figure S1‒S4, Supporting
Information).
Coordination-saturated Ru(II) polypyridyl complexes can bind
with DNA noncovalently.[15] On the contrary, cisplatin and its
derivatives can form irreversible covalent bonds with the N
atoms of the DNA bases. First, we studied the binding of Ru and
RuPt with calf thymus DNA (CT-DNA) by UV/Vis spectroscopy.
Upon the addition of DNA, the hypochromism in the MLCT
bands of Ru and RuPt reach about 58.9% and 61.0% at 422 nm,
respectively (Figure S6, Supporting Information). Meanwhile, an
obvious decline in the fluorescence of ethidium bromide (EB)
bound to CT-DNA was detected upon the addition of Ru and
RuPt (Figure S7, Supporting Information). The reduction in the
emission intensity is about 40.2% and 23.6% for RuPt and Ru at
a [Ru]/[DNA] ratio of 0.80, respectively.
Atomic force microscopy (AFM) was utilized to explore the
morphology of Ru- and RuPt-DNA adducts. Free pBR322 DNA
exists as small loose clews with a diameter of about 30 nm
(Figure 3a), which is the characteristic morphology of intact
DNA.[24] After pBR322 DNA is mixed with Ru, a dose-dependent
increase in the sizes of DNA adducts (from 100 nm to 400 nm)
can be detected, which may be attributed to electrostatic forces
between the negatively charged DNA and the positively charged
ruthenium complex.[25] Different morphological alternations,
consisting of regular local kinks and cross-links, are observed for
RuPt-DNA adducts. These results suggest that the DNA-binding
modes of Ru and RuPt are different, and RuPt can interact with
DNA through covalent binding.
Ru and RuPt exhibit intense absorption in phosphate-
buffered saline (PBS), acetonitrile and dichloromethane at 298 K
in the visible light range at approximately 350‒500 nm and 350–
650 nm, respectively (Figure S5, Supporting Information).[19] The
intense absorption band in the UV region of Ru and RuPt is
attributed to the dip ligand-based π‒π* and n‒π* transitions.
The broad lower energy band and the lowest band in the visible
region are assigned to the d(Ru)→bpm (π*) and d(Pt)→bpm (π*)
metal–to–ligand charge transfer (1MLCT) states, respectively.[20]
It can be seen that the absorption spectrum of RuPt is
moderately red-shifted as compared with that of Ru, which can
be attributed to the heavy metal effects caused by the
introduction of Pt(II) center onto N^N unit of pyrimidines.[21]
The emission spectra of Ru and RuPt were studied in
deoxygenated PBS, acetonitrile and dichloromethane at 298 K
and the emission data are summarized in Table S1 (Supporting
Information). Both complexes show very weak emission with
quantum yields fall between the range of 0.0009‒0.0265.
Singlet oxygen (1O2) generation
Upon irradiation, the photosensitized molecules cross over to a
triplet state and transfer energy to molecular oxygen (3O2) to
generate singlet oxygen (1O2), which is considered to be the
main cytotoxic species during PDT.[10] The efficiency of Ru and
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Information). As for whole cell measurement, Ru shows slightly
higher cellular uptake efficacy than RuPt. To verify the
subcellular distribution of Ru and RuPt, the mitochondrial,
cytosolic and nuclear fractions were isolated from human lung
adenocarcinoma epithelial (A549) cells treated with Ru and
RuPt. The content of ruthenium in mitochondria is much higher
than those obtained in cytosol and nuclei for cells treated with
Ru and RuPt. The result is reasonable as Ru and RuPt conform
to the molecular characteristics that can accumulate in the
mitochondria (electric charge Z > 0; 0 < log Po/w < +5).[26] In
addition, similar results are obtained for RuPt using two different
standards (101Ru and 195Pt), which demonstrates the association
of two metal moieties. Interestingly, the accumulation of the
metal content in mitochondria is more obvious for RuPt than that
measured for Ru. The results indicate that RuPt can
preferentially localize to mitochondria, which offer the possibility
for it to bind with mtDNA.
Cellular mtDNA binding and damage
As RuPt can irreversibly bind with DNA and accumulate in
mitochondria, we hypothesize that mtDNA may be an important
cellular target of RuPt. Firstly, we measured the platination of
mtDNA and nucDNA isolated from RuPt-treated cells (Figure 4a).
In RuPt-treated cells, the platination level of mtDNA shows a
time-dependent increase, which is much higher than that
obtained for nucDNA. In contrast, cisplatin can associate with
both mtDNA and nucDNA, with only a slight preference
observed for mtDNA.
Figure 3. a) AFM analysis of the impact of Ru and RuPt on the formation and
morphology of pBR322 plasmid DNA aggregates. The color code represents
the height (in nm) with respect to the bare surface. [DNA] = 5 μM. b) In vitro
pBR322 plasmid DNA damage caused by Ru and RuPt in dark or under
irradiation using agarose gel electrophoresis analysis. Irradiation condition:
450 nm, 17 mW cm‒2, 15 min.
PicoGreen is a DNA-intercalating fluorophore that can label
mtDNA.[27] The bright fluorescence of PicoGreen is partly
quenched in A549 cells treated with RuPt (Figure 4b). At the
same time, the formation of giant and bright nucleoids are
observed, which is known as nucleoid remodeling and
The binding and damage of plasmid DNA by Ru and RuPt
were examined using agarose gel electrophoresis in vitro both in
dark and under irradiation (450 nm, 15 J cm−2). As compared
with the control samples, plasmid DNA incubated with RuPt
considered to be
a
sign of mtDNA damage.[28] Gel
electrophoresis shows that the fluorescent bands of mtDNA
isolated from Ru- and RuPt-treated cells is greatly weakened,
which indicates the damage of mtDNA (Figure S9, Supporting
Information). Under light conditions (450 nm, 15 J cm−2), the
phenomenon is more obvious.
To investigate the selectivity of Ru and RuPt towards
mtDNA, we assessed the efficiency of PCR (polymerase chain
reaction) amplification of an 8.9 kb mitochondrial genomic
segment and a 12.2 kb nuclear genomic segment. Compared
with the controls, Ru and RuPt cause significant reductions in a
dose-dependent manner in amplification of mtDNA segment but
not nucDNA segment upon irradiation (Figure 4c and Figure S10,
Supporting Information).
shows
a decrease in the electrophoretic mobility of the
supercoiled form (Form I). The phenomenon may be attributed
to the covalent binding of RuPt to DNA, which is not observed
for Ru (Figure 3b). Enhanced cleavage is observed for DNA
samples treated with Ru and RuPt upon irradiation as
evidenced by the marked increase in the relative amount of
nicked circular form (Form II) and linear form (Form III) DNA,
which can be attributed to the DNA oxidation caused by singlet
oxygen or other ROS. These results indicate that two metal
centers may play synergistic roles in DNA damage: the Ru-
based PS generates oxidative stress while the Pt-based moiety
promotes DNA platination.
An immunofluorescence assay was further used to assess
the capacity of Ru and RuPt to induce mtDNA damage. Cells
treated with Ru and RuPt in the absence and presence of light
were immunostained with the antibody against 8-oxoguanine (8-
OG), a common DNA lesion resulted from the oxidation of
Lipophilicity and localization
The log Po/w (in which Po/w = the octanol/water partition
coefficient) values that represent the lipophilicity of Ru (0.63)
and RuPt (0.45) were determined by a classical shake-flask
method. As both ruthenium and platinum are exogenous
elements, the cellular uptake levels and subcellular distributions
of them are quantitatively determined by inductively coupled
plasma-mass spectrometry (ICP-MS, Figure S8, Supporting
guanine nucleotides.[29]
A
significant increase in 8-OG
fluorescence, localized exclusively in mitochondria, is detected
in cells treated with RuPt either in dark or upon irradiation
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Figure 4. a) Platination levels of nucDNA and mtDNA of A549 cells incubated with RuPt (10 μM) or cisplatin (10 μM) for 6 h or 12 h. b) Observation of mtDNA
stained with PicoGreen in A549 cells treated with RuPt (10 µM) at the indicated time intervals. c) Impact of Ru and RuPt on DNA amplification of an 8.9 kb
mtDNA segment and a 12.2 kb nucDNA segment. Cells were treated with Ru (4 µM) or RuPt (4 µM) with/without irradiation. d) Alteration of mtDNA copy number
in A549 cells treated with Ru or RuPt treatment with/without irradiation. (e and f) The impact of Ru (4 μM) and RuPt (4 μM) on the transcription of mitochondrial
genes
determined
by
RT-qPCR.
Housekeeping
gene:
gapdh.
Irradiation
condition:
450
nm,
17
mW
cm‒2
,
15
min.
(Figure S11, Supporting Information). In contrast, Ru can
effectively damage mtDNA only upon irradiation.
Compared with the unique nuclear genome, each cell
contains plentiful copies of mitochondrial genome. It has been
reported that mtDNA damage caused by anticancer agents, e.g.,
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decreased transcription caused by Ru- and RuPt-treatment may
greatly impact the related mitochondrial functions. These results
collectively indicate that RuPt can selectively damage mtDNA
rather than nucDNA, which may further lead to mitochondrial
dysfunction.
Photodynamic anticancer activity in vitro
The cytotoxicities of Ru and RuPt either in dark or under
irradiation were evaluated in A549, cisplatin-resistant A549
(A549R), human cervical cancer (HeLa), mtDNA-knockdown
HeLa (HeLa-ρ0), human prostate carcinoma (PC3), human
colorectal adenocarcinoma (HT-29), mouse colon carcinoma
(CT26) and human normal liver (LO2) cells by a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay after 48 h treatment (Table S2 and S3, Supporting
Information). RuPt shows very low cytotoxicity in the dark, with
most of the IC50 values being higher than 100 μM, which
indicates that it is suitable for PDT applications. The dark
cytotoxicity of Ru is higher than that of RuPt in all cell lines
tested.
Upon visible light irradiation (450 nm, 15 J cm−2), the
phototoxicity index (PI) values obtained for RuPt are much
higher than those obtained for Ru. The highest PI values for Ru
(PI = 73.6) and RuPt (PI > 119.0) are observed in A549 cells.
The phototoxicity of RuPt is higher than some of the ruthenium
PSs reported until now[32-33] but still lower than some of the
highly active PSs based on ruthenium.[34-35] In addition, RuPt
shows higher phototoxicity than Ru in A549 cells upon
irradiation with lower doses (Table S4, Supporting Information).
Moreover, the clinically approved PS 5-amino-levulinate acid (5-
ALA) displays lower phototoxicity and PI values than RuPt under
the similar conditions.
The high phototoxicities of Ru and RuPt are retained in
A549R cells, which indicates that the Ru- and RuPt-initiated
PDT strategy is able to bypass cisplatin resistance (Figure 5a
and 5b). As expected, cisplatin is much less effective in killing
cisplatin insensitive HT-29 cells (IC50 = 35.9 μM) than cisplatin
sensitive PC3 cells (IC50 = 6.1 μM). However, the phototoxicities
of Ru and RuPt obtained in HT-29 cells are generally
approximate to those measured in PC3 cells (Figure 5c and 5d),
which indicates that the anticancer spectra of Ru- and RuPt-
mediated PDT are different from that of cisplatin. Importantly,
knockdown of mtDNA in HeLa cells diminishes the cytotoxicity of
RuPt both in dark and under irradiation. RuPt shows
approximately 6-fold lower phototoxicity in HeLa-ρ0 cells than
that in HeLa cells (Figure 5e and 5f), which infers that mtDNA is
an important target for RuPt. Additionally, both Ru and RuPt
show approximately 5-fold lower photo-induced cytotoxicity in
LO2 cells than that in A549 cells. These results indicate that
RuPt-mediated PDT can overcome the intrinsic/acquired drug
resistance of cisplatin, and mtDNA plays a vital role in RuPt-
mediated PDT.
Figure 5. Dose-dependent curves showing the phototoxicity of RuPt and Ru
in a) A549 wt (wild-type), b) A549R (cisplatin resistant) cells, c) PC3 (cisplatin-
sensitive), d) HT29 (cisplatin-insensitive) cells, e) HeLa wt (wild-type) and f)
HeLa-ρ0 (mtDNA depletion) cells. Irradiation condition: 450 nm, 17 mW cm‒2
,
15 min.
nucleoside analogs and DNA cross-linking agents, can block
mtDNA replication and result in mtDNA depletion.[28] Thus, we
determined the mtDNA copy number relative to nucDNA in Ru-
and RuPt-treated A549 cells using a reverse transcription-
quantitative real-time PCR (RT-qPCR) method.[30] After 12 h
treatment of Ru and RuPt and immediate irradiation, a dose-
dependent decrease in mtDNA copy number is observed in
A549 cells as compared with the controls (Figure 4d).
Finally, we evaluated the effect of Ru and RuPt on the
transcriptional levels of 13 genes located in human
mitochondrial genome using RT-qPCR. These genes encode
essential polypeptides that belong to mitochondrial electron
transport chain (ETC) and the vital sectors of the oxidative
phosphorylation (OXPHOS) system.[31] On a standard of more
than two fold changes (FC > 2), most of these genes, especially
for dehydrogenase subunit genes (ND genes), show dose-
dependent decreased transcription in A549 cells treated with Ru
and RuPt. The impact of them on gene expression is more
obvious for RuPt than Ru either in dark or under irradiation
(Figure 4e, 4f, and Figure S12‒S14, Supporting Information).
Similar results are obtained by using two different housekeeping
genes, gapdh and β-actin (Figure S15 and S16, Supporting
Information). Considering these ND genes are involved in
mitochondrial glycolytic metabolism and respiration,[4] the
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Figure 6. a) Detection of MMP in cells by JC-1 staining and flow cytometry after PDT treatment with Ru and RuPt. A549 cells were incubated with vehicle, Ru
and RuPt for 12 h at 37 °C before irradiation (450 nm, 15 J cm‒2) and then stained with JC-1. λex = 488 nm. λem = 530 ± 30 nm (green) and 585 ± 30 nm (red). b)
Intracellular ATP levels in A549 cells. The cells were treated with the vehicle, Ru and RuPt at the indicated concentrations for 12 h at 37 °C before irradiation (450
nm, 17 mW cm‒2, 15 min). c) The respiratory profiles of A549 cells treated with Ru and RuPt. The cells were treated with the vehicle, Ru and RuPt the indicated
concentrations for 12 h at 37 °C before irradiation (450 nm, 5 J cm−2). OCR values were recorded after the sequential addition of oligomycin (1.0 μM), FCCP (0.8
μM), and a mixture of antimycin A and rotenone (0.5 μM) (R/A). d) Basal cell respiration corresponds to the difference between OCR before the injection of
oligomycin and OCR after the addition of a mixture of rotenone and antimycin A (R/A). e) ATP production values was obtained by subtracting OCR values after
the addition of oligomycin from OCR values before the injection of oligomycin (R/A). f) Maximal cell respiration corresponds to the difference between OCR after
the injection of FCCP and OCR after the addition of a mixture of rotenone and antimycin A (R/A). g) Non-mitochondrial cell respiration is the OCR values after the
addition of a mixture of rotenone and antimycin A (R/A).
Induction of mitochondrial dysfunction
After A549 cells are treated with Ru and RuPt in the dark, a
small portion of cells lose their MMP. Upon irradiation (450 nm,
15 J cm−2), a marked decrease in MMP can be observed in Ru-
and RuPt-treated cells, as evidenced by the fluorescence shift
from red to green. The percentages of cells with depolarized
mitochondria increase from 10.4 ± 1.1% to 70.7 ± 6.6% and 62.5
± 5.7% for Ru (4 μM) and RuPt (4 μM) in the presence of light,
As Ru and RuPt could be localized to mitochondria and cause
mtDNA damage effectively, their impact on mitochondrial
integrity was investigated. First, the changes in mitochondrial
membrane potential (MMP) was monitored by 5,5’,6,6’-
tetrachloro-1,1’-3,3’-tetraethyl-benzimidazolylcarbocyanine
iodide (JC-1) staining detected by flow cytometry (Figure 6a).
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respectively. In addition, RuPt induces more depolarized
mitochondria than Ru with lower irradiation doses (9 and 3 J
cm−2) (Figure S17, Supporting Information). Accordingly, Ru and
RuPt cause a significant dose-dependent decrease in ATP
production as compared with the control cells (Figure 6b). Upon
irradiation (450 nm, 15 J cm−2), the ability of Ru and RuPt to
reduce the ATP content in A549 cells is correlated with their
ability to depolarize MMP. At a concentration of 4-fold of IC50,
the ATP levels decrease by 78.3 ± 2.6% and 73.1 ± 2.3% in Ru-
and RuPt-treated cells, respectively. Similarly, RuPt causes
more pronounced impact on ATP production than Ru with lower
irradiation doses (9 and 3 J cm−2) (Figure S18, Supporting
Information).
The impact of Ru and RuPt on mitochondrial respiratory
capacity was investigated by evaluating the oxygen consumption
rate (OCR) using a Seahorse XF24 extracellular flux analyzer.
The assay uses several respiration modulators that target the
components of ETC to measure the key parameters of
mitochondrial OXPHOS (Figure 6c).[36] Compared with the
control group, Ru- and RuPt-mediated PDT cause a decrease in
basal OCR (Figure 6d), a dose-dependent decrease in ATP
production (Figure 6e), a decrease in the maximum respiration
(Figure 6f) and a decrease in non-mitochondrial respiration
(Figure 6g).
Elevation of intracellular ROS levels
Damage to mtDNA can produce ROS,[37] and intracellular ROS
elevation induced by Ru and RuPt upon irradiation was detected
by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining.
H2DCFDA is nonfluorescent and can be converted to the highly
fluorescent 2′,7′-dichlorofluorescein (DCF) by cellular ROS.[38]
Flow cytometric analysis shows a concentration-dependent
increase in DCF fluorescence in A549 cells treated with Ru or
RuPt under irradiation (Figure S19a, Supporting Information).
Compared with the control cells, a ca. 31- and 28-fold increase
in the mean fluorescence intensity (MFI) of DCF is observed in
A549 cells treated with Ru (4 μM) or RuPt (4 μM) in combination
with light (15 J cm−2), respectively. Additionally, RuPt induces
higher ROS levels than that of Ru upon irradiation with lower
doses. Moreover, pretreatment of cells with N-Acetyl-Cysteine
(NAC), an efficient quencher of ROS, leads to marked inhibition
of cell death (Figure S19b, Supporting Information). Meanwhile,
Figure 7. a) Mitochondrial ROS production caused by Ru- or RuPt-treatment.
A549 cells were incubated with Ru (1 μM) or RuPt (1 μM) for 12 h and then
irradiated with a 450 nm LED light (17 mW cm‒2, 5 min.). The cells were
stained with H2DCFDA and MTDR and analyzed by confocal microscopy. b)
Assessment of mitochondrial O2 production by MitoSox staining. A549 cells
were treated with Ru (4 μM) or RuPt (4 μM) for 12 h and then irradiated with a
450 nm LED light (17 mW cm‒2, 15 min.). Scale bars: 10 μm.
–
observed in Ru- and RuPt-treated cells upon irradiation (450 nm,
15 J cm−2). The percentage of cells in the G0/G1 phase
increases from 52.2 ± 2.6% (control) to 76.6 ± 1.9% and 70.8 ±
2.0% for Ru (4 μM) or RuPt (4 μM) in the presence of light,
respectively. However, cisplatin causes G2-M cell cycle arrest.
The changes in cell morphology upon Ru- and RuPt-
mediated PDT treatment were first examined by 2′-(4-
ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-
benzimidazole trihydrochloride (Hoechst 33342) staining (Figure
S21, Supporting Information). Control cells show normal overall
morphology and a homogeneous nuclear staining pattern. After
A549 cells are treated with Ru and RuPt, the proportion of cells
with typical apoptotic morphology increases. Meanwhile,
externally exposed phosphatidylserine, one of the characters of
apoptosis, was determined by annexin V labeling measured by
flow cytometry (Figure S22a, Supporting Information). Apoptotic
cells increase significantly in a PS concentration-dependent
manner upon PDT treatment. The percentage of apoptotic cells
increases from 5.3 ± 0.7% (control) to 57.2 ± 2.9% and 51.3 ±
3.6% after Ru- and RuPt-treatment (4 μM) in combination with
light at different irradiation doses. An approximately 2.5- and
1.8-fold increases in caspase-3/7 activity is observed in cells
a
high colocalization of the fluorescence of DCF and
MitoTracker Deep Red (MTDR) is observed in Ru- and RuPt-
treated cells (Figure 7a), indicating that ROS generated in PDT
treatment originates from mitochondria. In addition, MitoSox (a
–
mitochondrial O2 indicator) staining shows that RuPt induces
‒
significant increases in mitochondrial superoxide (O2 ) in A549
cells following PDT treatment, which is much higher that caused
by Ru-mediated PDT (Figure 7b).
Cell cycle arrest and induction of apoptosis
Mitochondria mediate essential cell functions such as cell-cycle
control and apoptosis.[39] Cell cycle arrest in A549 cells induced
by Ru and RuPt was analyzed by flow cytometry using
propidium iodide (PI) staining (Figure S20, Supporting
Information). A dose-dependent G0/G1 cell cycle arrest is
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PDT decrease the tumor volume by 65.7 ± 8.5% and 74.0 ±
9.3%, respectively (Figure 8a, 8b and Figure S23 in Supporting
Information). Under dark condition, Ru and RuPt can also inhibit
tumor growth, with RuPt shows higher potency than Ru.
Hematoxylin-eosin (H&E) staining of the tumor sections also
indicates that RuPt causes a higher level of tumor cell death
than Ru both in dark and upon irradiation (Figure S24,
Supporting Information). There is no mouse death or obvious
body weight loss during the experiment (Figure S25, Supporting
Information), which indicates Ru and RuPt have low side effects
and systematic toxicities under these treatments.
Conclusions
In summary,
a heterobimetallic complex RuPt has been
developed as the mtDNA-targeted PDT agent. RuPt can bind
with DNA more efficiently than the mononuclear complex Ru
because RuPt can form covalent and non-covalent interactions
with DNA. RuPt can cause platination and damage of mtDNA
rather than nucDNA, and it can influence the amplification, copy
number, nucleoid remodeling, and transcription of mtDNA in the
presence of light. As a consequence, in the presence of light,
RuPt can cause mitochondrial dysfunction, including MMP
collapse, ATP depletion, attenuation of mitochondrial energy
status and generation of massive mitochondrial ROS.
Furthermore, we show that RuPt can induce caspase-
dependent apoptosis upon light irradiation. Finally, RuPt-
mediated PDT can almost eliminate solid tumor. In all, our study
demonstrate that by rational molecular design, targeting mtDNA
may be an effective strategy to design effective metal-based
PDT agents. As RuPt has the potential to overcome the intrinsic
and acquired resistance of cisplatin, it may find further
application in clinical treatment, especially for superficial tumors.
Figure 8. a) Relative tumor volumes of mice after treatment with PBS, Ru and
RuPt (n = 4). The intratumoral injections were performed at day 1 and day 6.
Irradiation condition: 450 nm, 176 mW cm−2, 10 min. b) Tumors separated
from nude mice. The red dotted circles represent tumors completely
scavenged.
Experimental Section
Synthesis and characterization
treated with Ru or RuPt upon irradiation, respectively (Figure
S22b, Supporting Information). Furthermore, z-VAD-fmk, a pan-
caspase inhibitor, can efficiently inhibit cell death caused by Ru-
and RuPt-mediated PDT (Figure S22c, Supporting Information).
These results indicate that Ru- and RuPt-mediated PDT induce
cell death through caspase-dependent apoptosis.
cis-Ru(dip)2Cl2: The precursor was synthesized according to the literature
method.[40]
[Ru(dip)2(bpm)]Cl2 (Ru): Ligand bpm (63 mg, 0.43 mmol) and the
precursor cis-Ru(dip)2Cl2 (300 mg, 0.36 mmol) were suspended in 50 mL
75% (v/v) aqueous ethanol. After refluxed for 12 h under nitrogen, the
reaction mixture was stripped of solvent and the product was precipitated
as a Cl salt in water. The crude product was separated by column
chromatography (Al2O3) eluting the product as a deep red fraction with
acetonitrile and H2O. Ru was obtained as a deep red crystalline powder.
Yield: 280.0 mg (78%). 1H NMR (500 MHz, DMSO): δ = 9.23 (dd, J = 4.8,
1.9 Hz, 2H, H15), 8.61 (d, J = 5.5 Hz, 2H, H13), 8.30 − 8.21 (m, 6H, H5,
H12, H14), 8.19 (d, J = 5.5 Hz, 2H, H11), 7.92 (d, J = 5.5 Hz, 2H, H4), 7.76
(d, J = 5.5 Hz, 2H, H7), 7.73 − 7.66 (m, 10H, H3, H6, H8), 7.66 − 7.57 (m,
12H, H1, H2, H9, H10); ESI-MS: m/z 462.45 [M − 2Cl‒]2+; elemental
analysis calcd (%) for C56H38Cl2RuN8·0.5H2O: C 67.60, H 3.85, N 11.26;
found: C 67.20, H 3.88, N 11.28.
Inhibition of tumor growth by intratumoral injection
Finally, the PDT efficacy of Ru and RuPt was evaluated in vivo
(Figure 8). When the solid tumor grew to 80‒120 mm3, nude
mice implanted with A549 cells were randomly divided into six
groups (n = 4). Ru was dissolved in PET diluent (6%
polyethylene glycol 400, 3% ethanol, 1% Tween 80 and 90%
PBS) and RuPt was loaded on a block copolymer (PLGA-PEG)
at the indicated concentration. Light irradiation was conducted 3
h after injection. The mice of control (light) and PDT groups were
irradiated with a 450 nm laser at a power density of 176 mW
cm−2 for 10 min. After PDT treatment, Ru- and RuPt-mediated
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[Ru(dip)2(μ-bpm)PtCl2]Cl2 (RuPt): RuPt was synthesized using
a
MiniPrep kit, Corning, USA) according to the protocol. After that, 2 μL
template DNA was mixed with 2 μL mtDNA or nucDNA primer pair,
SYBR Green PCR buffer and ddH2O to a final volume of 25 μL. The RT-
qPCR experiment was performed using the following cycling conditions:
denature 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 62 °C
for 60 s, and 72 °C for 30 s. The primer sequences are listed in Table S4.
building block strategy (Scheme S1, Supporting Information). Ru (100
mg, 0.1 mmol) and cis-Pt(DMSO)2Cl2 (127 mg, 0.3 mmol) were heated at
reflux in 25 mL of ethanol for 24 h in the dark. The reaction mixture was
cooled to room temperature and the solid was washed by H2O,
acetonitrile and diethyl ether. RuPt was obtained as a dark green powder.
Yield: 45.3 mg (36%). 1H NMR (500 MHz, DMSO): δ = 9.59 (dd, J = 5.8,
1.2 Hz, 2H, H15), 8.86 (d, J = 5.5 Hz, 2H, H13), 8.50 (dd, J = 5.7, 1.2 Hz,
2H, H14), 8.30 − 8.18 (m, 4H, H5, H12), 8.14 (d, J = 5.6 Hz, 2H, H11), 7.98
(d, J = 5.5 Hz, 2H, H4), 7.94 (t, J = 5.8 Hz, 2H, H7), 7.77 (d, J = 5.6 Hz,
2H, H6), 7.70 (d, J = 4.2 Hz, 8H, H3, H8), 7.67 − 7.57 (m, 12H, H1, H2, H9,
H10); ESI-MS: m/z 594.40 [M − 2Cl−]2+, 803.65 [M − bpm − Pt2+ − Cl‒]+;
elemental analysis calcd (%) for C56H38Cl4RuPtN8·1.5H2O: C 52.22, H
3.21, N 8.70; found: C 52.17, H 3.24, N 8.74.
MtDNA gel electrophoresis assay
MtDNA was extracted and purified by
a commercial spin column
quantification kit (Quick-gDNATM MiniPrep kit, ZYMO Research, USA).
Purified mtDNA (100 ng) was incubated with Ru (4 μM) or RuPt (4 μM)
for 1 h in the absence or presence of light. Light irradiation was
performed at 45 min with a 450 nm LED array (17 mW cm−2, 15 J cm−2).
Gel electrophoresis was performed on a 1% agarose gel under 100 V
voltage for 1 h. The gel was stained with 2 μL Gel-Red and the images
were captured on FluorChem M (ProteinSimple, Santa Clara, CA).
DNA platination
A549 cells were seeded in 10 cm dishes and treated with these tested
complexes for 6 h or 12 h. NucDNA and mtDNA were extracted and
purified by commercial spin column quantification kits (nucDNA: AxyPrep
Blood gDNA MiniPrep kit; mtDNA: Quick-gDNATM MiniPrep kit). The
quantification and purity of DNA were measured by NanoDrop (MD2000,
Gene Company Limitied). Only highly purified samples (A260/A230 1.8
and A280/A260 2.0) were included for analysis to avoid any artifacts.
MtDNA transcriptional inhibition
RT-qPCR was performed in a Roche LightCycler 480 Detection System
(Roche, USA) using SYBR Green I Master (Roche, USA). A549 cells
were treated with Ru or RuPt for 12 h and irradiated with a 450 nm LED
array (17 mW cm−2, 15 J cm−2). Total RNA isolation (Trizol reagent, Life
technologies, USA) and cDNA synthesis (iScript cDNA synthesis kit, BIO-
RAD, USA) are performed according to the manufacturer’s instructions.
Each PCR sample (25 μL) contains 5 ng cDNA, 0.5 μM primers, SYBR
Green PCR buffer and ddH2O. Cycling conditions: denature 95 °C for 10
min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, 72 °C for 20 s.
GAPDH and β-actin are used as the housekeeping genes to normalize
the data. The primer sequences are listed in Table S5.
MtDNA nucleoids damage remodeling
A549 cells were pretreated with the PicoGreen reagent at 3 μL/mL
dilution in warm growth medium for 1 h. PBS was used to rinse the
excess dye. Cells were further incubated with RuPt (10 μM) in a
humidified incubator linked to the confocal microscope, which provided
an atmosphere of 5% CO2 and 95% air at 37 °C. Cell imaging was
carried out at different time intervals by confocal microscopy.
Mitochondrial bioenergetic analysis
Immunofluorescence (IF) staining
The XF Cell Mito Stress Test Kit with the Seahorse XFe24 analyzer
(Seahorse Bioscience, Billerica, USA) were used to measure the oxygen
consumption rate (OCR) to determine the key parameters of
A549 cells were seeded into 12 well plates with coverslips for 24 h and
then treated with Ru and RuPt at the indicated concentrations for 12 h.
After cells were irradiated (17 mW cm−2, 15 J cm−2), they were fixed with
methanol, treated with RNase, and fixed with 4% paraformaldehyde
again. Cells were incubated with the primary antibody against 8-
oxoguanine overnight at 4 oC and incubated with the secondary antibody,
DyeLight 549-conjugated anti-mouse (Abcam, USA), for 2 h at room
temperature. After that, cells were stained with Hoechst 33342. Cell
imaging was carried out by confocal microscopy.
mitochondrial function.
A series of preliminary experiments were
performed to determine the optimal cell density (1.5 × 104 cells per well)
and the appropriate concentrations of oligomycin (1.0 μM), FCCP (0.8
μM) and a mixture of antimycin A (0.5 μM) and rotenone (0.5 μM). A549
cells were treated with Ru and RuPt for 12 h at 37 °C under a 5% CO2
atmosphere and irradiated with a 450 nm LED array (17 mW cm−2, 5 J
cm−2). The experiment was carried out as described in literature.[43]
Colocalization assay of DCF and MTDR
MtDNA amplification by qPCR
A549 cells were seeded in 35 mm dishes and treated with Ru (1 μM) or
RuPt (1 μM) for 12 h. Cells were further incubated with 10 µM H2DCFDA
for 20 min at 37 °C in the dark and then irradiated with a 450 nm LED
light array (17 mW cm−2, 5 J cm−2). Then cells were co-incubated with
MTDR (150 nM) at 37 °C for another 0.5 h and washed three times with
PBS and visualized by a confocal microscope. MTDR was excited at 633
nm, with emission was collected at 683 ± 20 nm. DCF was excited at 488
nm with emission collected at 530 ± 20 nm.
Cells were treated with Ru and RuPt for 12 h at 37 °C and irradiated with
a 450 nm LED array (17 mW cm−2, 15 J cm−2). DNA was isolated from
cell pellets using the commercial spin column quantification kit (AxyPrep
Blood gDNA MiniPrep kit) according to the manufacturer’s instructions.
The Elongase long range PCR enzyme kit (Invitrogen, USA) was used to
amplify the 8.9 kb segment of mtDNA and the 12.2 kb segment of
nucDNA.[41] The amplified products were measured by Pico Green
staining.
Analysis of mitochondrial superoxide levels
Copy number of mtDNA
A549 cells were treated with Ru (4 μM) or RuPt (4 μM) for 12 h and
irradiated with a 450 nm LED light array (17 mW cm−2, 15 J cm−2). Cells
were washed with Hank’s buffered saline (HBS) and incubated with 5 μM
MitoSox reagent (Invitrogen) in HBS for 30 min in dark. Then cells were
washed again with HBS and analyzed by confocal microscopy.
A549 cells were seeded in 60 mm dishes overnight. Subsequently, cells
were treated with Ru or RuPt for 12 h and irradiated with a 450 nm LED
array (17 mW cm−2, 15 J cm−2). DNA was extracted and purified by a
commercial spin column quantification kit (AxyPrep Blood gDNA
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This study was supported by the National Natural Science
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the 973 program (Nos. 2014CB845604 and 2015CB856301),
the Guangdong Natural Science Foundation (2015A030306023),
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A ruthenium-platinum heterobimetallic
complex is reported as a
mitochondrial DNA-targeted
Yue Zheng, Dong-Yang Zhang, Hang
Zhang, Jian-Jun Cao, Cai-Ping Tan*,
Liang-Nian Ji and Zong-Wan Mao*
photodynamic anticancer agent to
overcome cisplatin resistance.
Page No. – Page No.
Photodamaging Mitochondrial DNA to
Overcome Cisplatin Resistance by a
Ru(II)-Pt(II) Bimetallic Complex
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